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Studies on the regulation of calcium transport in cardiac and skeletal muscle sarcoplasmic reticulum Eibschutz, Barry 1983

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STUDIES ON THE REGULATION OF CALCIUM TRANSPORT IN CARDIAC AND SKELETAL MUSCLE SARCOPLASMIC RETICULUM by BARRY EIBSCHUTZ B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES i n THE FACULTY OF PHARMACEUTICAL SCIENCES DIVISION OF PHARMACOLOGY AND TOXICOLOGY We accept t h i s t h e s i s as conforming to the r e q u i r e d standards THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 1983 © BARRY EIBSCHUTZ, 1983 In 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 of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t 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 study. 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 copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or 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 allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT The s a r c o p l a s m i c r e t i c u l u m (SR) membrane p l a y s a c r i t i c a l r o l e i n e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n both s k e l e t a l and c a r d i a c muscle. In an attempt to examine the f u n c t i o n and r e g u l a t i o n of SR, we examined the unique r o l e t h a t i t p l a y s i n both normal and d i s e a s e d t i s s u e . In our e l u c i d a t i o n of the r o l e of the SR i n normal dog heart muscle, we attempted to " p u r i f y " the crude microsomes e n r i c h e d i n SR a c c o r d i n g to the method of Jones e_t, a l (1979) , i n order to minimize contamination by sarcolemmal and m i t o c h o n d r i a l membranes; both of these membranes have been shown to c o n t a i n ATPase a c t i v i t y , and may c o n t r i b u t e to s p u r i o u s r e s u l t s . Both sarcolemmal and m i t o c h o n d r i a l membrane contamination were decreased i n the Ca-oxalate loaded, p u r i f i e d SR p r e p a r a t i o n . In a d d i t i o n , a 3 - 5 - f o l d enhancement of both Ca -uptake and (Ca -Mg )-ATPase a c t i v i t y was observed i n the p u r i f i e d p r e p a r a t i o n compared to crude. When analyzed on SDS-PAGE, the absence of a 95,000 MW p r o t e i n i n the p u r i f i e d p r e p a r a t i o n was e v i d e n t . R e g u l a t i o n of the p u r i f i e d SR p r e p a r a t i o n by cAMP-dependent p r o t e i n kinase (cAMP-PK) and c a l m o d u l i n (CAM) appeared i d e n t i c a l to the s t i m u l a t i o n t y p i c a l l y observed i n crude p r e p a r a t i o n s with the e x c e p t i o n of lower s t i m u l a t i o n of Ca -uptake at higher (1.0 ;JM) f r e e Ca c o n c e n t r a t i o n s . A comparison of the two p r e p a r a t i o n s with r e s p e c t to membrane p h o s p h o r y l a t i o n r e v e a l e d t h a t i n c u b a t i o n of e i t h e r p u r i f i e d or crude SR with cAMP-PK or the c a t a l y t i c (C) subunit of cAMP-PK r e s u l t e d i n s i m i l a r time-course p r o f i l e s . In the presence of CAM, the t o t a l l e v e l of phosphoprotein - i -i n c o r p o r a t i o n was decreased i n the crude p r e p a r a t i o n compared to the p u r i f i e d , and both the i n t e n s i t y and time to complete p h o s p h o r y l a t i o n of phospholamban were markedly d i m i n i s h e d i n p u r i f i e d SR. N e v e r t h e l e s s , both crude and p u r i f i e d SR were s i m i l a r i n t h a t CAM-dependent p h o s p h o r y l a t i o n was both slower and decreased compared to t h a t observed with e i t h e r cAMP-PK or C subunit i n c u b a t i o n . An examination of the r o l e of CAM i n normal r a b b i t s k e l e t a l SR was undertaken as a r e s u l t of the d i s p u t e d c l a i m s of the mode of CAM b i n d i n g to the s k e l e t a l SR membrane. Our s t u d i e s r e v e a l e d t h a t , u n l i k e the claims of Campbell and MacLennan (1982) , a c o n s i d e r a b l e amount of CAM remained bound to the SR membrane f o l l o w i n g e x t e n s i v e washing with EGTA. SDS-PAGE of a l l supernatants r e v e a l e d a number of bands which migrated i n the r e g i o n of CAM (15.5-19.5 K d a l t o n s ) ; f o l l o w i n g high-speed c e n t r i f u g a t i o n of a l l the supernatants, the molecular weight bands assumed to be CAM d i d not appear i n the EGTA-washed fSR or sSR supernatants. An RIA f o r CAM r e v e a l e d t h a t measureable l e v e l s of CAM were present i n a l l supernatants. Higher l e v e l s of CAM were r e l e a s e d i n t o the supernatant from sSR than from fSR. In a d d i t i o n , CAM was l i b e r a t e d from supernatants b o i l e d i n the presence or absence o f EDTA r e g a r d l e s s of p r i o r EGTA-washing, although the l e v e l s of CAM d e r i v e d from v e s i c l e s t h a t been p r e - t r e a t e d with EGTA were approximately 33% l e s s than the l e v e l s obtained from v e s i c l e s which were not t r e a t e d with EGTA. CAM RIA r e v e a l e d t h a t t h i s EGTA-extractable CAM was not present i n the - ii -supernatant of EGTA-washed SR. I t was concluded t h a t the s t i m u l a t o r p r e s e n t i n b o i l e d supernatants of f a s t and slow SR was, indeed, CAM, whereas the s t i m u l a t o r r e p o r t e d by Campbell and MacLennan (1982) was most l i k e l y CAM t h a t o r i g i n a t e d from the b o i l i n g of contaminant SR membrane fragments present i n EGTA-washed SR supernatants. The f i n a l aspect of SR r e g u l a t i o n i n v e s t i g a t e d was ++ Ca -uptake i n the s k e l e t a l muscle of s t r e p t o z o t o c i n - d i a b e t i c r a t s . I t was found t h a t , s i m i l a r to the r e p o r t s i n r a t c a r d i a c SR, s k e l e t a l SR from d i a b e t i c r a t s had s i g n i f i c a n t l y depresssed l e v e l s of Ca -uptake, and e l e v a t e d l e v e l s of f r e e c a r n i t i n e and l o n g - c h a i n a c y l c a r n i t i n e s , compared to c o n t r o l s . I t was concluded t h a t the d i a b e t i c s t a t e may r e s u l t i n g e n e r a l i z e d p a t h o p h y s i o l o g y , a r e s u l t of which may be the n o n - s p e c i f i c decrease i n C a + + - u p t a k e i n SR. Sidney Katz, Ph.D A s s o c i a t e P r o f . , Pharm S c i . ACKNOWLEDGEMENTS T h i s t h e s i s i s l a r g e l y the product of two i n d i v i d u a l s : my s u p e r v i s o r , Dr. Sidney Katz, and Dr. Gary Lopaschuk who, i n Dr. Katz's absence, played an i n s t r u m e n t a l r o l e i n ma i n t a i n i n g the c o n t i n u i t y and progress of t h i s work. The p a t i e n c e , guidance, and support o f both these i n d i v i d u a l s i s deeply acknowledged. I would a l s o l i k e to thank my committee members, Dr. K. MacLeod, Dr. J . Diamond, Dr. J.H. M c N e i l l , and Dr. D. Godin f o r t h e i r c o n s t r u c t i v e c r i t i c i s m and suggestions throughout the study. S p e c i a l thanks are extended to Dr M. Bridges and Dr. B.D. R o u f o g a l i s who served as s u p e r v i s o r s d u r i n g Dr. Katz's absence, and whose suggestions were p a r t i c u l a r l y a s t u t e . I would l i k e to g r a t e f u l l y acknowledge the Canadian Heart Foundation f o r f i n a n c i a l support d u r i n g the study p e r i o d . I a l s o wish t o thank a l l members of the f a c u l t y , s t a f f , and graduate student body i n the F a c u l t y of Pharmaceutical S c i e n e s , UBC, f o r having made t h i s masters programs the most enjoyable tenure I have spent at t h i s u n i v e r s i t y . F i n a l l y , I wish to extend the most s i n c e r e thank-you and best wishes to a l l my f r i e n d s and l a b o r a t o r y c o l l e a g u e s : Dr. Mic h a e l B r i d g e s , Miss Helen Arntsen, Dr. Dorothy J e f f e r y , Dr. Twum Ansah, Mr. Bruce A l l a n , Mrs. Peggy Cros, and Mrs Loan Hoang. Without t h e i r t e c h n i c a l a s s i s t a n c e and moral support, t h i s study would not have been completed. -W-TABLE OF CONTENTS page ABSTRACT i ACKNOWLEDGEMENTS i v TABLE OF CONTENTS V LIST OF FIGURES v i i i LIST OF TABLES X INTRODUCTION 1 I Morphology of the SR 1 II ( C a + + -Mg*4 ) -ATPase of the SR Membrane 3 A. S t r u c t u r e 4 B. K i n e t i c P r o p e r t i e s 5 C. R e a c t i o n Mechanism 6 D. Molecular Models of C a + + T r a n s p o r t 10 I I I Comparative Features of C a r d i a c and S k e l e t a l Muscle C o n t r a c t i l i t y 10 A. E x c i t a t i o n - C o n t r a c t i o n C o u p l i n g i n C a r d i a c and S k e l e t a l Muscle 10 B. M o r p h o l o g i c a l and B i o c h e m i c a l Charac-t e r i s t i c s of S k e l e t a l and C a r d i a c SR 14 IV Comparative Aspects of F a s t - t w i t c h and Slow-twitch S k e l e t a l Muscle 16 A. C h a r a c t e r i s t i c s of F a s t - t w i t c h and Slow-t w i t c h SR 17 V R e g u l a t i o n of the (Ca -Mg )-ATPase i n SR 19 A. R e g u l a t i o n of C a r d i a c SR 21 i) C y c l i c AMP-dependent P r o t e i n Kinase Reg-u l a t i o n of C a r d i a c SR 21 i i ) Calmodulin mediated R e g u l a t i o n of C a r d i a c SR 25 B. R e g u l a t i o n of S k e l e t a l SR 33 VI Disease S t a t e s and the SR 35 A. S k e l e t a l Muscle 35 B. C a r d i a c Muscle 40 OBJECTIVES OF THE STUDY 47 MATERIALS AND METHODS 50 - V -A. MATERIALS 50 (i) Animals 50 a) S t u d i e s u t i l i z i n g c a r d i a c microsomes en r i c h e d i n SR 50 b) S t u d i e s u t i l i z i n g s k e l e t a l microsomes en r i c h e d i n SR 50 c) S t u d i e s u t i l i z i n g s k e l e t a l microsomes en r i c h e d i n SR from d i a b e t i c animals 50 ( i i ) Chemicals 51 a) R a d i o i s o t o p e s 51 b) Reagents 51 ( i i i ) Apparatus 52 B. METHODS 54 1. P r e p a r a t i v e Methods 54 a) P r e p a r a t i o n o f s k e l e t a l muscle and c a r d i a c muscle microsomes e n r i c h e d i n SR 54 b) P r e p a r a t i o n o f a p u r i f i e d c a r d i a c microsomal p r e p a r a t i o n e n r i c h e d i n SR 54 c) P r e p a r a t i o n o f b o i l e d s k e l e t a l SR e x t r a c t s 55 d) P r e p a r a t i o n of EGTA-washed s k e l e t a l SR e x t r a c t s 56 e) P r e p a r a t i o n o f red c e l l membranes 58 i) Methods 1 and 2 58 2. A n a l y t i c a l Methods 59 a) Measurement of c a l c i u m uptake by s k e l e t a l and c a r d i a c muscle microsomes enr i c h e d i n SR 59 - c a l c u l a t i o n o f calcium-uptake a c t i v i t y 59 b) Assay o f (Ca** -Mg*4 )-ATPase a c t i v i t y i n c a r d i a c microsomes e n r i c h e d i n SR 60 c) P h o s p h o r y l a t i o n of c a r d i a c membrane v e s i c l e s 61 d) Measurement of "patent" and " l a t e n t " Na + ,K +-ATPase a c t i v i t y 62 e) Measurement of cytochrome c oxidase a c t i v i t y 62 f) Measurement of f r e e and lo n g - c h a i n a c y l c a r n i -t i n e s i n s k e l e t a l muscle SR i s o l a t e d from d i a b e t i c r a t s 63 g) Assay of (Ca**-Mg )-ATPase a c t i v i t y i n red blood c e l l membranes 65 h) Determination o f calm o d u l i n content of s k e l e t a l SR e x t r a c t s 66 3. E l e c t r o p h o r e t i c Methods 67 a) Sodium dodecyl s u l f a t e p o l y a c r y l a m i d e s l a b g e l e l e c t r o p h o r e t i c s e p a r a t i o n of p r o t e i n s 67 b) Sodium dodecyl s u l f a t e p o l y a c r y l a m i d e s l a b g e l e l e c t r o p h o r e s i s and autoradiography of phosphory-l a t e d c a r d i a c SR p r e p a r a t i o n s 67 - vi -4. M i s c e l l a n e o u s Methods 68 a) P r o t e i n assay 68 b) S t a t i s t i c a l a n a l y s i s 69 c) Determination of f r e e c a l c i u m c o n c e n t r a t i o n 69 RESULTS 70 A) C h a r a c t e r i z a t i o n of a p u r i f i e d c a r d i a c SR p r e p a r a t i o n 70 i ) Assessment of p u r i t y u s i n g marker enzyme assays 70 i i ) Calcium uptake and ATPase a c t i v i t y i n the p u r i f i e d c a r d i a c SR p r e p a r a t i o n 72 i i i ) R e g u l a t i o n of the p u r i f i e d c a r d i a c SR p r e p a r a t i o n 76 B) S t u d i e s on the r o l e of calmodulin i n s k e l e t a l muscle SR 85 i) I n d i r e c t measurement of calmodulin a c t i v i t y by Ca +*-ATPase a c t i v a t i o n 85 i i ) D i r e c t measurements of calmodulin by r a d i o -immunoassay 96 i i i ) SDS-PAGE 99 C) SR c a l c i u m t r a n s p o r t i n a c h r o n i c d i s e a s e s t a t e : e x p e r i m e n t a l l y - i n d u c e d d i a b e t e s 102 DISCUSSION 107 I R e g u l a t i o n of a p u r i f i e d c a r d i a c SR p r e p a r a t i o n 107 II S t u d i e s examining the presence of calmodulin i n f a s t and slow-twitch s k e l e t a l muscle SR 117 I I I The r e g u l a t i o n of s k e l e t a l SR i n a d i s e a s e s t a t e : c h r o n i c , e x p e r i m e n t a l l y - i n d u c e d d i a b e t e s 125 LIST OF FIGURES FIGURE page 1. Two molecular models f o r a c t i v e t r a n s p o r t of c a l c i u m 9 2. P o s s i b l e e f f e c t s o f ^ - a d r e n e r g i c agents on c o n t r a c t i l i t y , mediated by enhanced c a l c i u m uptake by the SR 23 3. The a c t i v a t i n g and r e l a x i n g e f f e c t s of c a t e c h o l -amines mediated by c a l c i u m r e l e a s e and phosphory-l a t i o n 26 4. G e n e r a l i z e d s t r u c t u r a l c h a r a c t e r i s t i c s of calmo d u l i n i n h i b i t o r s 29 5. Scheme d e p i c t i n g the known and p o s t u l a t e d r e g u l a t o r y mechanisms ^of c a r d i a c SR c a l c i u m t r a n s p o r t 31 6. P o s t u l a t e d sequence of steps i n v o l v e d i n he a r t f a i l u r e due to c a l c i u m o v e r l o a d or d e f i c i e n c y 44 7. Methodology o f p r e p a r a t i o n of supernatants used f o r s k e l e t a l SR s t u d i e s 57 8. SDS-PAGE of crude and p u r i f i e d c a r d i a c SR 73 9. Time-course of c a l c i u m uptake i n crude and p u r i f i e d SR 74 10. E f f e c t o f v a r i o u s c a l c i u m c o n c e n t r a t i o n s on c a l c i u m uptake i n crude and p u r i f i e d c a r d i a c SR 75 11. Calcium uptake a c t i v i t y at two d i f f e r e n t f r e e c a l c i u m c o n c e n t r a t i o n s (0.2 and 1.0 ;J.M) i n a p u r i f i e d p r e p a r a t i o n o f c a r d i a c SR i n the presence of c a l m o d u l i n (1 ,uM) or cAMP (1 ,UM) p l u s cAMP-dependent p r o t e i n kinase (0.5 mg/ml) 78 12. Calcium-dependent ATPase a c t i v i t y at two d i f f e r e n t f r e e c a l c i u m c o n c e n t r a t i o n s (0.2 and 1.0 JJM) i n a p u r i f i e d p r e p a r a t i o n o f c a r d i a c SR i n the presence of calmodulin (1 ;uM) , cAMP (1 ;JM) p l u s cAMP-dependent p r o t e i n kinase (0.5 mg/ml), or both calmodulin p l u s cAMP p l u s cAMP-dependent p r o t e i n k i n a s e 79 13. Time-course of p h o s p h o r y l a t i o n i n crude and p u r i f i e d c a r d i a c SR 81 -Vi i i — 14. ""P-Autoradiography time-course of crude c a r d i a c SR phosphorylated f o r v a r i o u s time i n t e r v a l s with e i t h e r cAMP-dependent p r o t e i n kinase or C subunit....82 15. ^"P-Autoradiography time-course of crude or p u r i f i e d SR phosphorylated i n * t h e presence of cal m o d u l i n 84 16. E f f e c t of calmodulin on red c e l l calcium-dependent ATPase a c t i v i t y 86 17. E f f e c t of b o i l e d or b o i l e d + EDTA-treated super-natants d e r i v e d from f a s t or slow SR on red c e l l ( C a + + -Mg^ ) -ATPase a c t i v i t y 88 18. E f f e c t s o f c a l m o d u l i n (60 nM) i n the presence and absence of t r i f l u o p e r a z i n e (60 ;LIM) on red c e l l calcium-dependent ATPase a c t i v i t y 90 19. E f f e c t s of Compound 48/80 on red blood c e l l c a l c i u m -dependent ATPase a c t i v i t y i n the presence and absence of calmodulin 93 20. Calcium-uptake a c t i v i t y of s k e l e t a l f a s t SR, slow SR, or 1 mM EGTA-washed f a s t or slow SR, i n the presence of ca l m o d u l i n (0.24 uM) 95 21. SDS-PAGE of v a r i o u s supernatants d e r i v e d from e i t h e r b o i l e d + EDTA-treated or 1 mM EGTA-washed s k e l e t a l SR 100 22. E f f e c t o f c h r o n i c d i a b e t e s (120 days) on s k e l e t a l SR c a l c i u m uptake at v a r i o u s c a l c i u m c o n c e n t r a t i o n s 104 LIST OF TABLES p a g e TABLE 1. Calmodulin-dependent enzymes and processes t h a t are i n h i b i t e d by p h e nothiazine a n t i -p s y c h o t i c s 28 2. Marker enzyme assays f o r d e t e r m i n a t i o n of the degree of m i t o c h o n d r i a l and sarcolemmal contamination of p u r i f i e d and crude c a r d i a c SR p r e p a r a t i o n s 71 3. Measurement of calcium-ATPase a c t i v i t y i n crude and p u r i f i e d c a r d i a c SR 77 4. L e v e l s of (Ca + + -Mg4* )-ATPase a c t i v i t y o b t ained f o l l o w i n g i n c u b a t i o n of b o i l e d + EDTA-treated f a s t s k e l e t a l SR e x t r a c t s i n the presence or absence of TFP (60 ^M) 89 5. Red c e l l membrane calcium-ATPase a c t i v i t y i n the presence of b o i l e d + EDTA-treated supernatants d e r i v e d from f a s t or slow s k e l e t a l SR and the e f f e c t of Compound 48/80 (10 jug/ml) 94 6. Measured l e v e l s o f red c e l l calcium-ATPase a c t i v i t y i n the presence of a) supernatants of 1 mM EGTA-washed f a s t or slow SR v e s i c l e s b o i l e d i n the presence of 0.2 mM EDTA, or b) EGTA-washed f a s t or slow SR supernatants, and the e f f e c t of TFP (60 jaM) 97 7. Radioimmunoassay f o r ca l m o d u l i n i n v a r i o u s p r e p a r a t i o n s of f a s t and slow s k e l e t a l SR 98 8. Molecular weights of some prominent bands below 20 Kdaltons i n r a b b i t s k e l e t a l SR e x t r a c t s as shown i n F i g u r e 21 101 9. Measurement of body weight, serum g l u c o s e , and serum i n s u l i n l e v e l s of c o n t r o l and d i a b e t i c rats..103 10. L e v e l s of c a r n i t i n e and l o n g - c h a i n a c y l c a r n i t i n e s i n c a r d i a c microsomal p r e p a r a t i o n s e n r i c h e d i n SR d e r i v e d from c o n t r o l and three-month d i a b e t i c r a t s 106 — x -In both heart and s k e l e t a l muscle, c o n t r a c t i o n and r e l a x a t i o n are c o n t r o l l e d by the i n t r a c e l l u l a r d i s t r i b u t i o n of Ca +* , r e g u l a t e d at l e a s t i n p a r t by a s p e c i a l i z e d membranous network surrounding the m y o f i b r i l s , the sarco p l a s m i c r e t i c u l u m (SR). Muscle r e l a x a t i o n i s b e l i e v e d t o occur when c y t o s o l i c 44 4+ Ca i s taken up by the SR, thus lowering the f r e e Ca c o n c e n t r a t i o n below t h a t needed to a c t i v a t e the c o n t r a c t i l e -7 p r o t e i n s (10 M). Muscle c o n t r a c t i o n , on the other hand, at l e a s t i n s k e l e t a l muscle, i s thought to r e q u i r e SR as i t s source of " a c t i v a t o r " C a + + f o r b i n d i n g to t r o p o n i n , the Ca*"* receptor o f the c o n t r a c t i l e p r o t e i n s . In c a r d i a c muscle, the i n t e r n a l Ca s t o r e has not been u n e q u i v o c a l l y l o c a l i z e d to the SR and may be a s s o c i a t e d with SR, sarcolemma, or both. Because of the unique r o l e of the SR i n mediating both c o n t r a c t i o n and r e l a x a t i o n , the k i n e t i c s of c a l c i u m s e q u e s t r a t i o n and r e l e a s e by t h i s o r g a n e l l e have been i n t e n s i v e l y i n v e s t i g a t e d . I Morphology of the SR The SR i s f r e q u e n t l y d i v i d e d i n t o two c a t e g o r i e s (Sommer and Waugh, 1976) which are d i s t i n c t m o r p h o l o g i c a l l y and, p o s s i b l y , f u n c t i o n a l l y : (1) j u n c t i o n a l SR and (2) l o n g i t u d i n a l SR. J u n c t i o n a l SR i s t h a t p o r t i o n o f SR which c l o s e l y abuts the plasmalemma at the l e v e l of the t r a n s v e r s e (T) tubules to form a t r i a d : two elements of j u n c t i o n a l SR form c o u p l i n g s ( t e r m i n a l c i s t e r n a e ) on o p p o s i t e s i d e s of a T t u b u l e . The T t u b u l e s , running p e r p e n d i c u l a r to the SR, are not p a r t of the SR but -1-are, r a t h e r , continuous with the s u r f a c e membrane. The t r i a d i s c o n s i d e r e d to be a key c o n t r o l p o i n t f o r at l e a s t two steps i n e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g : (1) the s i t e of storage and r e l e a s e of a c t i v a t o r c a l c i u m and (2) the s i t e at which the a c t i o n p o t e n t i a l i s t r a n s l a t e d i n t o a " t r i g g e r " f o r c a l c i u m r e l e a s e (Endo, 1977; F a b i a t o and F a b i a t o , 1977). L o n g i t u d i n a l or " f r e e " SR represents t h a t SR not a s s o c i a t e d with the T t u b u l e s : i t i s e l e c t r o n l u c e n t and forms the meshwork surrounding the A and I bands. The SR membrane i t s e l f i s a l i p i d b i l a y e r c o n s i s t i n g of s e v e r a l kinds of p h o s p h o l i p i d s : 60-75% p h o s p h a t i d y l c h o l i n e , 10-20% phosphatidylethanolamine, 5-10% p h o s p h a t i d y l s e r i n e , 10% p h o s p h a t i d y l i n o s i t o l , and s m a l l e r amounts of other l i p i d s (Meissner and F l e i s c h e r , 1971; MacLennan et a l , 1971). There are four major types of p r o t e i n embedded i n the membrane: (1) the 100,000 MW ATPase, which p a r t i c i p a t e s d i r e c t l y i n a c t i v e Ca t r a n s p o r t and accounts f o r 60-90% of the t o t a l p r o t e i n i n the s k e l e t a l SR membrane ( I n e s i , 1972; Meissner e t a l , 1973) and 35-40% of p r o t e i n i n c a r d i a c SR (Suko and Hasselbach, 1976), (2) c a l s e q u e s t r i n , a 66,000 MW (55,000 MW i n c a r d i a c SR; Campbell et a l , 1983) h i g h l y a c i d i c p r o t e i n able to bind 900 nmole Ca + +/mg p r o t e i n and thought to be r e s p o n s i b l e f o r c a l c i u m storage i n the SR lumen (MacLennan and Wong, 1971), (3) two h i g h - a f f i n i t y c a l c i u m b i n d i n g p r o t e i n s (MW = 53,000 & 160,000), and (4) a low molecular weight p r o t e o l i p i d (12,000 MW) . The r e l a t i v e content of these four types of p r o t e i n v a r i e s among the d i f f e r e n t p o p u l a t i o n s of v e s i c l e s i s o l a t e d by i s o p y c n i c -2-c e n t i f u g a t i o n and may e x p l a i n the d i s c r e p e n c y i n estimates of amount of a p a r t i c u l a r p r o t e i n . For example, Meissner (1975) d i s t i n g u i s h e d three d i f f e r e n t f r a c t i o n s of r a b b i t s k e l e t a l SR by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n : "heavy", " i n t e r m e d i a t e " , and " l i g h t " . Subsequent work has r e v e a l e d t h a t c a l s e q u e s t r i n i n v i t r o i s found almost e n t i r e l y i n the "heavy" f r a c t i o n (Caswell et a l , 1976; Campbell et a l , 1980); i n s i t u , i t i s found i n the t e r m i n a l c i s t e r n a e (Campbell et a l , 1980). T h i s has l e d to the c o n c l u s i o n t h a t "heavy" v e s i c l e s are d e r i v e d from the t e r m i n a l c i s t e r n a e . V e s i c l e s d e r i v e d from t h i s SR f r a c t i o n , then, may cause i n v e s t i g a t o r s to overestimate the amount of c a l s e q u e s t r i n as a p r o p o r t i o n of t o t a l p r o t e i n . I t i s of i n t e r e s t t h a t immunofluorescence l o c a l i z a t i o n s t u d i e s i n s k e l e t a l muscle i n s i t u have i d e n t i f i e d the (Ca -Mg )-ATPase throughout the l o n g i t u d i n a l SR but not i n t e r m i n a l c i s t e r n a e (Jorgensen et a l , 1979), suggesting t h a t the former may be l a r g e l y concerned with c a l c i u m uptake, whereas j u n c t i o n a l SR, with i t s l a r g e Ca - c a l s e q u e s t r i n s t o r e , may f u n c t i o n as the +4-s i t e of Ca r e l e a s e . II The (Ca -Mg )-ATPase of the SR membrane Much of the c u r r e n t understanding of the r o l e of SR i n mediating muscle r e l a x a t i o n stems from the d i s c o v e r y of an ATP-dependent s e q u e s t r a t i o n of c a l c i u m by v e s i c l e s d e r i v e d from s k e l e t a l muscle c e l l homogenates (Hasselbach and Makinose, 1961; 1962; 1963). V e s i c l e s d e r i v e d from s k e l e t a l SR demonstrated a Ca - s t i m u l a t e d , Mg -dependent ATPase a c t i v i t y -3-which was able to t r a n s p o r t 2 moles of C a t r inward f o r each mole of ATP hydrolyzed a g a i n s t a 1000-5000 f o l d C a ^ c o n c e n t r a t i o n g r a d i e n t (Hasselbach and Makinose, 1962). Two d i f f e r e n t ATPase a c t i v i t i e s were soon d i s t i n g u i s h e d : a ++ 44 Mg -dependent or " b a s a l " ATPase which r e q u i r e d o n l y Mg f o r i t s a c t i v a t i o n (Hasselbach, 1964) , and a Ca - s t i m u l a t e d 4-4-Mg -dependent ATPase whose a c t i v i t y was c o r r e l a t e d with c a l c i u m t r a n s p o r t . Only the l a t t e r w i l l be d i s c u s s e d here. (A) S t r u c t u r e The (Ca + +-Mg + + )-ATPase was f i r s t s u c c e s s f u l l y p u r i f i e d by MacLennan (1970) as a l i p o p r o t e i n complex. The enzyme has s i n c e been shown to be a s i n g l e p o l y p e p t i d e c h a i n o f 100,000 MW which can be cle a v e d by t r y p s i n i n t o fragments of 50,000 and 45,000 MW (Migala e t a l , 1973). The l a t t e r fragment i s cle a v e d by prolonged d i g e s t i o n i n t o two fragments of approximately 30,000 and 20,000 MW (Stewart et a l , 1976). Each of the three fragments has been shown to c o n t a i n d i f f e r e n t f u n c t i o n a l s i t e s ; o f p a r t i c u l a r note are the s i t e s of p h o s p h o r y l a t i o n ( the 30,000 MW fragment; Thorley-Lawson and Green, 1973) and Ca4"* b i n d i n g (the 20,000 d a l t o n fragment; Pick and Racker, 1979). The enzyme appears to be asymmetrically embedded i n the SR membrane, with one p a r t p r o t r u d i n g from the cy t o p l a s m i c s u r f a c e while the remainder r e s i d e s i n the l i p i d b i l a y e r (deMeis, 1981). In a d d i t i o n , i t i s thought t h a t the ATPase i s s e l f - a s s o c i a t e d i n the membrane as oligomers, with each ATPase p e p t i d e able to perform the e n t i r e c a t a l y t i c c y c l e o f ATP -4-h y d r o l y s i s and C a " t r a n s p o r t (Moller et a l , 1982) . Aggregation of the ATPase monomers i n t o the oligomer appears to be promoted by the p r e v a i l i n g l i p i d environment. T h i s was demonstrated i n d i r e c t l y by the f i n d i n g t h a t almost complete replacement of the enzyme's endogenous l i p i d s by s y n t h e t i c l i p i d (Warren e t  a l r 1974) or n o n i o n i c detergents (Dean and Tanford, 1978) r e s u l t e d i n a monomeric ATPase which r e t a i n e d f u l l a c t i v i t y . When detergent was removed (LeMaire et a l , 1978) and/or endogenous p h o s p h o l i p i d s r e p l a c e d , the ATPase p r o t e i n s e l f - a s s o c i a t e d i n t o l a r g e r aggregates. (B) K i n e t i c P r o p e r t i e s of the (Ca -Mg )-ATPase Hasselbach and Makinose (1962) found t h a t s k e l e t a l SR v e s i c l e s c a t a l y z e d an exchange of i n o r g a n i c phosphate (Pi) between ADP and ATP i n the presence of Ca and Mg . T h i s prompted s p e c u l a t i o n of a high-energy phosphoprotein i n t e r m e d i a t e (EP) formed d u r i n g the a c t i v e t r a n s p o r t of C a + + . K i n e t i c a n a l y s i s by Yamamoto and Tonomura (1967; 1968) showed t h a t a p r o t e i n of SR v e s i c l e p r e p a r a t i o n s was phosphorylated 32. when incubated with P-ATP and quenched with t r i c h l o r o a c e t i c a c i d . Phosphoprotein l e v e l s , a t steady s t a t e , depended on the ++ c o n c e n t r a t i o n s of Ca and ATP and p a r a l l e l e d the ++ Ca -dependent ATPase a c t i v i t y . When the pre-steady s t a t e of the ATPase was f o l l o w e d i n s k e l e t a l SR by a rapid-mixing d e v i c e , the r e a c t i o n i n t e r m e d i a t e EP was q u i c k l y formed i n the presence of suboptimal ATP and c a l c i u m c o n c e n t r a t i o n s , and reached a maximum w i t h i n 50 msec at 20 C ( F r o e l i c h and T a y l o r , -5-1976) . Inorganic phosphate, assumed to be l i b e r a t e d from EP, was monitored and shown to l a g i n i t i a l l y ( c o i n c i d i n g with the r a p i d i n c r e a s e i n EP formation) and then i n c r e a s e ( c o i n c i d i n g with the completion of EP f o r m a t i o n ) . Thus, i t i s now w e l l e s t a b l i s h e d t h a t EP i s the true i n t e r m e d i a t e of the ATPase r e a c t i o n . (c) R e a c t i o n Mechanism of the (Ca -Mg )-ATPase Based on the k i n e t i c analyses of EP formation and the r e l e a s e of the products ADP and P i , a simple r e a c t i o n mechanism which represented the c o u p l i n g between the ATPase and c a l c i u m t r a n s p o r t was suggested by Tada et a l (1978): where " i n " and "out" represent the i n s i d e and o u t s i d e of the SR membrane v e s i c l e s , r e s p e c t i v e l y . Two moles of Ca and one mole of ATP were thought to bind to one mole of the ATPase enzyme (E) at the outer s u r f a c e of the membrane, forming the 44. M i c h a e l i s complex E;ATP;Ca^. Calcium i s then t r a n s l o c a t e d i n s i d e the membrane when EP i s formed. Dephosphorylation of EP i s accompanied by the r e l e a s e of Ca from the enzyme i n t o the v e s i c l e i n t e r i o r . T h i s scheme has s i n c e been broadened by the f i n d i n g s of (1) at l e a s t two forms of EP, an A D P - s e n s i t i v e (E, P) and A D P - i n s e n s i t i v e (E^P) (Shikegawa et a l , 1978), which e x h i b i t d i f f e r e n t a f f i n i t i e s f o r Ca , and (2) high and low E + ATP + 2Ca(out) —E;ATP;Ca,(out) E + ADP + P i + 2Ca(in) E P;Ca,(in) + ADP -6-44-a f f i n i t y b i n d i n g s i t e s f o r Ca on the SR membrane. A r e v i s e d mechanism i s presented below (Tada and Katz, 1 9 8 2 ) : ATP E, ;Ca(out)- E, ;ATP;Ca(out): flPP •E ->-P;Ca(in) i .4+ E£;Mg; CO ( iv) E£;Mg;Pi ~7~ Ez—P;,Mg In t h i s scheme, EP i s now the sum of E^P and E^P and c a l c i u m i s t r a n s l o c a t e d from o u t s i d e to i n s i d e the membrane when E ~ P;Ca(in) i s formed and i s r e l e a s e d w i t h i n the v e s i c l e i n t e r i o r when E ~ P ; C a ( i n ) i s converted t o E -*P;Mg. The two major r a t e - l i m i t i n g steps are the con v e r s i o n of E^to E , ( s t e p i ) and the decomposition of E ~P (step iv) , under s a t u r a t i n g c o n c e n t r a t i o n s of Ca , ATP, and a l k a l i metals (Na* and K*). •44- 4 4 Both a l k a l i metals and d i v a l e n t c a t i o n s (Ca , Mg ) a c c e l e r a t e EP decomposition; i n the absence of a l k a l i metals, the r a t e - l i m i t i n g step becomes E^ ,P decomposition (step v) , and not step i v ) . A c t i v a t i o n of the s k e l e t a l SR pump d i s p l a y s a s i g m o i d a l dependence on the C a + + c o n c e n t r a t i o n i n the medium, with -M-half-maximal s a t u r a t i o n at 0.5 ;JM Ca (deMeis, 1 9 7 1 ) . The Km f o r c a l c i u m i n c a r d i a c SR i s somewhat higher ~ l - 2 J J M (Shikegawa et a l , 1 9 7 6 ) . Two h i g h - a f f i n i t y Ca* + b i n d i n g s i t e s have been shown to be exposed on the outer s u r f a c e of s k e l e t a l SR v e s i c l e s (Meissner and F l e i s c h e r , 1 9 7 3 ) and appear to operate through a c o o p e r a t i v e mechanism such t h a t b i n d i n g o f the f i r s t Ca i o n r e s u l t s i n a p r o t e i n c o n f o r m a t i o n a l change which then i n c r e a s e s the a f f i n i t y f o r b i n d i n g of a second Ca i o n ( I n e s i et a l , 1980) . Once the enzyme becomes phosphorylated, the h i g h - a f f i n i t y s i t e s are converted to a l o w - a f f i n i t y s t a t e (Ikemoto, 1974) , thereby a l l o w i n g Ca* + to be r e l e a s e d i n s i d e the v e s i c l e s . Mg has a l s o been shown to p l a y at l e a s t two important r o l e s (Yamamoto et a l , 1979) : (1) to a c c e l e r a t e the decomposition of EP formed d u r i n g the r e a c t i o n ( I n e s i et a l , 1974) , and (2) to form an equimolar complex with ATP and serve as the t r u e s u b s t r a t e f o r the Ca -dependent ATPase. T h i s +•*• l a t t e r r o l e of Mg was i n f e r r e d by a number of o b s e r v a t i o n s : Weber e t a l (1966) demonstrated t h a t Mg 4* c o n c e n t r a t i o n s t h a t exceeded those of ATP r e s u l t e d i n op t i m a l a c t i v i t y whereas the Ca -dependency p r o f i l e of the enzyme was not a l t e r e d over a 44 wide range of Mg c o n c e n t r a t i o n s . F r o e l i c h and T a y l o r (1975) found t h a t the i n i t i a l v e l o c i t y of EP formation was p r o p o r t i o n a l to the amount of Mg-ATP complex. In a d d i t i o n to the requirement of the enzyme f o r d i v a l e n t c a t i o n s , two p h y s i c a l parameters, pH and temperature, a l s o p l a y a c r i t i c a l r o l e . The t r a n s i t i o n temperature of 20 C (Johnson and I n e s i , 1969) appears to be r e l a t e d to the phase t r a n s i t i o n of the SR p h o s p h o l i p i d b i l a y e r , while enzymatic a c t i v i t y e x h i b i t s a b e l l - s h a p e d curve with an op t i m a l pH between 6.5-7.5. -8-\ 1 1 M 1 1 B Mo*.i. Port Maati FIGURE 1. 2 J W O m o l e c u l a r models f o r a c t i v e t r a n s -p o r t o f Ca , tak e n from Yamamoto e t _ a l (1979). In each model, X r e p r e s e n t s the Ca t o be t r a n s -l o c a t e d . (A) R o t a r y - c a r r i.er model. (B) M o b i l e -pore model. For e x p l a n a t i o n , see t e x t . -9-(D) Molecular Models of Calcium Transport In the process of h y d r o l y t i c cleavage of ATP by the SR ATPase, Ca i s t r a n s l o c a t e d from o u t s i d e to i n s i d e the membrane. A number of models of C a + * t r a n s p o r t have thus been suggested on the b a s i s of the known s t r u c t u r e of the enzyme, and can be d i v i d e d i n t o two c a t e g o r i e s (Yamamoto et a l , 1979): " r o t a r y " and "mobile pore" models (Figure 1 ) . "Rotary" models propose a r o t a t i o n w i t h i n the l i p i d b i l a y e r able to t r a n s l o c a t e bound c a l c i u m , while "mobile pore" models p o s t u l a t e t h a t the enzyme, through a c o n f o r m a t i o n a l change, forms a pore through which c a l c i u m i s t r a n s p o r t e d . Thus f a r , these models have had l i t t l e e xperimental support and have been deemed u n s a t i s f a c t o r y p r i m a r i l y due to thermodynamic o b j e c t i o n s (Tada et a l . 1978; Yamamoto et a l , 1979). I l l Comparative Features of C a r d i a c and S k e l e t a l Muscle C o n t r a c t i l i t y (A) E x c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n c a r d i a c and s k e l e t a l muscle E x c i t a t i o n - c o n t r a c t i o n (E-C) c o u p l i n g i s e s s e n t i a l l y the process by which e l e c t r i c a l a c t i v i t y at the sarcolemma (the a c t i o n p o t e n t i a l ) i s t r a n s l a t e d i n t o c a l c i u m r e l e a s e r e q u i r e d f o r c o n t r a c t i o n . R e l a x a t i o n , as d i s c u s s e d b r i e f l y e a r l i e r , r e s u l t s when the f r e e c a l c i u m c o n c e n t r a t i o n drops below the -7 10 M c o n c e n t r a t i o n r e q u i r e d to a c t i v a t e the c o n t r a c t i l e -10-p r o t e i n s . A p r i o r i , there i s no reason to expect much s i m i l a r i t y i n the E-C c o u p l i n g i n s k e l e t a l and c a r d i a c muscle. S k e l e t a l muscle, t y p i c a l l y , i s under n e u r a l c o n t r o l : f o r c e g e n e r a t i o n i s accomplished by the " v o l u n t a r y " a c t i v a t i o n of motor u n i t s . A s i n g l e neuron may c o n t r a c t as few as h a l f a dozen f i b r e s or as many as a few thousand. Neural " f i n e - t u n i n g " i s achieved v i a a complex i n t e r p l a y of a f f e r e n t and e f f e r e n t p o l y s y n a p t i c feedback l o o p s . The h e a r t , i n c o n t r a s t , does not have a " v o l u n t a r y " i n n e r v a t i o n system but c o n t r a c t s r h y t h m i c a l l y with i t s e n t i r e muscle mass a c t i v a t e d i n an " a l l - o r - n o n e " f a s h i o n . A c t i o n p o t e n t i a l s are i n i t i a t e d i n spontaneously d e p o l a r i z i n g pacemaker c e l l s i n the s i n o - a t r i a l node which are then propagated to adjacent c e l l s by low r e s i s t a n c e gap j u n c t i o n s i n the i n t e r c a l a t e d d i s c s (Noble, 1975). Free C a + * c o n c e n t r a t i o n does not r i s e as r a p i d l y as i t does i n f a s t s k e l e t a l c e l l s nor does i t s a t u r a t e the c o n t r a c t i l e p r o t e i n s to produce a 100% s t a t e of a c t i v a t i o n i n a s i n g l e t w i t c h . Incomplete a c t i v a t i o n can be viewed as advantageous s i n c e the h e a r t ' s f o r c e of c o n t r a c t i o n i s r e g u l a t e d by a number of f a c t o r s (which do not a f f e c t s k e l e t a l muscle c o n t r a c t i l i t y ) which allow f o r a reserve c o n t r a c t i l e c a p a c i t y . These i n c l u d e a c t i o n p o t e n t i a l d u r a t i o n , s t r e t c h ( S t a r l i n g ' s law), e x t e r n a l Ca and Na c o n c e n t r a t i o n , hormones and n e u r o t r a n s m i t t e r s (Chapman, 1979). N e v e r t h e l e s s , upon c l o s e r i n s p e c t i o n , p a r t i c u l a r l y at the l e v e l of the c o n t r a c t i l e p r o t e i n s and SR, the E-C c o u p l i n g mechanism i n the two muscle types appears fundamentally s i m i l a r . In both c a r d i a c and -11-s k e l e t a l muscle, e x c i t a t i o n i s i n i t i a t e d by a c t i v a t i o n of a sodium channel r e s u l t i n g i n r a p i d d e p o l a r i z a t i o n . In s k e l e t a l muscle, the d e p o l a r i z a t i o n i s q u i c k l y f o l l o w e d by r e p o l a r i z a t i o n as sodium c u r r e n t i s i n a c t i v a t e d and potassium c u r r e n t i s a c t i v a t e d . The t r a n s i e n t a c t i o n p o t e n t i a l " s p i k e " i s then t r a n s m i t t e d along the sarcolemma, down the T t u b u l a r network to the SR which, i n t u r n , r e l e a s e s Ca . At the c e s s a t i o n o f n e u r a l s t i m u l a t i o n , Ca begins to be pumped back i n t o the SR. In h e a r t , the sodium channel i s s i m i l a r l y i n a c t i v a t e d w i t h i n a few m i l l i s e c o n d s , but r e p o l a r i z a t i o n does not ensue immediately because of the a c t i v a t i o n of a slow inward c a l c i u m c u r r e n t ( i s i ) which r e s u l t s i n the c h a r a c t e r i s t i c p l a t e a u i n the a c t i o n p o t e n t i a l o f c a r d i a c muscle. In c a r d i a c muscle, u n l i k e s k e l e t a l muscle, the e n t r y o f ++ Ca from the e x t r a c e l l u l a r f l u i d i s e s s e n t i a l f o r the maintenance of c o n t r a c t i l i t y (Ringer, 1883). The estimated i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n present i n the heart with each beat (2 ;J.M) i s subt h r e s h o l d f o r d i r e c t a c t i v a t i o n of the myofilaments but has l e d to s p e c u l a t i o n t h a t an i n t e r n a l pool of a c t i v a t o r Ca i s p r e s e n t . Due to the r e l a t i v e s p a r s i t y of SR i n c a r d i a c muscle (see below) and an i n c r e a s e d percentage of s u r f a c e membrane components (e.g. SL and T tubules) compared to s k e l e t a l muscle, both SR and SL and i t s components have been proposed as the source of a c t i v a t o r C a * + . In s k e l e t a l muscle, the r o l e o f the SR as the source of •H a c t i v a t o r Ca f o r c o n t r a c t i o n remains l a r g e l y undisputed. -12-S t u d i e s by Huxley and T a y l o r (1958) demonstrated t h a t a d e p o l a r i z i n g s o l u t i o n administered i n t o the T t u b u l a r system of s k e l e t a l muscle r e s u l t e d i n a l o c a l i z e d c o n t r a c t i o n at the hemisarcomeres, suggesting t h a t d e p o l a r i z a t i o n at the t r i a d l e d ++ to Ca r e l e a s e by SR. In c a r d i a c muscle, however, the predominant r o l e of the SR i n Ca* 4 r e l e a s e remains c o n t r o v e r s i a l f o r the reasons d i s c u s s e d above. Lullman and P e t e r s (1977) , f o r example, have proposed the the sarcolemma ++ may serve as the s i t e of bound Ca which c o u l d be r e l e a s e d upon d e p o l a r i z a t i o n . Work by Langer and h i s a s s o c i a t e s (Langer and Frank, 1972; R i c h and Langer, 1975; Frank et a l , 1977) has suggested t h a t the g l y c o c a l y x , a l a y e r of g l y c o p r o t e i n on the c e l l s u r f a c e , f u n c t i o n s as an i n t e r m e d i a r y C a + + b i n d i n g s t o r e which i s i n r a p i d e q u i l i b r i u m with both the e x t r a c e l l u l a r space and the i n t e g r a l p r o t e i n s which p o s s i b l y serve as transmembrane +•+• 4-4-Ca channels. Any change i n bound Ca a l t e r s the amount of Ca which subsequently ent e r s the c e l l and a f f e c t s c o n t r a c t i l i t y . Langer's work, however, f a i l s to c o n s i d e r the importance of the slow inward C a + * c u r r e n t (Langer et a l , 1982), u n l i k e the work of F a b i a t o and F a b i a t o (1977) which has i n c o r p o r a t e d the i $ ^ as the t r i g g e r C a + + r e q u i r e d to induce a 11 more massive r e l e a s e of Ca from c a r d i a c SR. Although such c r i t i c i s m s as v a r i a t i o n s i n E-C c o u p l i n g between s p e c i e s and the use of i n a p p r o p r i a t e i n v i t r o models (e.g. " s k i n n i n g " of c a r d i a c muscle c e l l s ) have been l e v e l l e d at t h e i r work, the "calcium-induced c a l c i u m r e l e a s e " hypothesis i s now thought to most c l o s e l y model i n v i v o E-C c o u p l i n g i n the h e a r t (Wohlfart -13-and Noble, 1982) . The calcium-induced c a l c i u m r e l e a s e mechanism does not appear to be o p e r a t i v e i n s k e l e t a l muscle, as demonstrated by Thorens and Endo (1975) . The phenomenon cou l d be demonstrated o n l y i n the presence of an abnormally low Mg c o n c e n t r a t i o n , a high SR Ca p r e - l o a d , and a l a r g e (10 ;iM> C a + 4 " t r i g g e r " . (b) M o r p h o l o g i c a l and b i o c h e m i c a l c h a r a c t e r i s t i c s o f s k e l e t a l and c a r d i a c SR M o r p h o l o g i c a l d i f f e r e n c e s i n SR between c a r d i a c and s k e l e t a l muscle are apparent: u l t r a s t r u c t u r a l s t u d i e s i n mammals have r e v e a l e d t h a t the SR i s l e s s abundant i n c a r d i a c 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; t h i s comprises 9-30 times l e s s volume than t h a t found i n s k e l e t a l muscle (Chapman, 1979). In a d d i t i o n , the T t u b u l a r diameter i s much l a r g e r (5-20 nm) i n c a r d i a c muscle (Sommer and Waugh, 1976) . Apart from these two major d i s s i m i l a r i t i e s , the o v e r a l l "geometry" of the SR from both muscle types appears s i m i l a r . B i o c h e m i c a l s t u d i e s , however, have tended to d i s p u t e t h i s . Ca -uptake, Ca -dependent ATPase a c t i v i t y and l e v e l s of maximal p h o s p h o r y l a t i o n are c o n s i s t e n t l y 2-4 times lower i n c a r d i a c SR than i n the comparable a c t i v i t i e s o f f a s t s k e l e t a l muscle (Martonosi, 1972; Shikegawa et a l , 1976; L e v i t z k i et a l , 1982). A comparison of s k e l e t a l and c a r d i a c SR separated on SDS p o l y a c r y l a m i d e g e l s r e v e a l s two s t r i k i n g d i f f e r e n c e s : a much higher percentage of t o t a l p r o t e i n represented by the ATPase i n s k e l e t a l SR (up to 80% vs. 35-40% -14-i n c a r d i a c SR) and a number of a d d i t i o n a l p r o t e i n bands i n c a r d i a c SR not observed i n s k e l e t a l SR. The former r e s u l t has been l i n k e d to the f i n d i n g of a decreased number of ATPase p a r t i c l e s , as r e v e a l e d by f r e e z e - f r a c t u r e e l e c t r o n microscopy, on the cy t o p l a s m i c "P" faces of c a r d i a c muscle SR p r e p a r a t i o n s . The l a t t e r f i n d i n g has prompted s p e c u l a t i o n t h a t the unaccounted-for a d d i t i o n a l p r o t e i n s may p l a y a r o l e i n reducing c a r d i a c SR enzymatic a c t i v i t y (Jones and Besch, 1979). C o n s i d e r a b l e d i f f e r e n c e s have a l s o been observed i n the p h o s p h o l i p i d and f a t t y a c i d components of the the two SR p r e p a r a t i o n s (Hidalgo et a l , 1979) . I t i s g e n e r a l l y accepted ++ t h a t the major a c t i v i t i e s of the SR, Ca -ATPase, and c a l c i u m uptake, are markedly i n f l u e n c e d by the surrounding p h o s p h o l i p i d b i l a y e r . D i f f e r e n c e s noted i n c l u d e a g r e a t e r percentage of phosphatidylethanolamine, p h o s p h a t i d y l i n o s i t o l , p h o s p h a t i d y l s e r i n e , and sphingomyelin i n c a r d i a c SR, while p h o s p h a t i d y l c h o l i n e l e v e l s are decreased (Chamberlain et a l , 1983) . C a r d i a c SR a l s o has i n c r e a s e d l e v e l s o f f a t t y a c i d s with two or more double bonds ( L e v i t z k i et a l , 1982) . Other f a c t o r s have been suggested to account f o r the reduced enzymatic a c t i v i t i e s of c a r d i a c SR. These i n c l u d e (i) the l a b i l i t y of the heart muscle ATPase, p o s s i b l y due to the in c r e a s e d number of membranous and proteinaceous i m p u r i t i e s ( L e v i t z k i e t a l , 1976), and ( i i ) the simple t e l e o l o g i c a l e x p l a n a t i o n t h a t the c a r d i a c muscle c o n t r a c t i o n - r e l a x a t i o n c y c l e o f s e v e r a l hundred m i l l i s e c o n d s demands an ATPase which subserves somewhat d i f f e r e n t f u n c t i o n s than the enzyme found i n -15-s k e l e t a l muscle (Jones and Besch, 1979) . Although the amino a c i d composition of the Ca^-ATPase i n dog heart i s very s i m i l a r t o t h a t of r a b b i t s k e l e t a l SR (Tada e t a l , 1977), the calcium-pump p r o t e i n of the two muscle types was found to be d i s t i n c t a n t i g e n i c a l l y (DeFoor et a l , 1980) . Although a l l o f the above may c o n t r i b u t e to the d i s c r e p e n c y i n ATPase f u n c t i o n between c a r d i a c and s k e l e t a l SR, i t i s now widely accepted t h a t the p r i n c i p a l reason i s the lower d e n s i t y o f pump s i t e s i n c a r d i a c SR as opposed to f a s t s k e l e t a l muscle SR (Jones and Besch, 1979; Chamberlain et a l , 1983). IV. Comparative Aspects of Fa s t - T w i t c h and Slow-Twitch S k e l e t a l Muscle Throughout the preceding d i s c u s s i o n , comparison of the p r o p e r t i e s of c a r d i a c and s k e l e t a l muscle (and t h e i r r e s p e c t i v e SR) r e s t e d e x c l u s i v e l y on a comparison between c a r d i a c and f a s t - t w i t c h s k e l e t a l muscle. T h i s i s somewhat m i s l e a d i n g s i n c e mammalian s k e l e t a l muscle c o n s i s t s of both f a s t and slow f i b r e s , i n v a r i o u s p r o p o r t i o n s , and the d i f f e r e n c e s between slow s k e l e t a l and c a r d i a c muscle are l e s s marked than those between the two types of s k e l e t a l muscle (Ebashi et a l , 1974; Wang e t a l , 1979). As w i l l be d e s c r i b e d below, many of the d i f f e r e n c e s between f a s t and slow s k e l e t a l SR are s i m i l a r to the documented d i f f e r e n c e s between f a s t - t w i t c h and c a r d i a c SR. The two g e n e r a l c a t e g o r i e s o f s k e l e t a l muscle i n mammals, f a s t - t w i t c h and slo w - t w i t c h , have been e s t a b l i s h e d to d e s c r i b e f i r s t , the time to r e l a x a t i o n (short f o r f a s t - t w i t c h and long -16-f o r s l ow-twitch f i b r e s ; C l o s e , 1972) , and second, r e s i s t a n c e to f a t i g u e d u r i n g r e p e t i t i v e s t i m u l a t i o n (low f o r f a s t - t w i t c h f i b r e s and high f o r slow). Both of these p h y s i o l o g i c a l v a r i a b l e s have b i o c h e m i c a l and h i s t o c h e m i c a l c o r r e l a t e s (Eisenberg and Kuda, 1976): time to r e l a x a t i o n i s a s s o c i a t e d with myosin ATPase a c t i v i t y and the volume of SR, f o r example/ whereas f a t i g u a b i l i t y c o r r e l a t e s with o x i d a t i v e and g l y c o l y t i c c a p a c i t y . As summarized by B e l l et a l (1980), f a s t - t w i t c h f i b r e s have an i n c r e a s e d myosin ATPase a c t i v i t y , g r e a t e r SR volume, a b r i e f e r and f a s t e r propagating a c t i o n p o t e n t i a l , and l a r g e r amounts of glycogen and g l y c o l y t i c enzymes. Such muscle i s capable of s h o r t p e r i o d s of i n t e n s e a c t i v i t y but f a t i g u e s very r a p i d l y with d e p l e t i o n of glycogen s t o r e s . T h i s l a t t e r p o i n t has a l s o d i s t i n g u i s h e d between two types of f a s t - t w i t c h f i b r e s : those with high g l y c o l y t i c and o x i d a t i v e enzyme c o n c e n t r a t i o n s ("red" f a s t muscle, the red a s s o c i a t e d with a high myoglobin content) or those with predominantly g l y c o l y t i c enzymes ("white" f a s t muscle). As a consequence of the absence or presence of o x i d a t i v e enzymes, the f a s t muscle w i l l f a t i g u e sooner or l a t e r , r e s p e c t i v e l y . In c o n t r a s t , slow-twitch muscles are more e f f i c i e n t f o r a given work l o a d : they possess a higher d e n s i t y of c a p i l l a r i e s and t h e r e f o r e maintain a high r e s t i n g b l o o d flow. T y p i c a l l y , t h i s type of muscle i s red (again due to i t ' s high myoglobin content) and possesses a high c o n c e n t r a t i o n of o x i d a t i v e enzymes, such as s u c c i n i c dehydrogenase. (A) C h a r a c t e r i s t i c s of F a s t - T w i t c h and Slow-Twitch SR -17-As b r i e f l y mentioned i n the preceding s e c t i o n , the d e n s i t y of the SR membrane i s higher i n f a s t - t w i t c h than i n slow-twitch s k e l e t a l muscle ( L u f f and Atwood, 1971). P r e d i c t a b l y , the d e n s i t y of (Ca -Mg )-ATPase s i t e s , as determined u l t r a s t r u c t u r a l l y , i s dimi n i s h e d i n slow, as compared to f a s t s k e l e t a l muscle SR (Bray and Rayns, 1976; Jorgensen e t a l , 1982) which, as i n the case of c a r d i a c SR, may e x p l a i n the decreased Ca uptake and ATPase a c t i v i t y as w e l l as lower phosphoprotein l e v e l s compared to f a s t SR (Pette and Heilmann, 1979; Zubrzycka-Gaarn e t a l , 1982) . In a d d i t i o n , Borchman e t a l (1982) have suggested t h a t the decreased Ca 4 + -ATPase a c t i v i t y i n slow SR may be due to a decreased b i l a y e r f l u i d i t y : l e v e l s of c h o l e s t e r o l and sphingomyelin are s u b s t a n t i a l l y i n c r e a s e d whereas p h o s p h a t i d y l c h o l i n e l e v e l s are s i g n i f i c a n t l y decreased i n the slow SR membrane. U n l i k e the comparison between c a r d i a c and f a s t s k e l e t a l SR, th e r e are a few i n t r i g u i n g f e a t u r e s o f the f a s t versus slow system. F i r s t , although the d e n s i t y o f the c a l c i u m pump ++ p r o t e i n i s decreased i n slow SR, the b a s a l (Mg -ATPase) a c t i v i t y i s d r a m a t i c a l l y e l e v a t e d ( S r e t e r , 1969). The s i g n i f i c a n c e of t h i s i s unknown but may mirror the f i n d i n g of a mul t i t u d e o f p r o t e i n bands i n slow SR not observed i n f a s t SR (Pette and Heilmann, 1977) . Second, i t has been observed t h a t f a s t - t w i t c h muscle can be transformed i n t o a s l o w - t w i t c h - l i k e f i b r e when sub j e c t e d to c h r o n i c s t i m u l a t i o n resembling the f i r i n g frequency of a slow motoneuron (Salmons and Vrbova, 1969) . The r e s u l t i n g SR p r e p a r a t i o n obtained from such a f i b r e -18-resembles slow SR u l t r a s t r u c t u r a l l y and b i o c h e m i c a l l y (Pette and Heilmann, 1977) . Van Winkle and Entman (1979) suggest t h a t the t r a n s f o r m a t i o n may promote a l t e r e d gene e x p r e s s i o n such t h a t a d i f f e r e n t ATPase isozyme, with i t s a s s o c i a t e d membranous framework, i s manifested. •W- ++ V. R e g u l a t i o n of the (Ca -Mg )-ATPase i n SR The calcium-pump p r o t e i n and i t s a s s o c i a t e d p r o p e r t y of c a l c i u m t r a n s p o r t i s r e g u l a t e d by a v a r i e t y of f a c t o r s which appear to be s i m i l a r i n both s k e l e t a l and c a r d i a c SR. One prominent r e g u l a t o r i s MgATP. As d e s c r i b e d e a r l i e r , Mg p l a y s at l e a s t two important r o l e s i n ATP h y d r o l y s i s : the a c c e l e r a t i o n o f phosphorylated i n t e r m e d i a t e decomposition and i t s a s s o c i a t i o n with ATP to serve as t r u e s u b s t r a t e f o r the enzyme. ATP i s able to serve not o n l y as s u b s t r a t e but a l s o as a r e g u l a t o r c o n t r o l l i n g enzyme a c t i v i t y . Kanazawa et a l (1971) demonstrated t h a t s t e a d y - s t a t e ATP h y d r o l y s i s was a c c e l e r a t e d by ATP, i n t h a t Vmax and Km values i n c r e a s e d with ATP c o n c e n t r a t i o n . Dupont (1977) has found t h a t the ATPase of s k e l e t a l SR has high and low a f f i n i t y b i n d i n g s i t e s f o r ATP, the high a f f i n i t y s i t e can be phosphorylated while the low a f f i n i t y s i t e can serve as a r e g u l a t o r y s i t e . S t i m u l a t i o n of ATPase a c t i v i t y by high ATP c o n c e n t r a t i o n s appears to be due to an a c c e l e r a t e d c o n v e r s i o n of E2-E1 (deMeis and Vianna, 1979). Calcium ions are a l s o able to a l t e r t h e i r own t r a n s p o r t by SR. Increases i n c y t o p l a s m i c C a + + s t i m u l a t e ATP h y d r o l y s i s and 44 concomitant Ca t r a n s p o r t (Hasselbach, 1964) . Once t r a n s p o r t e d -19-i n t o the lumen of the SR v e s i c l e , C a T T a l s o i s able to decrease ATP h y d r o l y s i s and i t s own f u r t h e r uptake (Weber, 1971) . T h i s +•4" 4-4» e f f e c t of Ca appears to be r e l a t e d to a Ca -dependent accumulation of E,P i n h i b i t i n g the formation of E^P (Yamada and Tonomura, 1972) . Monovalent c a t i o n s have a marked s t i m u l a t o r y e f f e c t on 4-4-Ca t r a n s p o r t i n both s k e l e t a l (Shikegawa and P e a r l , 1976) and c a r d i a c (Jones et a l , 1977) muscle SR. The a l k a l i metals appear to e x e r t t h e i r e f f e c t by i n c r e a s i n g turnover of the pump, p o s s i b l y by s t i m u l a t i n g the decomposition r a t e of the A D P - s e n s i t i v e phosphoenzyme (E (P) (Shikegawa and Akowitz, 1979). S u b t l e d i f f e r e n c e s between c a r d i a c and s k e l e t a l muscle SR with repect to the i n f l u e n c e of monovalent c a t i o n s have been re p o r t e d (Wang et a l , 1981) . These authors r e p o r t t h a t i n 4-4- + Ca - d e f i c i e n t s k e l e t a l SR p r e p a r a t i o n s , p r e - m c u b a t i o n with K d i d not a f f e c t i n i t i a l p h o s p h o r y l a t i o n l e v e l s when C a + + and ATP were subsequently added; i n c a r d i a c SR, the phosphoenzyme l e v e l r o s e . In a d d i t i o n , K + was found to i n h i b i t EP formation i n 4-4-s k e l e t a l SR p r e p a r a t i o n s c o n t a i n i n g bound Ca , whereas i n h i b i t i o n was o n l y observed i n c a r d i a c SR p r e p a r a t i o n s at low 4+ (<1 ;uM) Ca c o n c e n t r a t i o n s . In v i v o , the SR i s bathed i n a 4-high K c o n c e n t r a t i o n (100-175 mM; S r e t e r , 1963); thus, the p h y s i o l o g i c a l r e l e v a n c e of r e g u l a t i o n by monovalent c a t i o n s i s not known. Thus f a r , I have d i s c u s s e d r e g u l a t o r y f e a t u r e s which appear to act i n p a r a l l e l i n both c a r d i a c and s k e l e t a l muscle. Fu r t h e r treatment of the r e g u l a t i o n of SR must now d i v e r g e i n t o -20-more i d i o s y n c r a t i c r e g u l a t o r y mechanisms u n d e r l y i n g the two muscle t y p e s . A. R e g u l a t i o n o f C a r d i a c SR The sa r c o p l a s m i c r e t i c u l u m of c a r d i a c muscle has been shown to possess at l e a s t two mechanisms which are not seen i n the SR of f a s t - t w i t c h (and p o s s i b l y slow t w i t c h ; see l a t e r ) muscle: s t i m u l a t i o n of c a l c i u m t r a n s p o r t , Ca -dependent ATPase a c t i v i t y , and phosphoprotein formation by (1) cAMP-dependent p r o t e i n kinase and (2) c a l m o d u l i n . (i) C y c l i c AMP-dependent p r o t e i n kinase r e g u l a t i o n of c a r d i a c SR Ever s i n c e the d i s c o v e r y t h a t catecholamines (CAs) a c t i v a t e d p a r t i c u l a t e adenylate c y c l a s e p r e p a r a t i o n s from heart i n accord with t h e i r i n v i v o potency (Sutherland et a l , 1968), i t has been p o s t u l a t e d t h a t the i n o t r o p i c a c t i o n of CAs was mediated by c y c l i c AMP (cAMP). C y c l i c AMP serves as the i n t r a c e l l u l a r second messenger f o r these r e g u l a t o r y processes i n t h a t beta a d r e n e r g i c a c t i v a t i o n o f a sarcolemmal adenylate c y c l a s e r e s u l t s i n an e l e v a t i o n o f i n t r a c e l l u l a r cAMP l e v e l s (Sutherland and R a i l , 1960) . T h i s e l e v a t i o n leads to s t i m u l a t i o n of cAMP-dependent p r o t e i n k i n a s e s , which c o n s i s t of a holoenzyme made up of two r e g u l a t o r y and two c a t a l y t i c s u b u n i t s (Rosen and Erlichman, 1975). When the r e g u l a t o r y s u b u n i t s bind cAMP, the c a t a l y t i c s u b u n i t s d i s s o c i a t e i n t o f r e e , a c t i v e forms (Erlichman et a l , 1971). C y c l i c -21-AMP-dependent p r o t e i n k i n a s e s , i n t u r n , c a t a l y z e the p h o s p h o r y l a t i o n o f a number of p r o t e i n s r e s u l t i n g i n f u n c t i o n a l m o d i f i c a t i o n s . The mechanical response of c a r d i a c muscle to agents which i n c r e a s e i n t r a c e l l u l a r cAMP l e v e l s i n c l u d e s (1) an enhanced l e v e l and r a t e of t e n s i o n development and (2) an i n c r e a s e d r a t e of r e l a x a t i o n (Katz et a l , 1975) . S u s p i c i o n s of the involvement of SR i n the cAMP-mediated i n o t r o p i s m were confirmed i n 1969 by Entman et a l who found a s t i m u l a t i o n o f SR Ca +* t r a n s p o r t by the c y c l i c n u c l e o t i d e . Subsequent work by K i r c h b e r g e r et a l (1972; 1974) and Tada et a l (1975) confirmed t h a t both c a l c i u m uptake and the Ca -dependent ATPase of c a r d i a c SR were s t i m u l a t e d t h r e e - f o l d i n the presence o f cAMP and exogenously added cAMP-dependent p r o t e i n kinase and were c l o s e l y c o r r e l a t e d with the p h o s p h o r y l a t i o n of a 22,000 MW p r o t e i n termed phospholamban (Figure 2 ) . The molecular weight of the phosphoprotein has r e c e n t l y been d i s p u t e d with the suggestion t h a t i t i s e i t h e r an 11,000 MW dimer (LePeuch e t a l , 1979), a tr i m e r of 11,000, 8000, and 4000 d a l t o n s (Louis et a l , 1982) or a 5500 MW tetramer (Kirchberger and Antonetz, 1982a). Phospholamban has p r o p e r t i e s c h a r a c t e r i s t i c o f a phosphoester ( i . e . the P i i s i n c o r p o r a t e d p r i m a r i l y i n t o s e r i n e ) and t h e r e f o r e e a s i l y d i s t i n g u i s h e d from the a c y l phosphate (EP) inte r m e d i a t e o f the ATPase (Tada et a l , 1975). S t i m u l a t i o n of c a l c i u m t r a n s p o r t i n c a r d i a c SR as a consequence of phospholamban p h o s p h o r y l a t i o n appears to be due to three interdependent f a c t o r s : (1) e f f e c t s on the -22-l-adranarftc •rtanylata C K l w ATP cAMP R cAMP / C (aclnratad prota /non-acti.atae' j RC phoaahcHmttan p h o i e r w r l a t a d aftoaptiuiawman artftancad Ca-ATPata ana f«n*«or-t I C T H I M Ca «i SR. 1 « ' M M O Ca t v u u it cenuactiont [ anhancn contract.H, | FIGURE 2. A p o s s i b l e mechanism f o r fl-ad rene rg i c effects on c o n t r a c t i l i t y , med ia t ed by enhanced Ca uptake by the s a r c o p l a s m i c r e t i c u l u m (SR) . RC r e p r e s e n t s p r o t e i n k i n a s e in s o l u b l e form o r in a s s o c i a t i o n w i t h SR membranes. R r e p r e s e n t s the r e g u l a t o r y s u b u n i t , C the c a t a l y t i c s u b u n i t o f the p r o t e i n k i n a s e . Taken from Tada et a 1 (1982). - 2 3 -i n t e r a c t i o n between the two Ca b i n d i n g s i t e s on the ATPase (Hicks e t a l , 1979), (2) d i r e c t e f f e c t s on the a c y l phosphate in t e r m e d i a t e (Tada and Katz, 1982), and (3) a p o s s i b l e i n t e r a c t i o n with phosphorylated Troponin I (Kranias and S o l a r o , 1982) . Hicks e t a l (1979) proposed t h a t phospholamban p h o s p h o r y l a t i o n r e s u l t s i n a c o n f o r m a t i o n a l change i n the ATPase enzyme such t h a t the p o s i t i v e c o o p e r a t i v i t y between the two Ca b i n d i n g s i t e s i s decreased. The r e s u l t i n g i n c r e a s e i n Ca s e n s i t i v i t y of the enzyme (Tada et a l , 1974) s t i m u l a t e s 44, Ca t r a n s p o r t . More recent work by Tada et a l (1979; 1982) suggests t h a t the a l t e r a t i o n i n Ca k i n e t i c s may be more the r e s u l t of phosphorylated phospholamban i n t e r a c t i n g with key elementary steps of the ATPase r e a c t i o n mechanism. These workers found t h a t s t e a d y - s t a t e EP l e v e l s were not a l t e r e d by phospholamban p h o s p h o r y l a t i o n at s a t u r a t i n g c o n c e n t r a t i o n s of Ca and ATP. However, below 10 /iM Ca , EP l e v e l s were reduced while the r a t e of P i l i b e r a t i o n was i n c r e a s e d , suggesting the the r a t e - l i m i t i n g step of EP decomposition was enhanced. Among the steps a t which EP i s decomposed (steps i v , v, and v i i n equation 1 ) , step i v appears to be the one a c c e l e r a t e d s i n c e the r a t e of decay of E^P was found to be enhanced (Tada et a l , 1982). Based on a number of o b s e r v a t i o n s , Tada et a l (1982) have proposed a model of the a c t i o n of phospholamban p h o s p h o r y l a t i o n ++ on the ATPase. During Ca t r a n s p o r t , the ATPase subunit c o n t a i n i n g the a c t i v e s i t e (the 30,000 MW fragment) undergoes a -24-c o n f o r m a t i o n a l change which promotes the t r a n s l o c a t i o n o f the C a + + b i n d i n g subunit (the 20,000 MW fragment) from o u t s i d e to i n . Phospholamban p h o s p h o r y l a t i o n i s proposed to a c c e l e r a t e the r a t e at which the 30,000 MW fragment undergoes i t s c o n f o r m a t i o n a l change, r e s u l t i n g i n an i n c r e a s e d r a t e of t r a n s l o c a t i o n of the Ca b i n d i n g s u b u n i t . The t h i r d p o s s i b i l i t y i s t h a t phospholamban p h o s p h o r y l a t i o n i n t e r a c t s with phosphorylated Troponin I ( T n l ) , a component of the r e g u l a t o r y complex of the t h i n f i l a m e n t s . T h i s s u g g e s t i o n has a r i s e n from the f i n d i n g of a cAMP-dependent p h o s p h o r y l a t i o n of T n l (England, 1975) mediating a decrease i n the Ca s e n s i t i v i t y of Troponin C and of c a r d i a c actomyosin ATPase a c t i v i t y (Solaro et a l , 1976). The p h y s i o l o g i c a l e f f e c t of a reduced a f f i n i t y of the t r o p o n i n complex to C a + + would be an enhanced r a t e of d i s s o c i a t i o n of C a + + from the c o n t r a c t i l e p r o t e i n s , r e s u l t i n g i n an a c c e l e r a t e d r a t e of r e l a x a t i o n . K r a n i a s and S o l a r o (1982) t h e r e f o r e propose t h a t both phospholamban and T n l p h o s p h o r y l a t i o n act i n c o n c e r t to enhance the r a t e of r e l a x a t i o n by SR (Figure 3 ) . N e v e r t h e l e s s , the p h y s i o l o g i c a l r e l e v a n c e of T n l p h o s p h o r y l a t i o n remains i n q u e s t i o n f o l l o w i n g the f i n d i n g of p h o s p h o r y l a t i o n extending beyond the time r e q u i r e d f o r a catecholamine-mediated i n o t r o p i c e f f e c t (England, 1976) . ( i i ) Calmodulin mediated r e g u l a t i o n of c a r d i a c SR +4 Calmodulin, a h e a t - s t a b l e a c i d i c Ca b i n d i n g p r o t e i n with a molecular weight of approximately 16,800 (Klee and Vanaman, -25-C A T E C H O L A M I N E S I • C A M P \ • P R O T E I N K I N A S E S Tnl-P Tnl S R - P S R F O R C E a n d A T P a s e P H O S P H A T A S E S C a U P T A K E C a * - * - T I M E t C a R E L E A S E B Y S R • C a I N A C T I V A T I N G P O O L R E L A X I N G E F F E C T S A C T I V A T I N G E F F E C T S FIGURE 3. A scheme descr ib ing poss ib le mechanisms for the a c t i v a t i ng and re lax ing e f f e c t s of c a t e c h o l -amines on the heart . Taken from Kranias 6 Solaro (1983). 1982) has been i s o l a t e d from a number of t i s s u e s , i n c l u d i n g b r a i n , h e a r t , and t e s t i s . D i s c o v e r e d independently by Cheung and K a k i u c h i and Yamazaki i n 1970 as an a c t i v a t o r of phosphodiesterase, c a l m o d u l i n (CAM) has s i n c e been shown to serve as a m u l t i f u n c t i o n a l i n t r a c e l l u l a r modulator of a number of Ca f*-dependent enzymes as shown i n Table 1. I t appears t h a t each CAM molecule binds 4 Ca i o n s , with d i s s o c i a t i o n c o nstants i n the range of 1-100 ;uM (Wolff et a l , 1977) . The b i n d i n g of Ca : a l t e r s the conformation of CAM, i n c r e a s i n g i t s h e l i c a l content (Dedman et a l , 1977) and exposing hydrophobic regions (LaPorte et a l , 1980). In t h i s conformation, the +f Ca -CAM complex can bind to t a r g e t enzymes ( N i g g l i et a l , 1977) and, through an unknown mechanism, i n c r e a s e t h e i r a c t i v i t i e s . W i t h in the l a s t ten y e a r s , a number of seemingly d i v e r s e p h a r m a c o l o g i c a l agents, among them: the phenothiazine t r i f l u o p e r a z i n e (TFP) and the histamine r e l e a s e r , compound 48/80, have been shown to i n h i b i t the CAM-induced a c t i v a t i o n of a v a r i e t y of enzymes. A l l of these "anti-CAM" ( V i n c e n z i 1981) agents show s e v e r a l common s t r u c t u r a l c h a r a c t e r i s t i c s , namely a l a r g e hydrophobic r e g i o n and a charged amino group at p h y s i o l o g i c a l pH (Figure 4 ) . The charged amino group i s presumed to i n t e r a c t with negative charges on the h i g h l y a c i d i c CAM, while the hydrophobic regions uncovered when Ca binds to CAM are thought to i n t e r a c t with the l i p o p h i l i c r e g i o n s on the anti-CAM agent ( P r o z i a l e c k and Weiss, 1982) . Although c l a i m s of s p e c i f i c i t y have been made (Levin and Weiss, 1977), i t i s now -27-TABLE 1. Calmodulin-dependent enzymes and processes that are inh ib i t ed by phenothiazine a n t i p s y c h o t i c s . Taken from Weiss et a l (1982) Enzymes Adenylate cyclase (Ca3' + Mg'*)-ATPase 15-Hydroxyproslaglandin dehydrogenase* Myosin light chain kinase NAD kinase Phosphodiesterase Phospholipase Aj Phosphorylase b kinase Protein kinase Tryptophan hydroxylase Processes ADH-mediated water transport (••-Adrenergic responses Calcium uptake Catecholaminergic (unction Chloride secretion in intestine DNA synthesis Endocytosis Exocytosis Insulin release Leukocyte function Neuromuscular transmission Neurotransmitter release Phospholipid methylation Platelet function Release of trichocyst in Paramecium Smooth muscle contraction * Calmodulin inhibits 1 5 - h ) d r o x y p r o s t a g l a n d i n d e r n d r o j - e n a < . e . - 2 8 -F IGURE 4 . G e n e r a l i z e d s t r u c t u r a l c h a r a c t e r i s t i c s o f c a l m o d u l i n i n h i b i t o r s . T h i s g e n e r a l i z e d s t r u c t u r e d e p i c t s a p o s i t i v e l y - c h a r g e d g r o u p (N ) a t t a c h e d t o t w o h y d r o p h o b i c g r o u p s . The h y d r o p h o b i c g r o u p s n e e d n o t n e c e s s a r i l y be a t t a c h e d t o e a c h o t h e r a t t h e r i n g . X r e p r e -s e n t s a m o i e t y t h a t i n c r e a s e s t h e h y d r o p h o b i c i t y o f t h e r i n g . F o r h i g h p o t e n c y n s h o u l d be a t l e a s t t h r e e c a r b o n s l o n g . T a k e n f r o m P r o z i a l e k & W e i s s ( 1 9 8 2 ) . -29-w e l l accepted t h a t the h i g h l y l i p o p h i l i c anti-CAM agents a l s o a c t n o n s p e c i f i c a l l y ( R o u f o g a l i s , 1981) i n t h a t they i n h i b i t b a s a l , CAM-independent enzyme a c t i v i t y (Luthra, 1982) p o s s i b l y by membrane p e r t u r b i n g e f f e c t s (Raess and V i n c e n z i , 1980; Ho et  a l , 1983) . Thus, c a u t i o n must be exerted when i n f e r r i n g the presence of a CAM-dependent process s o l e l y by the use of these agents. The f i r s t o b s e r v a t i o n t h a t CAM p l a y s a r o l e i n r e g u l a t i n g ++ Ca f l u x e s across membranes was the o b s e r v a t i o n by Bond and Clough (1973) t h a t a p r o t e i n i n human red c e l l s a c t i v a t e d the e r y t h r o c y t e (Ca 4* -Mg + + ) -ATPase, such t h a t C a + + was a c t i v e l y t r a n s p o r t e d from the c e l l . Our l a b o r a t o r y was the f i r s t to ++• demonstrate a s t i m u l a t i o n by CAM of both ATP-dependent Ca 4+ 4H-t r a n s p o r t (Katz and Remtulla, 1978) and (Ca -Mg )-ATPase a c t i v i t y (Lopaschuk et a l , 1980) i n c a r d i a c SR. T h i s work has subsequently been reproduced by a number of workers (LePeuch et a l , 1979; K r a n i a s et a l , 1980; K i r c h b e r g e r and Antonetz, 1982b). The calmodulin-dependent r e g u l a t o r y system r e q u i r e s Ca and a l s o depends on a s p e c i f i c kinase which, as f i r s t shown by LePeuch et a l (1979), a l s o phosphorylates phospholamban but at a s i t e d i s t i n c t from t h a t phosphorylated by the cAMP-dependent system; an a d d i t i v e i n c r e a s e i n the amount of p h o s p h o r y l a t i o n was observed when c a r d i a c SR was incubated i n the presence of both CAM and cAMP-dependent p r o t e i n k i n a s e . The mechanism of s t i m u l a t i o n of t r a n s p o r t and ATPase a c t i v i t y appears somewhat s i m i l a r to t h a t seen with cAMP-dependent p r o t e i n k i n a s e : an i n c r e a s e i n ATPase turnover -30-F I G U R E 5 S c h e m e d e p i c t i n g t h e k n o w n a n d p o s t u l a t e d r e g u l a t o r y m e c h a n i s m s o f c a r d i a c SR C a t r a n s p o r t . C y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e p h o s p h o r y l a t e s p h o s p h o l a m b a n w h i c h t h e n , p e r h a p s t h r o u g h a s e r i e s o f i n t e r m e d i a t e s t e p s , s t i m u l a t e s b o t h c a l c i u m t r a n s p o r t a n d ( C a - M g ) - A T P a s e a c t i v i t y o f t h e SR c a l c i u m p u m p p r o t e i n . C a l m o d u l i n , i n t h e p r e s e n c e o f C a , a l s o p h o s p h o r y l a t e s p h o p h o l a m b a n b y p r e s u m a b l y i n t e r a c t i n g w i t h a m e m b r a n e - b o u n d k i n a s e . T h e p o s s i b i l i t y r e m a i n s t h a t C a - c a l m o d u l i n c i r c u m v e n t s p h o s p h o l a m b a n t o a c t d i r e c t l y o n t h e A T P a s e o r o n a n , a s y e t , u n i d e n t i f i e d i n t e r m e d i a t e s t e p . - 3 1 -cAMP-dependent p r o t e i n k i n a s e P i phospholamban c a r d i a c SR ( C a 2 + + M g 2 + ) - A T P a s e C a 2 + - C a 1 m o d u l i n P i membrane-bound k i n a s e -31a-r a t e v i a enhancement of the r a t e of d e p h o s p h o r y l a t i o n of the acylphosphate (EP) i n t e r m e d i a t e (Lopaschuk e t a l , 1980) . The p o s s i b i l i t y s t i l l e x i s t s , a c c o r d i n g to Katz (1980), t h a t CAM a c t s d i r e c t l y on EP, bypassing the kinase phospholamban system (Figure 5 ) . The p h y s i o l o g i c a l r e l e v a n c e of CAM-mediated s t i m u l a t i o n of the (Ca -Mg )-ATPase i n the c a r d i a c SR membrane i s unknown. Rece n t l y , a sarcolemmal (Ca -Mg )-ATPase has been i d e n t i f i e d i n myocardium and p o s t u l a t e d to handle a p a r t of the e x t r u s i o n of Ca from the c a r d i a c c e l l along with the Na/Ca exchanger (Caroni and C a r a f o l i , 1981). The enzyme i s s t i m u l a t e d by CAM (Caroni and C a r a f o l i , 1981) and may serve the i n v i v o f u n c t i o n of r e g u l a t i n g the r i s e of i n t r a c e l l u l a r Ca t h a t occurs with each h e a r t beat. R e g u l a t i o n of the SR ATPase by CAM, however, can not be as e a s i l y e x p l a i n e d . C l e a r l y , an enhancement of the SR c a l c i u m pump w i l l mediate an enhanced r a t e of r e l a x a t i o n , s i m i l a r to t h a t seen i n the cAMP-dependent p r o t e i n kinase mediated system. Yet, s p e c u l a t i o n s range from CAM having an important r o l e as a modulator of the beat-to-beat r i s e and f a l l i n Ca with each c o n t r a c t i l e c y c l e (Kirchberger and Antonetz, 1982b) to a r o l e as a back-up mechanism i n the event of p a t h o l o g i c a l a l t e r a t i o n s (Lopaschuk et a l , 1980; L o u i s and M a f f i t t , 1982) , to i t s being a u b i q u i t o u s contaminant of c a r d i a c membrane p r e p a r a t i o n s (Hartweg and Bader, 1983) . C l e a r l y , more i n f o r m a t i o n w i l l have to be a c q u i r e d b e f o r e a d e f i n i t i v e r o l e f o r CAM i n c a r d i a c SR i s determined. -32-B. R e g u l a t i o n of S k e l e t a l SR U n l i k e the r e g u l a t o r y mechanisms documented i n c a r d i a c SR, s k e l e t a l muscle SR r e g u l a t i o n i s not n e a r l y as w e l l c h a r a c t e r i z e d and has been the s u b j e c t of ongoing c o n t r o v e r s y . Schwartz e t a l (1976) and Bornet e t a l (1977) r e p o r t e d t h a t cAMP-dependent p r o t e i n kinase s t i m u l a t e d Ca t r a n s p o r t i n f a s t or s l o w - t w i t c h s k e l e t a l muscle SR. F a b i a t o and F a b i a t o (1978), u s i n g skinned f a s t s k e l e t a l f i b r e s of the c a t , a l s o r e p o r t e d a cAMP-mediated r e l a x a t i o n o f t e n s i o n . On the other hand, a number of other l a b o r a t o r i e s , i n c l u d i n g our own, have been unable to r e p l i c a t e the f i n d i n g s of a cAMP-mediated i n c r e a s e i n 44 f a s t - t w i t c h s k e l e t a l SR Ca t r a n s p o r t (Kirchberger and Tada, 1976; C a s w e l l et a l , 1978; Katz and Wong, 1982), although K i r c h b e r g e r and Tada (1976) repo r t e d a s t i m u l a t i o n of slow s k e l e t a l SR by cAMP-dependent p r o t e i n k i n a s e . The r e g u l a t i o n of s k e l e t a l SR i s f u r t h e r complicated by r e p o r t s of p h o s p h o r y l a t i o n of a 100,000 MW membrane p r o t e i n by cAMP-dependent p r o t e i n kinase (Kranias et a l , 1980; 1983) or phosphorylase b kinase (Schwartz et a l , 1976). The cAMP-mediated p h o s p h o r y l a t i o n of the 100,000 MW p r o t e i n has been found by K r a n i a s e t a l (1983) to s t i m u l a t e both the i n i t i a l r a t e s of EP formation and decomposition. N e v e r t h e l e s s , the f i n d i n g of phosphorylase b kinase p h o s p h o r y l a t i o n suggests, perhaps, t h a t both kinases may be g l y c o g e n o l y t i c contaminants of s k e l e t a l SR p r e p a r a t i o n s . As y e 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 a cAMP-dependent s t i m u l a t i o n of s k e l e t a l muscle remains un c l e a r s i n c e the e f f e c t s of catecholamines on -33-c o n t r a c t i l i t y i n f a s t s k e l e t a l muscle, i n p a r t i c u l a r , are much lower than those observed i n heart and have been p o s t u l a t e d to be mediated p u r e l y by l o c a l v a s o c o n s t r i c t i o n (Bowman and Zaimis, 1958). R e c e n t l y , a calmodulin-dependent p h o s p h o r y l a t i o n has been noted i n f a s t s k e l e t a l SR ( C h i e s i and C a r a f o l i , 1982; Campbell and MacLennan, 1982; K i r c h b e r g e r and Antonetz, 1982c). The p r o t e i n s phosphorylated i n c l u d e a 20 and 57-60 kDalton p r o t e i n , the former, an a c i d i c p r o t e o l i p i d t h a t i s not i d e n t i c a l with phospholamban ( C h i e s i and C a r a f o l i , 1983) . Work i n our l a b o r a t o r y (Katz and Wong, 1982) and by others ( C a r a f o l i , 1980) has confirmed a lac k of s t i m u l a t i o n of C a + f t r a n s p o r t by CAM. The r o l e of CAM-mediated p h o s p h o r y l a t i o n , t h e r e f o r e , remains even l e s s understood than i n c a r d i a c muscle SR and has prompted a number of s p e c u l a t i o n s of the r o l e of CAM i n C a + + r e l e a s e from the SR (Campbell and MacLennan, 1982; C h i e s i and C a r a f o l i , 1982). -34-VI Disease S t a t e s and the Sarcoplasmic Reticulum P a t h o l o g i c a l changes i n s a r c o p l a s m i c r e t i c u l u m Ca t r a n s p o r t a b i l i t y are suspected t o p l a y important r o l e s i n the pathogenesis of a number of d i s o r d e r s i n both heart and s k e l e t a l muscle. Whether these changes re p r e s e n t primary or secondary events i n the development of the d i s e a s e process i s , however, not known (He f f r o n , 1979). A. S k e l e t a l Muscle One of the s t r o n g e s t cases f o r the primary involvement of s k e l e t a l SR i n the pathogenesis of a d i s e a s e s t a t e i s i n Duchenne Muscular Dystrophy (DMD), the most common of the human muscular d y s t r o p h i e s . DMD i s a p r o g r e s s i v e , c r i p p l i n g d i s e a s e of young males c h a r a c t e r i z e d by r e c e s s i v e s e x - l i n k e d i n h e r i t a n c e and an e a r l y onset of symptoms which i n c l u d e proximal muscle weakness and atrophy, a myocardial involvement, hypertrophy of the c a l v e s , muscular c o n t r a c t i o n s , mental r e t a r d a t i o n and death due to r e s p i r a t o r y or c a r d i a c f a i l u r e u s u a l l y b e f o r e t h i r t y years of age (Walton and Gardner-Medwin, 1974) . One of the e a r l i e s t b i o c h e m i c a l s i g n s of DMD i s an i n c r e a s e i n serum enzymes d e r i v e d from muscle, e s p e c i a l l y c r e a t i n e k i n a s e , which are thought to enter the c i r c u l a t i o n e i t h e r from n e c r o t i c muscle or due to a l t e r e d p e r m e a b i l i t y of muscle SL (Furukawa and P e t e r , 1978). I n i t i a l l y , the SL was suspected as being important i n the e a r l y pathogenesis of the d i s e a s e due to the e a r l y appearance of c r e a t i n e k i n a s e , but a number of o b s e r v a t i o n s have s i n c e c ontested the primacy of SL - 3 5 -d e f e c t s and p l a c e d the burden, i n s t e a d , on the SR network. These o b s e r v a t i o n s i n c l u d e : (1) u l t r a s t r u c t u r a l evidence o f morph o l o g i c a l changes i n the SR pre c e d i n g those o f the SL ( C u l l e n and F u l t h o r p e , 1975), (2) although c r e a t i n e kinase may be leaked through the SL, c a r n i t i n e , a much smaller molecule, appears to be r e t a r d e d (DiMauro and Rowland, 1976), and (3) a rep o r t e d e l e v a t i o n (a suspected e a r l y event) of i n t r a c e l l u l a r Ca l e v e l s i n DMD s k e l e t a l muscle (Oberc and Engeel, 1977; Maunder et a l , 1977). T h i s l a t t e r o b s e r v a t i o n , i n p a r t i c u l a r , has d i r e c t e d a t t e n t i o n towards the SR as a r e s u l t of the p r o p o s i t i o n o f a "calcium h y p o t h e s i s " of DMD pathogenesis (Ebashi and S u g i t a , 1979; C u l l e n et a l , 1979). B r i e f l y , the c a l c i u m hypothesis suggests t h a t a d e f e c t i n c a l c i u m r e g u l a t i o n by s k e l e t a l muscle c e l l s , r e s u l t i n g i n a s u b s t a n t i a l , s u s t a i n e d 44 e l e v a t i o n o f i n t r a c e l l u l a r Ca , may e l i c i t d i r e c t or i n d i r e c t p a t h o l o g i c a l a l t e r a t i o n s i n the s k e l e t a l muscle c e l l . D i r e c t 44 damage by the i n c r e a s e d i n t r a c e l l u l a r Ca may be due to h y p e r c o n t r a c t i o n o f sarcomeres i n the immediate v i c i n i t y of the e l e v a t e d [ C a ] i i n a d d i t i o n to s t r e t c h i n g and r u p t u r i n g of sarcomeres i n neighbouring m y o f i b r i l l a r r e g i o ns of the c e l l ; a l l of these changes pr e d i s p o s e to n e c r o s i s ( C u l l e n and F u l t h o r p e , 1975). I n d i r e c t damage by the e l e v a t e d [ C a ] i may be 44 the a c t i v a t i o n of i n t r a c e l l u l a r Ca -dependent p r o t e a s e s , r e s u l t i n g i n h y d r o l y s i s o f m y o f i b r i l l a r elements and other o r g a n e l l e s (Ebashi and S u g i t a , 1979) . Perhaps the c l e a r e s t phenomenon of DMD i m p l i c a t i n g a r o l e of SR i s the f i n d i n g o f a number of independent l a b o r a t o r i e s of -36-a d e f e c t (a decrease compared to c o n t r o l ) i n SR Ca t r a n s p o r t i n both human (Takagi e t a l , 1973) and animal ( S r e t e r e t a l , 1967; Martonosi, 1968; D h a l l a and Sulakhe, 1973) muscular dystrophy. To exclude the p o s s i b i l i t y t h a t these apparent f u n c t i o n a l changes r e f l e c t SR v e s i c l e contamination by membranes of i n f i l t r a t i n g f a t and con n e c t i v e t i s s u e c e l l s , c a f f e i n e - i n d u c e d t r a n s i e n t i s o m e t r i c t e n s i o n response s t u d i e s were performed i n s i n g l e "skinned" DMD s k e l e t a l muscle f i b r e s with the f i n d i n g of a d e f e c t i n SR Ca r e g u l a t i o n (Wood e t a l , 1978). A d e f e c t i n the a b i l i t y of d y s t r o p h i c s k e l e t a l muscle SR ++-to r a p i d l y sequester and/or s t o r e i n t r a c e l l u l a r Ca may e x p l a i n the delayed r e l a x a t i o n time and p a r t i a l l o s s of normal t e n s i o n development observed i n d y s t r o p h i c muscle (Roc et a l , 1967) and the p o s s i b l e calcium-mediated c y t o t o x i c i t y brought about by the s u s t a i n e d i n c r e a s e i n [ C a l i . The p r e c i s e nature of the p u t a t i v e SR d e f e c t of s k e l e t a l muscle remains to be demonstrated. P o s s i b l e s i t e s of the SR d e f e c t i n c l u d e the SR ATPase p r o t e i n and/or p r o t e i n s a s s o c i a t e d with the pump (Ve r j o v s k i - A l m e i d a and I n e s i , 1979), the documented r e g u l a t o r s of the ATPase a c t i v i t y ( ca l m o d u l i n , cAMP-dependent p r o t e i n ++ k i n a s e , e t c . ), SR p r o t e i n s a s s o c i a t e d with Ca r e l e a s e or 44 f a c i l i t a t i n g Ca storage (e.g. c a l s e q u e s t r i n ) (Etienne et a l , 1980), and a l t e r a t i o n s i n the l i p i d environment of the ATPase p r o t e i n i n the SR membrane (Hanna et a l , 1981). Malignant hyperthermia (MH) i s another d i s e a s e e n t i t y i n 44 which an SR Ca handl i n g d i s o r d e r may p l a y a prominent r o l e . -37-As summarized by Gronert (1980) , i n MHf s k e l e t a l muscle i n response to c e r t a i n a n e s t h e t i c s (halothane, s u c c i n y l c h o l i n e ) and drugs suddenly and unexpectedly i n c r e a s e s i t s a e r o b i c and anaerobic metabolism, r e s u l t i n g i n an inte n s e p r o d u c t i o n of heat, C02, and l a c t a t e . Muscular r i g i d i t y , i n c r e a s e d muscle p e r m e a b i l i t y ( i . e . e l e v a t e d serum c r e a t i n e kinase) and sympathetic s t i m u l a t i o n then ensue with m o r t a l i t y o c c u r r i n g i n almost 30% of re p o r t e d cases. I t i s c u r r e n t l y b e l i e v e d t h a t c o n t r o l of i n t r a c e l l u l a r (Cal i s l o s t i n MH ( G a l l a n t et a l , 1979) , the r e s u l t a n t i n c r e a s e i n metabolism i s symptomatic of the body's attempt to re v e r s e the abnormally high Ca** c o n c e n t r a t i o n s . The r e s u l t a n t damage to muscle and other o r g a n e l l e s may be presumed, as i n DMD, to r e s u l t from the s u s t a i n e d Ca l e v e l s and consequent t o x i c accumulation i n o r g a n e l l e s . Claims f o r the primary abnormality of SR i n MH have a r i s e n on the b a s i s of the prominent c l i n i c a l o b s e r v a t i o n of muscle r i g i d i t y and the i n c r e a s e d s e n s i t i v i t y of MH muscle to develop c a f f e i n e - i n d u c e d c o n t r a c t u r e s (Kalow et a l , 1972); c a f f e i n e i s thought to s p e c i f i c a l l y induce Ca r e l e a s e from the SR (Weber, 1968) . Ca t r a n s p o r t by SR appears to a l s o be decreased i n both human and p o r c i n e models of MH (Gronert et a l , 1979; Hidalgo e t  a l , 1982) but the d i f f e r e n c e s noted are not dramatic (Gronert, 1980) . Gronert (1980) suspects t h a t the noted decrease i n t r a n s p o r t a b i l i t y i s more of a secondary e f f e c t s i n c e (1) the decrease i n t r a n s p o r t i s not severe enough to account f o r the -38-e n t i r e MH syndrome, (2) with c l i n i c a l c o n c e n t r a t i o n s of halothane, Ca b i n d i n g ( i . e . oxalate-independent) was i n c r e a s e d i n both MH and normal SR, whereas at higher c o n c e n t r a t i o n s of halothane, Ca b i n d i n g and Ca - o x a l a t e uptake i n both types of SR were depressed (Gronert et a l , 1979), and (3) the s p e c i f i c a c t i o n of d a n t r o l e n e sodium i n p r e v e n t i n g and r e v e r s i n g MH along with the f i n d i n g of an m c r e a s e d s e n s i t i v i t y of MH muscle to c a f f e i n e i m p l i c a t e s Ca r e l e a s e (and the t e r m i n a l c i s t e r n a e of the SR) as opposed to ++ the Ca uptake process (Nelson, 1978). Somewhat u n r e l a t e d to the f i n d i n g of impaired SR f u n c t i o n due to presumed p a t h o l o g i c a l i n c r e a s e s i n i n t r a c e l l u l a r Ca i s ++• the noted decrease i n s k e l e t a l SR Ca t r a n s p o r t observed i n a v a r i e t y of e x p e r i m e n t a l l y - i n d u c e d v i t a m i n D d e f i c i e n t s t a t e s . Muscle weakness and atrophy are prominent f e a t u r e s of n u t r i t i o n a l v i t a m i n D d e f i c i e n c y or d i s e a s e s i n which the metabolism of v i t a m i n D i s a l t e r e d (e.g. uremia) (Smith and S t e r n , 1969). S p e c u l a t i o n has t h e r e f o r e a r i s e n t h a t the abnormal muscle f u n c t i o n may be the r e s u l t of impaired muscle c e l l C a + * turnover (Stanbury, 1965), perhaps at the l e v e l of E-C c o u p l i n g (Curry et a l , 1974) . The noted decrease i n SR Ca** t r a n s p o r t i n s t a t e s of both n u t r i t i o n a l v i t a m i n D d e f i c i e n c y (Curry et a l , 1974; Pointon et a l , 1979) and experimental uremia (Heimberg e t a l , 1976; Boland et a l , 1983) , and r e v e r s a l of such impaired SR f u n c t i o n by i n v i v o a d m i n i s t r a t i o n of c a l c i t r i o l ( 1 , 2 5 - d i h y d r o x y c h o l e c a l c i f e r o l ) (Matthews et_ al.,1977; Boland et a l , 1983) , the a c t i v e m e t a b o l i t e of v i t a m i n -39-D, have i m p l i c a t e d the SR as an important t a r g e t organ f o r v i t a m i n D. Three o b s e r v a t i o n s , however, suggest t h a t both the i n h i b i t i o n of C a + f t r a n s p o r t and r e v e r s a l of such impaired f u n c t i o n by c a l c i t r i o l may be more the r e s u l t of systemic metabolic changes than a primary SR d e f e c t . F i r s t , Ca -ATPase a c t i v i t y i n SR d e r i v e d from both c o n t r o l s and v i t a m i n D d e f i c i e n t animals remains unchanged (Curry et a l , 1974; Boland et a l , 1983b). Second, d e s p i t e c l e a r evidence of d e f e c t i v e m i n e r a l i z a t i o n i n r a t s t r e a t e d with ethane 1-hydroxy 1,1 diphosphonate (EHDP), an i n h i b i t o r of c a l c i t r i o l f ormation i n the kidney ( H i l l et a l , 1973), no change i n Ca +*-uptake a c t i v i t y was observed i n s k e l e t a l muscle SR of these animals (Pointon et a l , 1979) . T h i r d , muscle t i s s u e has been shown not to c o n t a i n s p e c i f i c c a l c i t r i o l r e c e p t o r s (Boland e t a l , 1983b) thus d i s c o u n t i n g such t i s s u e as a s p e c i f i c t a r g e t organ f o r v i t a m i n D. B. C a r d i a c Muscle S i m i l a r to the f i n d i n g s i n s k e l e t a l muscle SR c i t e d above, c e r t a i n p a t h o l o g i c a l s t a t e s of the myocardium are c o r r e l a t e d ++ with an i n h i b i t i o n of the h e a r t ' s SR Ca t r a n s p o r t system; whether the change re p r e s e n t s a primary or secondary event i n the development of the d i s e a s e process i s , a g a i n , u n c l e a r , but most i n v e s t i g a t o r s t h e o r i z e t h a t the a l t e r a t i o n of t r a n s p o r t i s a secondary m a n i f e s t a t i o n of the d i s e a s e ( D h a l l a , 1976; Katz, 1975) . The a l t e r a t i o n s i n SR c a l c i u m uptake and r e l a t e d changes -40-i n E-C c o u p l i n g have been most thoroughly i n v e s t i g a t e d i n ischemic heart d i s e a s e ( D h a l l a et a l , 1982). Ischemic heart d i s e a s e r e f e r s to a spectrum of c l i n i c a l d i s o r d e r s of the heart r e s u l t i n g from imbalance between the myocardial need f o r oxygen and the adequacy of i t s supply (Robbins and Cotran, 1979) . At l e a s t 95% of a l l cases examined prese n t a t h e r o s c l e r o t i c narrowing of the coronary a r t e r i e s ( H i l l i s and Braunwald, 1977). Involvement of the SR i s thought to begin as a c o r r e l a t e of the r a p i d l o s s of c o n t r a c t i l i t y (Scwartz et a l , 1973) accompanying i n t e r r u p t i o n of myocardial blood flow. The mechanism r e p o n s i b l e f o r the d e p r e s s i o n of c o n t r a c t i l e performance i s not known, although t h i s has not stopped i n v e s t i g a t o r s from proposing a p l e t h o r a of p o s s i b i l i t i e s , of which I w i l l b r i e f l y d i s c u s s f o u r : c e l l u l a r d e p l e t i o n of h i g h energy phosphate compounds, a c i d o s i s , C a + + o v e r l o a d , and the accumulation of l o n g - c h a i n f a t t y a c i d s . C e l l u l a r d e p l e t i o n of high energy phosphate compounds (e.g. ATP, c r e a t i n e phosphate) i s b e l i e v e d to occur i n the ischemic myocardium e i t h e r as a r e s u l t of the i n h i b i t o r y e f f e c t ++ of e l e v a t e d Ca c o n c e n t r a t i o n s (see below) on m i t o c h o n d r i a l o x i d a t i v e p h o s p h o r y l a t i o n ( D h a l l a et a l , 1982) or through the s e l e c t i v e l o s s of the adenine r i n g as ATP degrades to ADP, AMP, and adenosine, the l a t t e r r a p i d l y d i f f u s i n g through the c a r d i a c c e l l membrane ( O l i v e r , 1978). Although some workers i n s i s t t h a t such l o s s of high energy phosphates i s the major cause of c a r d i a c c e l l death and the r e s u l t i n g d e p r e s s i o n of c o n t r a c t i l i t y (Guyton, 1981), o t h e r s (Katz, 1975; D h a l l a et a l , -41-1978) h o l d t h a t ATP d e p l e t i o n i s not a c r i t i c a l f a c t o r , at l e a s t i n i t i a l l y . I n s o f a r as a d e p r e s s i o n of the SR Ca + +-ATPase i s concerned, the average c e l l u l a r ATP l e v e l s i n the f a i l i n g myocardium s t i l l f a l l w e l l above the Km of 0.1 mM, thus ++ e x c l u d i n g an impairment of the Ca pump f o l l o w i n g ATP " d e p l e t i o n " (Katz, 1975). The f a l l i n i n t r a c e l l u l a r pH t h a t occurs when the myocardium becomes ischemic i s a l s o thought to be a l i k e l y e x p l a n a t i o n f o r the reduced c a r d i a c c o n t r a c t i l i t y (Steenbergen et a l , 1977) . Under r e s t i n g c o n d i t i o n s , c a r d i a c muscle u t i l i z e s f a t s mainly f o r i t s energy, with approximately 70% of the normal metabolism being d e r i v e d from f a t t y a c i d s ( O l i v e r , 1978). Under anaerobic or ischemic c o n d i t i o n s , however, the m e t a b o l i c demands of the h e a r t are d r a m a t i c a l l y a l t e r e d such t h a t anaerobic g l y c o l y s i s becomes the primary energy source. Under these c o n d i t i o n s , H + w i l l be produced d u r i n g the metabolism of glucose to l a c t a t e . The f a l l i n pH i n f l u e n c e s the c a r d i a c c e l l n e g a t i v e l y i n a number of ways (Mandel et a l , 1982), a f f e c t i n g mitochondria, myosin ATPase a c t i v i t y , and the b i n d i n g of Ca to t r o p o n i n C and sarcolemma. Of p a r t i c u l a r *+ r e l e v a n c e , however, i s the noted decrease i n SR Ca -uptake a c t i v i t y (Nakamura and Schwartz, 1972; Sorenson and deMeis, 1977; F a b i a t o and F a b i a t o , 1978) under a c i d i c c o n d i t i o n s , suggesting a p o s s i b l e e x p l a n a t i o n of the decrease i n c o n t r a c t i l i t y as a s t a t e of a c i d o s i s develops i n the ischemic myocardium. The mechanism of the e f f e c t of pH on SR Ca t r a n s p o r t has r e c e n t l y been i n v e s t i g a t e d by Mandel et a l (1982) -42-who found t h a t formation and decomposition of E-P became depressed under a c i d i c c o n d i t i o n s . The r e t a r d a t i o n of these two r a t e - l i m i t i n g steps i n the r e a c t i o n sequence of the 4-4- ++ (Ca -Mg )-ATPase may, then, be one of the c o n t r i b u t i n g f a c t o r s to the i n h i b i t i o n of myocardial f u n c t i o n i n ischemia. Another f a c t o r thought to be important i n the ischemic ++ myocardium i s a p a t h o l o g i c a l r i s e i n i n t r a c e l l u l a r Ca ( ( C a ] i ) , termed c a l c i u m o v e r l o a d ( D h a l l a et a l , 1978). As d i s c u s s e d i n the s e c t i o n on p a t h o l o g i c a l a l t e r a t i o n s i n s k e l e t a l SR, a r i s e i n [ C a ] i appears to be c l o s e l y c o r r e l a t e d ++ with muscle t i s s u e n e c r o s i s . The concept of a Ca -induced myocardial n e c r o s i s ( F l e c k e n s t e i n et a l , 1974) has a l s o been in t r o d u c e d to account f o r the d e c l i n e i n c o n t r a c t i l i t y of the ischemic myocardium. In the o r i g i n a l f o r m u l a t i o n by F l e c k e n s t e i n e t a l (1974) , i t was suggested t h a t m i t o c h o n d r i a l 4-4-damage r e s u l t i n g from Ca o v e r l o a d , coupled with the a c t i v a t i o n of Ca^-dependent ATPases, would r e s u l t i n sharp decreases i n high energy phosphates, i m p a i r i n g c e l l u l a r f u n c t i o n . D h a l l a (1976) has suggested t h a t p a t h o l o g i c a l s t a t e s such as ischemia a l t e r c a r d i a c membrane s t r u c t u r e and f u n c t i o n . 4-4-T h i s , then, leads e i t h e r to a Ca o v e r l o a d or d e f i c i e n c y (Figure 6) both of which are d r a s t i c circumstances i n the c a r d i a c c e l l and r e s u l t i n c o n t r a c t i l e f a i l u r e and i n h i b i t i o n 4-4- f+ of Ca t r a n s p o r t v i a any of the Ca - a s s o c i a t e d p a t h o l o g i c a l 4-+ consequences such as Ca -induced i n h i b i t i o n of m i t o c h o n d r i a l o x i d a t i v e p h o s p h o r y l a t i o n , t i s s u e n e c r o s i s , or the r e l e a s e of 4-4-Ca - a c t i v a t e d p r o t e a s e s , p hospholipases, and lysosomal enzymes -43-DIFFERENT ETiOLOOlCAL FACTORS J MEMBRANE DEFECTS S A R C O L E M M A S A R C O P L A S M I C R E T I C U L U M S A R C O P L A S M I C R E T I C U L U M S A R C O L E M M A 2* I N F L U X MITOCHONDRIA I C a 2 * RELEASE I N T R A i E L L U L A R C a z > P E r i c i E N C Y f*'taJ I ATP T PRODUCTION 2+ I N F L U X 1 Ca 2 + uPTAKt 1 ATP PRODUCTION J I N T R A C E L L U L A R C a 2 + O V E R L O A l > I A T P * UTILIZATION ACTIVATION OF B t S T R U C T l V e MECHANISMS T k ATP ' U T I L I Z A T I O N AVAILABILITY OF ENERGY FOR [CEU.UUR INTIMITY AND FUNCTION CONTRACTILE FAILURE FIGURE 6 . P o s t u l a t e d sequence o f s t e p s in a scheme i n v o l v i n g membrane d e f e c t s in h e a r t f a i l u r e due to i n t r a c e l l u l a r c a l c i u m d e f i c i e n c y o r i n t r a c e l l u l a r c a l c i u m o v e r l o a d . Taken f rom D h a l l a ( 1 9 7 6 ) . -44-( D h a l l a et a l , 1982). The newest p o s t u l a t e invoked to e x p l a i n c o n t r a c t i l e f a i l u r e i n ischemic myocardium i s the suggestion o f lo n g - c h a i n f a t t y a c i d (and t h e i r d e r i v a t i v e s ) accumulation i n the he a r t (Katz and Messineo, 1981). I t i s proposed t h a t m i t o c h o n d r i a l f a t t y a c i d o x i d a t i o n becomes i n h i b i t e d i n the ischemic myocardium, p o s s i b l y due to a b u i l d - u p o f i n t r a c e l l u l a r ( C a l , with the r e s u l t a n t accumulation of lo n g - c h a i n f a t t y a c i d s and t h e i r a c y l c a r n i t i n e d e r i v a t i v e s (Idell-Wenger and Neely, 1978). Of p a r t i c u l a r i n t e r e s t i s the r a p i d accumulation of p a l m i t y l c a r n i t i n e , a potent i n h i b i t o r o f both sarcolemmal Na, K 44- 4r ATPase and sa r c o p l a s m i c r e t i c u l u m (Ca -Mg )-ATPase ( P i t t s et a l , 1978; Adams et a l , 1979). The s t r o n g l y charged c a r n i t i n e moiety i n a s s o c i a t i o n with l o n g - c h a i n (hydrophobic) f a t t y a c i d s imparts a h i g h l y a m p i p h i l i c c h a r a c t e r to the molecule, a l l o w i n g f o r i t s i n t e r c a l a t i o n i n t o the membrane and a b i l i t y to d i s r u p t enzymatic a c t i v i t y , p o s s i b l y due to i t s d e t e r g e n t - l i k e a c t i o n (Adams e t a l , 1979). I t has t h e r e f o r e been suggested t h a t the e l e v a t e d l e v e l s of p a l m i t y l c a r n i t i n e and consequent i n h i b i t i o n 4+ of SR Ca t r a n s p o r t may be a major c o n t r i b u t i n g f a c t o r i n the f a l l of c o n t r a c t i l i t y i n ischemic myocardium. Lopaschuk et a l (1983) have r e c e n t l y invoked the n o t i o n of an accumulation o f lon g - c h a i n a c y l c a r n i t i n e s to e x p l a i n the mechanism whereby both c o n t r a c t i l e f u n c t i o n (Fein et a l , 1980; 4-+ Vadlamudi et a l , 1982) and c a r d i a c SR Ca t r a n s p o r t (Penpargkul e t a l , 1981; Lopaschuk et a l , 1983) are depressed i n the c h r o n i c e x p e r i m e n t a l l y - i n d u c e d d i a b e t i c r a t . In - 4 5 -d i a b e t e s , the heart e x t r a c t s i t s energy almost e x c l u s i v e l y from the o x i d a t i o n of f a t t y a c i d s (Randle, 1978), a by-product of which are e l e v a t e d l e v e l s of a c y l c a r n i t i n e s (Feuvray et a l , 1979) which serve as i n t e r m e d i a t e s i n the t r a n s p o r t of a c y l CoA i n t o the inner m i t o c h o n d r i a l matrix ( F r i t z , 1959). Lopaschuk et a l (1983) found t h a t as the course of the d i a b e t i c s t a t e p r o g r e s s e d , the l e v e l s of l o n g - c h a i n a c y l c a r n i t i n e s rose. T h i s c o r r e l a t e d with the corresponding time-dependent i n h i b i t i o n of heart f u n c t i o n and SR Ca t r a n s p o r t . I t i s of i n t e r e s t t h a t l e v e l s of long c h a i n a c y l c a r n i t i n e s are a l s o e l e v a t e d i n the s k e l e t a l muscles of d i a b e t i c r a t s (Stearns, 1980) . A corresponding decrease i n SR C a + + t r a n s p o r t and s k e l e t a l muscle c o n t r a c t i l i t y , i f observed, may suggest a r o l e f o r the involvement of a c y l c a r n i t i n e d e r i v a t i v e s i n the muscle pathophysiology of d i a b e t e s . -46-O b j e c t i v e s of the Study The main o b j e c t i v e s of t h i s c u r r e n t study were (i) to f u r t h e r e x p l o r e the nature of the c a l m o d u l i n and cAMP-dependent p r o t e i n kinase modulation of c a r d i a c SR c a l c i u m t r a n s p o r t , ( i i ) , to determine the r o l e , i f any, of calmodulin i n s k e l e t a l 44 muscle SR systems and ( i i i ) , study Ca t r a n s p o r t i n s k e l e t a l muscle SR obtained from a d i s e a s e model where a l t e r a t i o n s i n c a l c i u m t r a n s l o c a t i o n have been noted. The f o l l o w i n g i s the r a t i o n a l e f o r these s t u d i e s : 1) Much of the work on c a r d i a c microsomes e n r i c h e d i n SR has been plagued by doubts about p u r i t y . In c o n t r a s t to s k e l e t a l muscle, where the SR i s the more e x t e n s i v e l y developed membrane system, i n c a r d i a c SR, the sarcolemma c o n t r i b u t e s a much gre a t e r percentage of the t o t a l membrane mass. In p r e p a r i n g the SR v e s i c l e s , contamination by SL and m i t o c h o n d r i a l membranes, 44 44 both of which c o n t a i n (Ca -Mg )-ATPase a c t i v i t y , i s of r e a l concern and may l e a d to s p u r i o u s r e s u l t s . We t h e r e f o r e undertook a p u r i f i c a t i o n of c a r d i a c SR f o r the sake of i n v e s t i g a t i n g of the e f f e c t s of the indigenous r e g u l a t o r s 44 (e.g., cAMP-dependent p r o t e i n k i n a s e , calmodulin) on Ca 44 44 t r a n s p o r t , (Ca -Mg )-ATPase a c t i v i t y and membrane p h o s p h o r y l a t i o n i n a system t h a t i s l e s s contaminated with other o r g a n e l l e s . 2) The groups of C a r a f o l i ( C h i e s i and C a r a f o l i , 1982) and -47-MacLennan (Campbell and MacLennan, 1982) have r e c e n t l y i n v e s t i g a t e d the r o l e of calmodulin i n s k e l e t a l muscle SR. Both groups have determined the presence of CAM-dependent p r o t e i n p h o s p h o r y l a t i o n which appears not to r e s u l t i n the s t i m u l a t i o n ++ of Ca t r a n s p o r t . Our l a b o r a t o r y had assumed t h a t the _i \ i n a b i l i t y of CAM to s t i m u l a t e Ca t r a n s p o r t was r e l a t e d to i t s high endogenous l e v e l i n SR membranes. C h i e s i and C a r a f o l i (1982) demonstrated the presence of CAM i n f a s t s k e l e t a l SR by f i r s t b o i l i n g the membranes and then p a s s i n g the denatured suspension down a CAPP-Sepharose c a l m o d u l i n - a f f i n i t y column. They concluded t h a t CAM was t i g h t l y bound to the membrane and o n l y removable by harsh treatment such as b o i l i n g . I t was of g r e a t i n t e r e s t , t h e r e f o r e , t h a t Campbell and MacLennan (1982) s t a t e d t h a t CAM was indeed bound to the membrane but was e a s i l y d i s l o d g e d by treatment with EGTA. T h e i r s u p p o s i t i o n was p a r t l y based on the o r i g i n a l o b s e r v a t i o n s of MacLennan (1972) t h a t c e n t r i f u g a t i o n and re-suspension of f a s t SR v e s i c l e s three times i n the presence of 1 mM EGTA r e s u l t e d i n a decreased a b i l i t y of the SR p e l l e t to t r a n s p o r t Ca . When the r e s u l t a n t supernatant was added back to the SR t r a n s p o r t system, Ca uptake c o u l d be p a r t i a l l y r e s t o r e d . The more recent work by MacLennan (Campbell and MacLennan, 1982) confirmed t h a t the supernatant of 1 mM EGTA-washed fSR contained CAM on the b a s i s of two o b s e r v a t i o n s . F i r s t , EGTA-washed fSR demonstrated an enhanced l e v e l of p h o s p h o r y l a t i o n i n the presence of CAM compared to "unwashed" SR v e s i c l e s (presumably due to the d e p l e t i o n of CAM by EGTA), and second, supernatant d e r i v e d from -48-1 mM EGTA-washed SR s t i m u l a t e d p h o s p h o r y l a t i o n of fSR. Our work, then, was an attempt to r e s o l v e the c o n t r o v e r s y of the nature of the b i n d i n g of CAM to the s k e l e t a l SR membrane. +-+-3) I t has been proposed t h a t the d e p r e s s i o n of Ca -uptake a c t i v i t y noted i n c a r d i a c SR obtained from c h r o n i c a l l y d i a b e t i c r a t s may be due to the accumulation of l o n g - c h a i n a c y l c a r n i t i n e s which a c t as d i s r u p t i v e ampiphiles i n the l i p i d environment of the Ca pump p r o t e i n (Lopaschuk e t a l , 1983) . As t o t a l t i s s u e l e v e l s of l o n g - c h a i n a c y l c a r n i t i n e s have a l s o been r e p o r t e d to be e l e v a t e d i n c h r o n i c a l l y d i a b e t i c r a t s (Stearns, 1980), i t was of i n t e r e s t to i n v e s t i g a t e whether the 44-Ca t r a n s p o r t a b i l i t y of s k e l e t a l muscle SR was a l s o a f f e c t e d and, i f so, what r o l e the a c y l c a r n i t i n e d e r i v a t i v e s might p l a y i n the p a t h o p h y s i o l o g y o f d i a b e t e s and r e g u l a t i o n of both s k e l e t a l and c a r d i a c SR f u n c t i o n . -49-MATERIALS AND METHODS A) MATERIALS (i) Animals: a) S t u d i e s u t i l i z i n g c a r d i a c microsomes e n r i c h e d i n s a r c o p l a s m i c r e t i c u l u m . Dogs (10-30 kg) of e i t h e r sex were used f o r p r e p a r a t i o n of c a r d i a c microsomes enr i c h e d i n sarcoplasmic r e t i c u l u m . Hearts were removed from p e n t o b a r b i t a l - a n e s t h e t i z e d dogs and p l a c e d i n i c e - c o l d normal s a l i n e . The v e n t r i c l e s were cut i n t o 2-4 g p i e c e s , and e i t h e r used immediately f o r p r e p a r a t i o n of s a r c o p l a s m i c r e t i c u l u m , or q u i c k - f r o z e n i n 2-methylbutane on o dry i c e , and s t o r e d a t -80 C u n t i l use. b) S t u d i e s u t i l i z i n g s k e l e t a l microsomes e n r i c h e d i n sarcoplasmic r e t i c u l u m . New Zealand White r a b b i t s of e i t h e r sex were used throughout the study. Animals, some of which were i n j e c t e d with 500 U of h e p a r i n 10 minutes p r i o r to s a c r i f i c e , were k i l l e d by a sharp blow to the base of the s k u l l . Vastus l a t e r a l i s ( f a s t ) and s o l e u s (slow) muscle were e x c i s e d from the hind limbs and p l a c e d i n i c e - c o l d 10 mM t r i s maleate, pH 6.8 f o r microsomal SR p r e p a r a t i o n . c) S t u d i e s u t i l i z i n g s k e l e t a l microsomes enr i c h e d i n -50-s a r c o p l a s m i c r e t i c u l u m d e r i v e d from d i a b e t i c animals. Female Wistar r a t s (Charles R i v e r Canada ) between 150-200 g were used. Diabetes was induced by a s i n g l e t a i l - v e i n i n j e c t i o n of s t r e p t o z o t o c i n (50 mg/kg) d i s s o l v e d i n c i t r a t e b u f f e r , pH 4.0. C o n t r o l animals were i n j e c t e d with c i t r a t e b u f f e r alone. Animals were then housed f o r 90 days d u r i n g which time food and water were a v a i l a b l e ad l i b i t u m . Rats were s a c r i f i c e d at the end of the 90 day p e r i o d and the t i b i a l i s a n t e r i o r ( f a s t ) muscle was i s o l a t e d from both hindlimbs, and p l a c e d i n i c e - c o l d 10 mM t r i s maleate b u f f e r , pH 6.8 f o r p r e p a r a t i o n of microsomes e n r i c h e d i n sarcoplasmic r e t i c u l u m . At the time of s a c r i f i c e , serum samples were withdrawn and assayed f o r i n s u l i n [Becton-Dickinson I n s u l i n 1 I-Radioimmunoassay K i t ] and glucose [Ames Reagent K i t f o r R Blood G l u c o s e ] . ( i i ) Chemicals a. R a d i o i s o t o p e s : ( ^ * C a ) C l t (10 Ci/mmole) was purchased from * i v r Amersham r a d i o c h e m i c a l s . I-RIA k i t f o r c a l m o d u l i n was obtained from New England Nuclear.. b. Reagents: the f o l l o w i n g chemicals were purchased from R Sigma Chemical Company: ammonium b i c a r b o n a t e , L - a s c o r b i c a c i d , bromphenol b l u e , c i t r i c a c i d , compound 48/80, d e o x y c h o l i c a c i d , EDTA (disodium s a l t ) , EGTA, F o l i n and C i o c a l t e u ' s phenol reagent, g l y c e r o l , g l y c i n e , N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N 1 - 2 - e t h a n e s u l f o n i c a c i d (HEPES), -51-magnesium c h l o r i d e , potassium c h l o r i d e , s i l v e r n i t r a t e , sodium a z i d e , sodium carbonate, sucrose, N,N,N',N'-tetrarnethylethylenediamine (TEMED), t r i c h l o r o a c e t i c a c i d , Trizma adenosine t r i p h o s p h a t e , Trizma base, Trizma h y d r o c h l o r i d e , Trizma maleate, and Trizma o x a l a t e . - Sodium dodecyl s u l f a t e was purchased from B i o r a d . g - Sodium c h l o r i d e was purchased from Amachem. - The f o l l o w i n g reagents were purchased from F i s h e r : g l a c i a l a c e t i c a c i d , g l u t a r a l d e h y d e , & c e l l o - s e a l . - B i o r a d e l e c t r o p h o r e t i c reagents i n c l u d e d acrylamide, ammonium p e r s u l f a t e , b i s a c r y l a m i d e , and High and Low Molecular Weight Standards. - Calmodulin used was purchased from e i t h e r Calbiochem or Sigma Chemical Co. - Calcium c h l o r i d e and sodium f l u o r i d e were obtained from H A n a l a r . - T r i f l u o p e r a z i n e d i h y d r o c h l o r i d e was a generous g i f t of Smith, K l i n e , & French. - Aquasol s c i n t i l l a t i o n f l u i d was purchased from New England a Nuclear. Z - 2-Methylbutane was obtained from MCB Manufacturing Chemists. ( i i i ) Apparatus - The assay of i n o r g a n i c phosphate was performed s p e c t r o p h o t o m e t r i c a l l y at 660 nm using a Technicon Autoanalyzer model I and recorded on a Technicon c h a r t r e c o r d e r . -52-- Immersible-Cx separator f i l t e r s were obtained from M i l l i p o r e Co. - E l e c t r o p h o r e s i s was performed with a s l a b - g e l apparatus on s l a b s of 1.5-mm t h i c k n e s s and 15-cm l e n g t h . Constant c u r r e n t was s u p p l i e d by a Pharmacia power supply (Model EPS 500/400). f? Gels were d r i e d under vacuum using a Bio-Rad g e l d r y e r , model 224. -53-B. METHODS (1) P r e p a r a t i v e Methods (a) P r e p a r a t i o n of S k e l e t a l Muscle and C a r d i a c Muscle Microsomes Enr i c h e d i n Sarcoplasmic Reticulum Rabbit f a s t and slow s k e l e t a l muscle and dog c a r d i a c muscle microsomes e n r i c h e d i n sarcoplasmic r e t i c u l u m were prepared by a m o d i f i c a t i o n of the method of Sumida et a l o (1978) . A l l p r e p a r a t i o n was performed at 4 C. Muscle was homogenized with a t e f l o n p e s t l e f o r 15 seconds at 1500 rpm i n 10 mM t r i s maleate, pH 6.8. The homogenate was then c e n t r i f u g e d at 4000g f o r 10 min, and the supernatant passed through c h e e s e c l o t h . F o l l o w i n g r e - c e n t r i f u g a t i o n at 15,000g f o r 20 min, the supernatant was again passed through c h e e s e c l o t h . T h i s supernatant was then c e n t r i f u g e d at 40,000g f o r 80 min. The r e s u l t i n g p e l l e t was then re-suspended i n 10 mM t r i s maleate, pH 6.8, c o n t a i n i n g 0.6M KC1 and c e n t r i f u g e d at 40,000g f o r 100 min. The f i n a l p e l l e t was suspended i n 10 mM t r i s maleate c o n t a i n i n g 40% sucrose, q u i c k - f r o z e n i n 2-methylbutane on dry o i c e , and s t o r e d at -70C u n t i l use. (b) P r e p a r a t i o n of a P u r i f i e d C a r d i a c Microsomal P r e p a r a t i o n E n r i c h e d i n Sarcoplasmic Reticulum C a r d i a c microsomes enr i c h e d i n sarcoplasmic r e t i c u l u m prepared by the method of Sumida e t a l (1978) were s u b j e c t to f u r t h e r p u r i f i c a t i o n u s ing the method of Jones et a l (1979) -54-with m o d i f i c a t i o n s . F r e s h l y prepared or f r o z e n a l i q u o t s of crude 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 (10-25 mg) were incubated at 37°C f o r 5 minutes i n a medium c o n t a i n i n g 50 mM h i s t i d i n e - C l pH 6.8, 15 mM C a C l ^ , 5 mM t r i s - o x a l a t e , 16 mM t r i s - E G T A pH 6.8, 5 mM MgCl^, 100 mM KC1, and 5 mM NaN^. Loading of the v e s i c l e s with c a l c i u m o x a l a t e was i n i t i a t e d by the a d d i t i o n of 5 mM ATP. A f t e r 5 minutes, a second a l i q u o t of 5 mM t r i s - o x a l a t e was added and the r e a c t i o n terminated a f t e r 5 minutes by p l a c i n g the r e a c t i o n tubes on i c e . The sample was then c e n t r i f u g e d a t 4°C a t 40,000xg f o r 90 minutes, and the p e l l e t suspended i n a medium c o n t a i n i n g 0.25 M sucrose, 0.30 M KC1, and 0.1 M t r i s base, pH 7.2, and l a y e r e d onto a d i s c o n t i n u o u s sucrose d e n s i t y g r a d i e n t c o n s i s t i n g of c o n s e c u t i v e l a y e r s of 0.6 M, 1.0 M, and 1.5 M sucrose ( a l l c o n t a i n i n g 0.3 M KC1 and 0.1 M t r i s base, pH 7.2). The p r e p a r a t i o n was then c e n t r i f u g e d f o r 2 hours at 27,000 rpm i n an SW 27 r o t o r (Beckman Instruments). The p e l l e t sedimenting through the 1.5 M sucrose l a y e r was then re-suspended i n 10 mM t r i s maleate, pH 6.8 and e i t h e r used f r e s h l y prepared or q u i c k - f r o z e n i n 2-methylbutane on dry i c e o and s t o r e d a t -80 C u n t i l use. (c) P r e p a r a t i o n of B o i l e d S k e l e t a l Sarcoplasmic Reticulum E x t r a c t s Approximately 3-8 ml of f a s t or slow sarcoplasmic o r e t i c u l u m v e s i c l e s (1-3 mg/ml) were p l a c e d i n a 95 water bath and incubated f o r f i v e minutes i n e i t h e r the presence or absence of 0.2 mM EDTA. Samples were then c e n t r i f u g e d at -55-40,000xg f o r 30 minutes and the c l e a r supernatant was i s o l a t e d . When EDTA was pre s e n t , 0.2 mM C a C ^ was added i n order to c h e l a t e i t . Samples were p l a c e d i n d i a l y s i s membrane tubing (Spectrapor 1) and d i a l y z e d a g a i n s t 10 mM NH^HCO^ pH 7, o f o r 48 hours at 4 C. E x t r a c t s were then l y o p h i l i z e d , re-suspended i n a sm a l l volume of 10 mM t r i s maleate, pH 6.8, and f r o z e n u n t i l use. (d) P r e p a r a t i o n of EGTA-Washed S k e l e t a l Sarcoplasmic Reticulum E x t r a c t s I s o l a t i o n of supernatant from f a s t and slow sarcoplasmic r e t i c u l u m washed i n 1 mM EGTA was performed a c c o r d i n g to the method of MacLennan (1972). A l i q u o t s of f a s t and slow SR (3-8 ml of 1-3 mg/ml) were incubated at 4 C with 1 mM EGTA f o r 15 minutes and then c e n t r i f u g e d a t 40,000xg f o r 50 min. The p e l l e t was suspended and g e n t l y homogenized with a T e f l o n p e s t l e and r e - c e n t r i f u g e d at 40,000xg f o r 50 min. T h i s process was repeated a t h i r d time. F o l l o w i n g the f i n a l c e n t r i f u g a t i o n , the supernatant was withdrawn and the p e l l e t homogenized and suspended i n approx. 2 ml of 10 mM t r i s maleate pH 6.8, c o n t a i n i n g 40% sucrose. The p e l l e t (EGTA-washed SR) was then used f o r p r e p a r a t i o n of b o i l e d e x t r a c t s (see above). C a C l ^ (1 mM) was added to the supernatant f o r c h e l a t i o n f o l l o w i n g which t h i s e x t r a c t was d i a l y z e d a g a i n s t 10 mM NH,HCOa pH 7, f o r 48 o hours at 4 C, l y o p h i l i z e d , and the p e l l e t re-suspended i n a sm a l l volume of 10 mM t r i s maleate b u f f e r . A summary of the p r e p a r a t i o n o f the v a r i o u s e x t r a c t s i s presented i n F i g u r e 7. -56-F a s t o r S l ow SR b o i l e d i n p r e s e n c e o r absence o f 0.2 mM EDTA wash 3x i n 1 mM EGTA s u p e r n a t a n t s u p e r n a t a n t + 0.2 mM CaCl2 d i a l y z e and c o n c e n t r a t e + 1 mM CaCl2 d i a l y z e and c o n c e n t r a t e F i g . 7 : M e t h o d o l o g y f o r p r e p a r a t i o n o f s u p e r n a t a n t s used f o r s k e l e t a l SR s t u d i e s . - 5 7 -(e) P r e p a r a t i o n of Red C e l l Membranes Human blood, not more than four days o l d , was obtained from the Canadian Red C r o s s . E r y t h r o c y t e s , c o l l e c t e d by c e n t r i f u g a t i o n a t 2500xg f o r 5 min., were then prepared by one o of two methods ( a l l p r e p a r a t i o n performed at 4 C ) : (i) Method 1 F o l l o w i n g removal of the plasma and b u f f y coat, the red blood c e l l s were washed twice i n two volumes of normal s a l i n e . Osmotic l y s i s was i n i t i a t e d by the a d d i t i o n of 10 volumes of i c e - c o l d d i s t i l l e d , d e - i o n i z e d water. A f t e r 30 minutes, the c e l l s were c e n t r i f u g e d a t 38,000xg f o r 20 minutes. The supernatant was a s p i r a t e d and the f o l l o w i n g washes were i n i t i a t e d (each wash c o n s i s t i n g of a 38,000xg x 20 min. c e n t r i f u g a t i o n f o l l o w e d by a s p i r a t i o n of s u p e r n a t a n t ) : twice i n 1 mM t r i s - E D T A , pH 7.4, once i n 10 mM t r i s - E D T A , pH 7.4, three times i n 2 mM t r i s - C l , pH 7.4, and the f i n a l white p e l l e t suspended i n 2 mM t r i s - C l , pH 7.4 c o n t a i n i n g 4 ;uM EDTA. Membranes were q u i c k - f r o z e n i n 2-methylbutane on dry i c e and o s t o r e d f o r one week at -80 C. ( i i ) Method 2 Red c e l l membranes were prepared by the method of N i g g l i et a l (1981) with some m o d i f i c a t i o n . E r y t h r o c y t e s were washed twice i n f i v e volumes of 130 mM KC1 and 20 mM t r i s - C l , pH 7.4. The c e l l s were then hemolyzed i n 5 volumes of 1 mM K-EDTA, 10 mM t r i s - C l , pH 7.4, and c e n t r i f u g e d a t 18,000xg f o r 10 minutes. T h i s s t e p was repeated f i v e times, f o l l o w i n g which the -58-membranes were washed i n 10 mM HEPES, pH 7.4, and c e n t r i f u g e d at 18,000xg f o r 10 min. T h i s l a t t e r step was repeated four times. The ghosts were r e t a i n e d i n 10 mM HEPES, pH 7.4, q u i c k - f r o z e n i n 2-methylbutane on dry i c e , and s t o r e d f o r 1 week at -80°C. (2) A n a l y t i c a l Methods (a) Measurement of Calcium Uptake by S k e l e t a l and C a r d i a c Muscle Microsomes Enriched i n Sarcoplasmic Reticulum. 44-ATP-dependent Ca -uptake i n t o s k e l e t a l and c a r d i a c SR was measured by the method of Tada et a l (1974) with a few •H* m o d i f i c a t i o n s . O x a l a t e - f a c i l i t a t e d Ca -uptake was determined i n an i n c u b a t i o n medium c o n t a i n i n g 10-30 ug SR p r o t e i n , 40 mM h i s t i d i n e - H C l , pH 6.8, 5 mM MgCl^, 5 mM t r i s ATP, 2.5 mM t r i s •H-o x a l a t e , 110 mM KC1 and a v a r i e t y of f r e e Ca c o n c e n t r a t i o n s . ++-The d e s i r e d f r e e Ca c o n c e n t r a t i o n s ( c o n t a i n i n g 10 Ci/mmole At CaCl^) were maintained by the a d d i t i o n of ethylene g l y c o l b i s ( aminoethyl ether)-N,N' t e t r a a c e t a t e (EGTA), and determined by the F o r t r a n program, Mult.S (see below). Samples were o p r e i n c u b a t e d f o r 7 minutes at 30 C and the r e a c t i o n i n i t i a t e d 45" by the a d d i t i o n of C a C l ^ . The r e a c t i o n was terminated a f t e r 5 min, i n the case of c o n t r o l s k e l e t a l and c a r d i a c muscle SR, and 1 min., i n the case of p u r i f i e d c a r d i a c and d i a b e t i c s k e l e t a l muscle SR, by f i l t e r i n g an a l i q u o t of the r e a c t i o n mixture through a 0.45 jum M i l l i p o r e f i l t e r (HA 45, M i l l i p o r e Co.). The f i l t e r was then washed twice with Aquasol (New England N u c l e a r ) , and counted f o r r a d i o a c t i v i t y i n a l i q u i d s c i n t i l l a t i o n counter. - C a l c u l a t i o n o f Calcium Uptake A c t i v i t y by S k e l e t a l Muscle Microsomes En r i c h e d i n Sarcoplasmic Reticulum The r a t e of c a l c i u m uptake by the microsomal p r e p a r a t i o n i s expressed i n nmoles of C a + + taken up per mg p r o t e i n per minute. T h i s i s determined by the f o l l o w i n g c a l c u l a t i o n : (sample counts - blank) x d i l u t i o n f a c t o r x t o t a l c a l c i u m ( t o t a l counts - blank) x i n c u b a t i o n time x mg p r o t e i n where: AC sample counts= Ca counts (dpm) obtained per sample t o t a l counts= t o t a l Ca counts (dpm) present i n the i n c u b a t i o n media blank= Ca counts (dpm) obtained i n the absence of microsomal p r o t e i n d i l u t i o n f a c t o r = c o r r e c t i o n f o r i n c u b a t i o n volume sampled (= 1.21) i n c u b a t i o n time= l e n g t h of time (5 min) microsomal p r o t e i n i s incubated i n the presence of C a C l ^ t o t a l calcium= t o t a l amount of c a l c i u m present i n the i n c u b a t i o n medium (= 62.5 nmoles) mg pr o t e i n = weight of microsomal p r o t e i n present i n the i n c u b a t i o n medium ++ 4+ (b) Assay of (Ca -Mg )-ATPase A c t i v i t y i n C a r d i a c Microsomes En r i c h e d i n Sarcoplasmic Reticulum -60-, ++ ++ (Ca -Mg )-ATPase a c t i v i t y was measured by a m o d i f i c a t i o n of the method of Katz and B l o s t e i n (1975) using the i n c u b a t i o n c o n d i t i o n s of Tada e t a l (1979). P u r i f i e d 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 (0.6-3.6 )iq) was pr e - i n c u b a t e d i n 40 mM h i s t i d i n e - C l b u f f e r pH 6.8, 5 mM MgCl^, 110 mM KC1, 10 mM NaF, and 5.7 pM A23187 a t 30 C f o r 5 minutes. The f r e e c a l c i u m c o n c e n t r a t i o n was maintained by the a d d i t i o n of Ca-EGTA. The r e a c t i o n was s t a r t e d by the a d d i t i o n of 200 pn ATP c o n t a i n i n g P-ATP (10 Ci/mmole; 100,000 dpm/sample) and terminated i n one minute by the a d d i t i o n of i c e - c o l d 5% TCA c o n t a i n i n g 5 mM Na 0ATP and 2 mM * ft KH^PO^. A suspension of a c t i v a t e d c h a r c o a l (1.5 g F i s h e r N o r i t A/10 ml) was added to each sample, vortexed, and incubated at o 4 C f o r 15 minutes with o c c a s i o n a l v o r t e x i n g . Samples were then c e n t r i f u g e d at 1500xg f o r 5 minutes, and an a l i q u o t of the c l e a r supernatant counted f o r r a d i a c t i v i t y . R e s u l t s are expressed as nmoles P i r e l e a s e d / mg p r o t e i n / min. (c) P h o s p h o r y l a t i o n of C a r d i a c Membrane V e s i c l e s Crude and p u r i f i e d (10-40 jug p r o t e i n ) microsomes enr i c h e d o i n s a r c o p l a s m i c r e t i c u l u m were preincubated at 30 C f o r 10 minutes i n a medium c o n t a i n i n g 40 mM h i s t i d i n e - C l pH 6.8, 110 mM KC1, 4 mM MgCl^, 10 mM NaF, and 5.7 JJM A23187. Calmodulin (1 ;uM) or c y c l i c AMP (1 juM) and cAMP-dependent p r o t e i n kinase (20 ;i g ) , or the c a t a l y t i c subunit of cAMP-dependent p r o t e i n kinase (0.5 /JM) were present i n some r e a c t i o n tubes. The r e a c t i o n was ML i n i t i a t e d by the a d d i t i o n of 0.2 mM ATP c o n t a i n i n g P-ATP (10 6 Ci/mmole; 1-2 x 10 dpm/sample) and terminated a f t e r v a r i o u s -61-time i n t e r v a l s with a quenching s o l u t i o n of 5% TCA, 5 mM Na^ATP, and 2 mM KH^PO^. Bovine Serum Albumin (0.125% f i n a l c o n c e n t r a t i o n ) was then added to each sample, the tubes mixed, and c e n t r i f u g e d at 1500xg f o r 5 minutes. The supernatant was decanted and the p e l l e t a p p l i e d onto g l a s s m i c r o f i b r e f i l t e r s rt (Whatman GF/A), washed with 30 ml of the TCA quench s o l u t i o n , d r i e d , and counted f o r r a d i o a c t i v i t y . + +• (d) Measurement of "patent" and " l a t e n t " (Na -K )-ATPase a c t i v i t y (Na +-K +)-ATPase a c t i v i t y measured e i t h e r i n the absence ("patent") or presence ("latent") of detergent was determined i n accordance with the methods of Besch et a l (1976) and Jones et a l (1979) with m o d i f i c a t i o n . L atent Na +-K +ATPase a c t i v i t y was "unmasked" by p r e i n c u b a t i n g c a r d i a c SR membranes (0.15-0.20 mg/ml) i n the presence of T r i t o n X-100 (0.05%) f o r twenty o minutes a t room temperature. A c t i v i t y was then assessed a t 37 C by adding membrane p r o t e i n (15-20 jug/ml) to a medium c o n t a i n i n g 50 mM h i s t i d i n e , pH 7.4, 3 mM MgCl^, 1 mM t r i s - E G T A , 100 mM NaCl, 10 mM KC1, and 3 mM t r i s ATP. The r e l e a s e o f i n o r g a n i c phosphate was then measured using the semi-automated method of F i s k e and Subbarow (1925) as modified by Raess and V i n c e n z i (1980) (see below). (Na +-K +)-ATPase a c t i v i t y was taken as th a t a c t i v i t y i n h i b i t e d by 1 mM ouabain. (e) Measurement of Cytochrome C Oxidase A c t i v i t y Cytochrome C Oxidase a c t i v i t y was measured -62-s p e c t r o p h o t o m e t r i c a l l y u sing the method of Wharton and T z a g a l o f f (1967). To each of 2 c u v e t t e s , 100 p i of 10 pM potassium phosphate b u f f e r , pH 7.0, 70 p i 1% ferrocytochrome C, and 0.83 ml of water was added. The ferrocytochrome C was reduced with a s c o r b i c a c i d and d i a l y z e d o v e r n i g h t p r i o r to i t s a d d i t i o n to the r e a c t i o n medium. Ten pi of 0.1 M potassium f e r r o c y a n a t e was added to the "blank" c u v e t t e i n order to o x i d i z e the ferrocytochrome p r e s e n t . Both c u v e t t e s were o incubated f o r 10 minutes at 37 C at which time the r e a c t i o n was i n i t i a t e d by the a d d i t i o n of 10 pi c a r d i a c SR (approximately 5 jug p r o t e i n ) to the " r e a c t i o n " c u v e t t e . The decrease i n absorbance a t 550 nm was used as an index of the r a t e of o x i d a t i o n of ferrocytochrome C and was monitored f o r 3 minutes. Cytochrome C Oxidase a c t i v i t y of the SR p r e p a r a t i o n was determined u s i n g the f o l l o w i n g f i r s t order r a t e equations: k = l n (absorbance a t time 0) min (absorbance at 1 minute) S p e c i f i c A c t i v i t y = k ( c o n c e n t r a t i o n of cytochrome c) ( c o n c e n t r a t i o n of SR) The s p e c i f i c a c t i v i t y was expressed as nmole of cytochrome c o x i d i z e d per mg SR p r o t e i n per minute. (f) Measurement of Free and Long-Chain A c y l c a r n i t i n e s i n S k e l e t a l Muscle Sarcoplasmic Reticulum I s o l a t e d From D i a b e t i c Rats A l i q u o t s of microsomal SR ( c o n t a i n i n g 1-4 mg p r o t e i n ) were e pooled and c e n t r i f u g e d at 40,000xg f o r 45 min. at 4C. The p e l l e t was then suspended i n c o l d 6% p e r c h l o r i c a c i d . A 100 ;ul -63-a l i q u o t was n e u t r a l i z e d with 2M t r i s base and used to determine the l e v e l s of t o t a l c a r n i t i n e p r e s e n t . The remainder of the sample was then c e n t r i f u g e d at 12,000g f o r 10 min. A 200 p i a l i q u o t of the subsequent supernatant was aso n e u t r a l i z e d with t r i s base and was used to measure the l e v e l s of a c i d - s o l u b l e f r e e c a r n i t i n e . The p e l l e t obtained was washed with 6% p e r c h l o r i c a c i d and was used to determine the l e v e l s of ( a c i d i n s o l u b l e ) l o n g - c h a i n a c y l c a r n i t i n e s . T o t a l c a r n i t i n e and l o n g - c h a i n a c y l c a r n i t i n e s were assayed as f r e e c a r n i t i n e f o l l o w i n g a l k a l i n e h y d r o l y s i s of the samples by i n c u b a t i o n at o 70C f o r 1 hour i n the presence of 1 M t r i s base and 0.4 N KOH (pH approx. 13). F o l l o w i n g i n c u b a t i o n , samples were n e u t r a l i z e d with 0.6 N HC1 and were assayed f o r c a r n i t i n e s . Free c a r n i t i n e was measured u s i n g the r a d i o i s o t o p i c procedure developed by McGarry and F o s t e r (1976) , which uses 14 C-acetyl-CoA, c a r n i t i n e a c e t y l t r a n s f e r a s e , and sodium t e t r a t h i o n a t e . The samples were incubated f o r t h i r t y minutes f o l l o w i n g which a 0.3 ml a l i q u o t of Dowex 1X8-400 anion exchange r e s i n was added and the samples vortexed and p l a c e d on i c e . The samples were vortexed twice a t 10 min i n t e r v a l s , and c e n t r i f u g e d a t 3000g f o r 5 min. A 0.7 ml a l i q u o t of the ft supernatant was then p l a c e d i n Aquasol and measured f o r r a d i o a c t i v i t y i n a l i q u i d s c i n t i l l a t i o n counter. The amount of 14 C-acetyl-CoA i n the supernatant f r a c t i o n i s s t o i c h i o m e t r i c a l l y r e l a t e d to the amount of c a r n i t i n e present i n the sample. L e v e l s of f r e e c a r n i t i n e and l o n g - c h a i n -64-a c y l c a r n i t i n e s are expressed as nmoles/mg sarc o p l a s m i c r e t i c u l u m . (g) Assay of (Ca -Mg )-ATPase A c t i v i t y i n Red Blood C e l l Membranes EDTA-washed red c e l l membranes (50-250 jug p r o t e i n ) were o freeze-thawed twice and then incubated at 37 C f o r 30 minutes i n the presence of 44 mM t r i s - C l , pH 7.4, 4.0 mM MgCl^, 0.1 mM ouabain, 0.17 mM C a C l ^ , and 0.15 mM EGTA r e s u l t i n g i n a f r e e C a + + c o n c e n t r a t i o n of 10 ^M as determined by MULT-S. The r e a c t i o n was i n i t i a t e d by the a d d i t i o n of 2 mM ATP, allowed to proceed f o r 15 minutes, and stopped by the a d d i t i o n of 0.5 ml 2% SDS to y i e l d a f i n a l r e a c t i o n volume of 0.71 ml. The a n t i - c a l m o d u l i n agents, t r i f l u o p e r a z i n e d i h y d r o c h l o r i d e (60 ;uM) and Compound 48/80 (1 pq/ml), when used, were f r e s h l y prepared and assayed i n the dark ( R o u f o g a l i s , 1981). When calmodulin (60 nm) or the e x t r a c t s d e r i v e d from s k e l e t a l muscle SR were to be assayed, they were added to membranes 10 minutes p r i o r to the s t a r t of the r e a c t i o n . +4* 4*4" (Ca -Mg )-ATPase a c t i v i t y was measured by the r e l e a s e of i n o r g a n i c phosphate (Pi) using the semi-automated procedure of F i s k e and Subbarow (1925) as modified by Raess and V i n c e n z i (1980). Samples were analyzed u s i n g a Technicon Autoanalyzer Pump I with a 16 channel manifold and a Technicon Sampler I I f i t t e d with a 40-2/1 cam h a n d l i n g 40 samples per hour. Two l a r g e mixing c o i l s p r o v i d e d f o r adequate mixing between reagents (see below) and samples. Output of the Autoanalyzer -65-was connected to a flow-through c e l l l o c a t e d i n a Technicon Spectrophotometer. Absorbance was monitored at 660 nm and recorded on a Technicon c h a r t r e c o r d e r . The reagents used were: i) a c i d molybdate s o l u t i o n (130 ml cone. H^SO^, 25 g ammonium molybdate, made up to 1L), i i ) SDS: 6% and 2% (w/v) , i i i ) 9% a s c o r b i c a c i d (w/v) A phosphate standard curve ranging from 0-250 nmoles Pi/ml KHjjPO^. was run with each experiment and found to be l i n e a r over the range of c o n c e n t r a t i o n s used. Ca + +-dependent ATPase a c t i v i t y was determined by 11 s u b t r a c t i n g the a c t i v i t y obtained i n the presence of Ca and EGTA from t h a t obtained with EGTA alone. (h) Determination of calmodulin content of s k e l e t a l SR e x t r a c t s The l e v e l s of c a l m o d u l i n present i n s k e l e t a l SR e x t r a c t s were determined by radioimmunoassay using a c a l m o d u l i n IT- $ K [ I]-RIA k i t (New England Nuclear ) i n c o r p o r a t i n g the method of Chafouleas e t a l (1979). The radioimmunoassay k i t i s a c o m p e t i t i v e i n h i b i t i o n system employing iodine-125 l a b e l l e d c a lmodulin as the t r a c e r and a s p e c i f i c sheep a n t i - c a l m o d u l i n antibody as the b i n d e r . A l i q u o t s (100 }\1) of s k e l e t a l SR e x t r a c t s were compared to standard calmodulin c o n c e n t r a t i o n s (0.31 - 20 ng /100 pi) and l e v e l s of c a l m o d u l i n expressed as ng / s t a r t i n g mg of SR p r o t e i n . -66-(3) E l e c t r o p h o r e t i c Methods (a) Sodium Dodecyl S u l f a t e P o l y a c r y l a m i d e Slab Gel E l e c t r o p h o r e t i c S e p a r a t i o n of P r o t e i n s P o l y a c r y l a m i d e s l a b g e l s (12.5%) o f 1.5 mm t h i c k n e s s were c a s t a c c o r d i n g to the method of Laemmli and Favre (1973) , u s i n g a 5% s t a c k i n g g e l . T y p i c a l l y , 75 ^ul of e x t r a c t d e r i v e d from s k e l e t a l SR was b o i l e d f o r 90 seconds i n 25 p i of a medium c o n s i s t i n g of ( f i n a l c o n c e n t r a t i o n ) : 62.5 mM t r i s - C l , pH 6.8, 2% SDS, 10% g l y c e r o l , 5%^-mercaptoethanol, and 0.002% bromphenol b l u e . Samples were c e n t r i f u g e d at 10,000xg f o r 5 minutes and 50-55 ;ul a p p l i e d per w e l l . Gels were run a t 35 mA constant c u r r e n t f o r 6 hours, s i l v e r s t a i n e d a c c o r d i n g to the procedure of M o r r i s s e y (1981) , and d r i e d . The p r o t e i n standards used f o r e s t i m a t i o n of molecular weights ( i n d a l t o n s ) were myosin (200,000) , f-galactosidase (116,250), phosphorylase b (92,500), bovine serum albumin (66,200), ovalbumin (45,000), c a r b o n i c anhydrase (31,000), soybean t r y p s i n i n h i b i t o r (21,500), and lysozyme (14,400). (b) Sodium-Dodecyl-Sulfate Polyacrylamide Gel E l e c t r o p h o r e s i s and Autoradiography of Phosphorylated C a r d i a c SR P r e p a r a t i o n s Phosphorylated samples were run concomitantly f o r a n a l y s i s by SDS-gel e l e c t r o p h o r e s i s and autoradiography. The i n c u b a t i o n c o n d i t i o n s were i d e n t i c a l to those d e s c r i b e d above except f o r the t e r m i n a t i o n of the r e a c t i o n by an "SDS stop s o l u t i o n " c o n s i s t i n g of ( f i n a l c o n c e n t r a t i o n ) : 0.083 mM t r i s - C l , pH 6.8, -67-2.5% SDS, 0.12 M sucrose, 0.50 M 2-mercaptoe'thanol, 0.0067% bromphenol b l u e , 1.7 mM Na^ATP, and 3.3 mM KH^PO^. Samples were heated a t 95 C f o r 90 seconds, c e n t r i f u g e d a t 1500xg f o r 5 minutes and then a p p l i e d onto the g e l . E l e c t r o p h o r e s i s was performed as p r e v i o u s l y d e s c r i b e d except t h a t some g e l s were c a s t as a 5-20% acrylamide g r a d i e n t i n a d d i t i o n to the u s u a l 12.5% acrylamide c o n c e n t r a t i o n . F o l l o w i n g e l e c t r o p h o r e s i s , g e l s were s t a i n e d with 0.25% Coomassie Blue i n 50% methanol/10% a c e t i c a c i d , d e s t a i n e d o v e r n i g h t i n 7.5% a c e t i c a c i d / 5 % o methanol, and then d r i e d under vacuum at 80 C f o r 2 hours. The standard p r o t e i n s were then spotted with P-ATP (50 dpm//Ul) and the d r i e d g e l p l a c e d i n c o n t a c t with X-ray f i l m ( Kodak X-OMAT AR) along with i n t e n s i f y i n g screens (Cronex L i g h t n i n g R © Plus) f o r 7-16 days at -80 C f o l l o w i n g which the f i l m was developed. (4) M i s c e l l a n e o u s Methods (a) P r o t e i n Assay Microsomal SR or red c e l l membranes (10-150 yug p r o t e i n ) were suspended i n d i s t i l l e d water to a f i n a l volume of 1.5 ml. To t h i s was added 12.5 yx\ of a 2% deoxycholate s o l u t i o n . F o l l o w i n g a ten minute i n c u b a t i o n at room temperature, 0.5 ml of c o l d t r i c h l o r o a c e t i c a c i d (24%) was added to p r e c i p i t a t e any p r o t e i n . The suspension was c e n t r i f u g e d a t 3000 x g f o r t h i r t y minutes, the supernatant a s p i r a t e d , and the p e l l e t assayed f o r p r o t e i n u s i n g the standard Lowry (1951) p r o t e i n assay. Bovine -68-serum albumin was used as the standard p r o t e i n . (b) S t a t i s t i c a l A n a l y s i s When two samples were compared, s t a t i s t i c a l a n a l y s i s was performed u s i n g the unpaired Student's t - t e s t . A p r o b a b i l i t y of p<0.05 was used as the l e v e l o f s i g n i f i c a n c e . (c) Determination of Free Calcium C o n c e n t r a t i o n Free c a l c i u m values were c a l c u l a t e d u sing the Mult.S F o r t r a n program w r i t t e n by Mr. Roland Burton. The program c a l c u l a t e s the apparent a s s o c i a t i o n constant of c a l c i u m and ++• ++ EGTA f o r the pH s e l e c t e d . B i n d i n g of both Mg and Ca to ATP and EGTA were c a l c u l a t e d by s o l v i n g a s e t of simultaneous equations d e s c r i b i n g the b i n d i n g . Log a s s o c i a t i o n constants used were f i r s t to f o u r t h proton a s s o c i a t i o n with EGTA: 9.461, 8.851, 2.679, 2.000; CaEGTA: 10.650; HATP: 6.970; CaATP: 4.4 98; and MgATP: 4.944. - 6 9 -RESULTS A) C h a r a c t e r i z a t i o n of a p u r i f i e d c a r d i a c SR p r e p a r a t i o n o E i t h e r a f r e s h l y prepared or p r e v i o u s l y f r o z e n (-80 C) p r e p a r a t i o n of crude c a r d i a c v e s i c l e s were subjected to f u r t h e r p u r i f i c a t i o n u s i n g the method of Jones et a l (1979) which employs Ca-oxalate l o a d i n g f o l l o w e d by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . T y p i c a l l y , 20-25 mg of crude SR y i e l d e d 4-5 mg of p u r i f i e d SR. (i) Assesment of p u r i t y u s i n g marker enzyme assays A summary of the marker enzyme assays used to determine the presence of membranes of other than SR o r i g i n i n the two p r e p a r a t i o n s i s shown i n Table 21 P u r i f i e d SR demonstrated at l e a s t a t h r e e - f o l d decrease i n cytochrome c oxidase a c t i v i t y , a measure of m i t o c h o n d r i a l membrane contamination. I n i t i a l experiments s t r i c t l y f o l l o w e d the p r o t o c o l of Jones et a l (1979) which d i d not i n c l u d e the m i t o c h o n d r i a l Ca -uptake i n h i b i t o r , sodium a z i d e . Values of cytochrome c oxidase, t h e r e f o r e , were i n i t i a l l y i d e n t i c a l i n crude and p u r i f i e d SR p r e p a r a t i o n s (data not shown). Only when the a z i d e was i n c l u d e d was the decrease i n cytochrome c oxidase apparent. Sarcolemmal membrane contamination was determined by measurement of "patent" and " l a t e n t " Na ,K -ATPase a c t i v i t y . As shown i n Table 2, both crude and p u r i f i e d SR had approximately equal "patent" o u a b a i n - i n h i b i t a b l e Na ,K -ATPase a c t i v i t y . F o l l o w i n g i n c u b a t i o n with 0.05% T r i t o n X-100, however, a much gr e a t e r degree of " l a t e n t " a c t i v i t y was -70-T a b l e 2 M a r k e r enzyme a s s a y s p e r f o r m e d as d e s c r i b e d i n Methods f o r d e t e r m i n a t i o n o f the degree o f m i t o c h o n d r i a l and sa r co lemmal c o n t a m i n a t i o n o f p u r i f i e d and c r u d e 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 p r e p a r a t i o n s . R e s u l t s shown a r e t y p i c a l o f two e x p e r i m e n t s p e r f o r m e d on d i f f e r e n t SR p r e p a r a t i o n s . A s s a y Crude SR Pu re SR 1. Cy tochrome C o x i d a s e a c t i v i t y (nmole o f c y t C o x i d i z e d / m g SR/min) 1 2 . 8 4 .0 2 . N a + , K + - A T P a s e a c t i v i t y (nmol/mg/min) i ) " P a t e n t " o u a b a i n (1 mM) i n h i b i t e d 88 80 i i ) " L a t e n t " o u a b a i n (1 mM) i n h i b i t e d 242 88 recorded i n the crude p r e p a r a t i o n , suggesting the g r e a t e r number of sarcolemmal enzyme s i t e s i n the crude p r e p a r a t i o n . F u r t h e r comparison of crude and p u r i f e d SR u t i l i z i n g SDS-polyacrylamide g e l e l e c t r o p h o r e s i s (SDS-PAGE) re v e a l e d a marked s i m i l a r i t y i n p r o t e i n composition with one notable e x c e p t i o n : the absence of a 95,000 d a l t o n p r o t e i n i n the p u r i f i e d p r e p a r a t i o n (Figure 8 ) . The absence of the 95 Kdalton p r o t e i n i n p u r i f i e d SR was confirmed using e i t h e r Coomassie Blue or s i l v e r s t a i n i n g techniques (data not shown). i i ) Calcium Uptake and ATPase a c t i v i t y i n the P u r i f i e d C a r d i a c SR P r e p a r a t i o n 4+ Measurement of Ca -uptake a c t i v i t y i n the p u r i f i e d SR p r e p a r a t i o n r e v e a l e d a 3-5 f o l d enhancement over the a c t i v i t y t y p i c a l l y o b tained with crude SR. Because of t h i s enhanced a c t i v i t y , o n l y 3-5 J j g p r o t e i n per i n c u b a t i o n tube of the p u r i f i e d p r e p a r a t i o n was r e q u i r e d as opposed to 20-30 pq of crude SR. The time course of C a + + uptake by p u r i f i e d SR i s shown i n F i g u r e 9. In c o n t r a s t to the crude p r e p a r a t i o n , which 4-4-t r a n s p o r t e d Ca as a l i n e a r f u n c t i o n of time f o r 10 minutes, p u r i f i e d SR maintained l i n e a r i t y f o r 1 minute, f o l l o w i n g which Ca uptake d e v i a t e d from l i n e a r i t y . F u r t h e r t r a n s p o r t s t u d i e s u t i l i z i n g p u r i f i e d SR t h e r e f o r e used 1 minute as the assay d u r a t i o n i n order to i n s u r e l i n e a r i t y . 44-At a l l f r e e Ca c o n c e n t r a t i o n s t e s t e d (0.1 - 2.0 /iM) , 44-p u r i f i e d SR demonstrated an enhanced l e v e l of Ca -uptake a c t i v i t y compared to crude SR (Figure 10). The e f f e c t appeared -72-FIGURE 8 Sodium dodecyl s u l f a t e - p o l y a c r y l a m i d e (12.5%) g e l e l e c t r o p h o r e t i c p a t t e r n of two d i f f e r e n t p r e p a r a t i o n s of crude and p u r i f i e d c a r d i a c sarcoplasmic r e t i c u l u m , performed as d e s c r i b e d i n Methods. Approximately 3 pq of p r o t e i n were a p p l i e d of each sample and s i l v e r s t a i n e d a c c o r d i n g to the procedure of M o r r i s s e y (1981). -73-MW standards crude S R 200,000 1 16,250 92,500 31,000 66,200 45,000 (I pure S R 21,500 $ 14,400 -73a-FIGURE 9 Time c o u r s e o f c a l c i u m uptake i n a crude and p u r i f i e d p r e p a r a t i o n o f dog 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 . C a l c i u m uptake was measured i n t h e presence o f 1 jaM f r e e C a + + as d e s c r i b e d i n Methods, i n a crude (• •.) and p u r i f i e d (o o) SR p r e p a r a t i o n . - 7 4 -FIGURE 1 0 4-4-E f f e c t of various calcium concentrations on Ca uptake a c t i v i t y in p u r i f i e d ( ° o) and crude (O Q) cardiac sarcoplasmic reticulum preparations. Calcium uptake and free 4-4 Ca concentrations were determined as described in Methods. Result shown i s a t y p i c a l experiment. - 7 5 -44 to be one of Vmax, s i n c e the Km f o r Ca i n both p r e p a r a t i o n s was the same (0.3 ;JM f r e e ) . 4 4 +4 (Ca -Mg )-ATPase a c t i v i t y was a l s o enhanced more than t h r e e - f o l d i n p u r i f i e d SR compared to crude (Table 3 ) , 44-p a r a l l e l m g the f i n d i n g s of an enhanced Ca -uptake a c t i v i t y i n 4-4' the former p r e p a r a t i o n ; Ca -ATPase a c t i v i t y s t i m u l a t i o n was approximately two-fold i n p u r i f i e d SR. i i i ) R e g u l a t i o n of the P u r i f i e d C a r d i a c SR P r e p a r a t i o n The e f f e c t o f two of the documented r e g u l a t o r s of c a r d i a c SR, cal m o d u l i n (CAM) and cAMP-dependent p r o t e i n kinase (cAMP-PK) was i n v e s t i g a t e d i n the p u r i f i e d SR p r e p a r a t i o n . As shown i n F i g u r e 11, both CAM (1 ;uM) and cAMP-PK (0.5 mg/ml) 4-4-s t i m u l a t e d Ca uptake a c t i v i t y at both 0.2 and 1.0 ;uM f r e e c a l c i u m . As has been p r e v i o u s l y shown i n crude SR (Tada et a l , 1975; K i r c h b e r g e r and Antonetz, 1982b; Plank et a l , 1983) the degree of s t i m u l a t i o n over b a s a l a c t i v i t y by both CAM and cAMP-PK was more marked at the lower f r e e C a + + c o n c e n t r a t i o n ; enhancement by CAM of C a + + t r a n s p o r t , f o r example, was 73% at 0.2 ;uM f r e e C a f + and 40% at 1.0 JUM f r e e Ca*"*'. E s s e n t i a l l y , the same f i n d i n g s , with r e s p e c t to s t i m u l a t i o n by CAM, were t+ obtained when we examined Ca -dependent ATPase a c t i v i t y ( F i gure 12). S t i m u l a t i o n by cAMP-PK, however, was more marked 4"+ 44. at 1.0 ;uM f r e e Ca than at the lower f r e e Ca . When both CAM and cAMP-PK were present i n the i n c u b a t i o n , s t i m u l a t i o n of ATPase a c t i v i t y was a d d i t i v e at 0.2 /iM f r e e Ca , but there was 4/4-no f u r t h e r s t i m u l a t i o n at the higher f r e e Ca -76-T a b l e 3 Measurement o f C a 2 + - A T P a s e a c t i v i t y i n c rude o r p u r i f i e d dog 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 . M g 2 + - A T P a s e ( C a 2 + - M g 2 + ) - A T P a s e C a 2 + - A T P a s e A c t i v i t y A c t i v i t y A c t i v i t y C o n d i t i o n (umole/mg/nrin) (umole/mg/min) (umole/mg/min) c r u d e SR 0 . 1 6 0 0 .215 0 . 0 5 5 p u r i f i e d SR 0 .634 0 . 7 4 0 0 .106 ( C a 2 + - M g 2 + ) - A T P a s e a c t i v i t y was measured a t 10 uM f r e e C a 2 + as d e s c r i b e d i n M e t h o d s . R e s u l t shown i s a t y p i c a l e x p e r i m e n t . FIGURE 11 Calcium uptake a c t i v i t y at two d i f f e r e n t f r e e C a 4 * c o n c e n t r a t i o n s (0.2 and 1.0 JJM) i n a p u r i f i e d p r e p a r a t i o n of dog 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 i n the presence of 1 /iM calmodulin (CAM) or 1 ,uM cAMP p l u s 0.5 mg/ml cAMP-dependent p r o t e i n kinase (cAMP). Free c a l c i u m c o n c e n t r a t i o n s and Ca uptake were determined as d e s c r i b e d i n Methods. R e s u l t shown i s a t y p i c a l experiment. -78-500-1 400 H c E CT> E 300-o E c J 200-a ZD + CM o U 1O0H 0-0 CAM cAMP 0.2 uM calcium 0 C A M cAMP 1.0 I 'M calcium -78a-FIGURE 12 Calcium-dependent ATPase a c t i v i t y at two d i f f e r e n t f r e e Ca c o n c e n t r a t i o n s (0.2 and 1.0 juM) i n a p u r i f i e d p r e p a r a t i o n of dog 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 i n the presence of 1 ;aM calmo d u l i n (CAM), 1 /iM cAMP p l u s 0.5 mg/ml cAMP-dependent p r o t e i n kinase (cAMP), or both calmodulin and cAMP p l u s cAMP-dependent p r o t e i n kinase (cAMP/CAM). Calcium ATPase a c t i v i t y and f r e e Ca c o n c e n t r a t i o n s were determined as d e s c r i b e d i n Methods. R e s u l t shown i s a t y p i c a l experiment. -79-1200n 1000H 800H 600 H 400 H 200 H 0 0 0 CAM cAMPcAMP CAM 0.2 uM calcium CAM cAMP cAMP CAM 1.0 uM calcium -7 9a-A f u r t h e r study of the r e g u l a t o r y mechanisms of the p u r i f i e d p r e p a r a t i o n was c a r r i e d out by an examination of t o t a l l e v e l s of p r o t e i n p h o s p h o r y l a t i o n . F i g u r e s 13 (a) & (b) show the time-course of p h o s p h o r y l a t i o n of crude and p u r i f i e d © 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 v e s i c l e s performed at 30 C f o l l o w i n g p r e i n c u b a t i o n of SR with e i t h e r cAMP-PK (0.1 mg/ml), the c a t a l y t i c C C ) subunit of cAMP-PK (0.5 ;iM) , or CAM (1 juM) i n the presence o f 2.0 jaM f r e e Ca**. In both crude and p u r i f i e d p r e p a r a t i o n s , i n c o r p o r a t i o n of phosphate i n c r e a s e d with time, the h i g h e s t l e v e l s of i n c o r p o r a t i o n i n both SR p r e p a r a t i o n s a t t a i n e d i n the presence of cAMP-PK. The maximum l e v e l s of cAMP-PK and C subunit p h o s p h o r y l a t i o n , however, i n the p u r i f i e d p r e p a r a t i o n exceeded t h a t of crude SR by 30% and 55%, r e s p e c t i v e l y . Although the degree of p h o s p h o r y l a t i o n by C subunit was not as marked as t h a t seen with cAMP-PK, the h a l f - t i m e of p h o s p h o r y l a t i o n by both r e g u l a t o r s was s i m i l a r , estimated at 20-30 seconds. In c o n t r a s t , CAM-dependent p h o s p h o r y l a t i o n i n both crude and p u r i f i e d SR was slower (t 1/2 = 45-60 sec) and lower compared to cAMP-PK and C subunit p h o s p h o r y l a t i o n . U n l i k e the higher l e v e l s of cAMP-dependent p h o s p h o r y l a t i o n noted, CAM-dependent phosphate i n c o r p o r a t i o n was decreased by 50% i n p u r i f i e d SR as compared to crude SR. ++• The maximum a t t a i n a b l e l e v e l of Ca -dependent p h o s p h o r y l a t i o n was a l s o decreased i n p u r i f i e d SR. Autoradiograms of phosphorylated SR p r o t e i n s s u b j e c t e d to SDS-PAGE r e v e a l e d a marked s i m i l a r i t y i n the time-course p a t t e r n i n both crude and p u r i f i e d SR. As shown i n F i g u r e 14, -80-FIGURE 13 Time-course of p h o s p h o r y l a t i o n i n both crude (Figure a) and p u r i f i e d ( F igure b) dog c a r d i a c sarcoplasmic r e t i c u l u m . Sarcoplasmic r e t i c u l u m v e s i c l e s (5-15 pq) were preincubated at 30 C f o r 10 minutes i n the presence of 40 mM h i s t i d i n e - C l , pH 6.8, 110 mM KC1, 4 mM MgCl^, 10 mM NaF, 5.7 jiM A23187, and e i t h e r EGTA (0.1 mM) (not shown), CaCl,(2.0 uM free) (• • ) , CaC l ^ p l u s CAM (1 pM) (* * ) , CAMP (1 JiM) p l u s cAMP-dependent p r o t e i n kinase (0.1 mg/ml) (• • ) , or the c a t a l y t i c subunit o f cAMP-dependent p r o t e i n kinase (0.5 /iM) (° ° ) . The r e a c t i o n was i n i t i a t e d by the a d d i t i o n of 0.2 39- 6 mM ATP c o n t a i n i n g P-ATP (1-2 x 10 dpm/ sample). L e v e l s of u p h o s p h o r y l a t i o n and f r e e Ca were determined as d e s c r i b e d i n Methods. Values are t y p i c a l of two experiments performed on d i f f e r e n t SR p r e p a r a t i o n s . -81-FIGURE 14 P-Autoradiography of crude dog c a r d i a c SR phosphorylated with P-ATP and su b j e c t e d to autoradiography as d e s c r i b e d i n Methods. R e s u l t shown i s t y p i c a l of two d i f f e r e n t crude or p u r i f i e d SR p r e p a r a t i o n s phosphorylated f o r v a r i o u s i n c u b a t i o n times (10 sec, 30 sec, e t c . ) . The time-course was performed e i t h e r i n the presence of cAMP-dependent p r o t e i n kinase or the c a t a l y t i c (C) subunit of cAMP-dependent p r o t e i n k i n a s e . The molecular weight ( i n dalton s ) of phosphorylated bands i s shown on the o r d i n a t e . -82-10 sec 30MC 1m*n 2 m*t 5 min I O M C 30s«c 1 rrHn 2min 5min cAMP-dependerrt PK C Subunit of PK -82 a-cAMP-PK phosphorylated a number of p r o t e i n bands, many of which were absent from the p a t t e r n observed i n the presence o f C subun i t . These i n c l u d e d p r o t e i n s of 66, 57, and 51 Kdaltons, of which the 66 Kdalton p r o t e i n decreased i n phosphate i n c o r p o r a t i o n with time, while the other two p r o t e i n s showed a time-dependent i n c r e a s e i n p h o s p h o r y l a t i o n . Of p a r t i c u l a r r e l e v a n c e was the marked p h o s p h o r y l a t i o n of a 9-11 Kdalton p r o t e i n b e l i e v e d to be phospholamban (LePeuch et a l , 1979) i n the presence o f both cAMP-PK or C su b u n i t . The time-course of phopholamban p h o s p h o r y l a t i o n v a r i e d depending on the presence of cAMP-PK or C su b u n i t . The presence of C subunit r e s u l t e d i n almost immediate, complete p h o s p h o r y l a t i o n of phospholamban, a c h i e v i n g maximum i n t e n s i t y by 30 sec. In c o n t r a s t , the presence o f cAMP-PK t y p i c a l l y r e s u l t e d i n complete p h o s p h o r y l a t i o n by 2 min. Un l i k e the r e s u l t s with cAMP-PK or C su b u n i t , a marked d i f f e r e n c e i n the p h o s p h o r y l a t i o n p a t t e r n between crude and p u r i f i e d SR was observed i n the presence of 1 uM CAM. As shown i n F i g u r e 15, i n crude SR, p h o s p h o r y l a t i o n of phospholamban was complete by 30 sec. whereas the l e v e l of phosphate i n c o r p o r a t i o n i n the p u r i f i e d p r e p a r a t i o n appeared to s t e a d i l y i n c r e a s e with time and never p l a t e a u . The l e v e l o f CAM-dependent p h o s p h o r y l a t i o n achieved a f t e r 5 minutes i n p u r i f i e d SR was not as in t e n s e as t h a t seen at s t e a d y - s t a t e i n crude SR, although s i m i l a r amounts of p r o t e i n (4 /iq) were a p p l i e d to the electrophoretogram. A number of p r o t e i n s unique to the crude SR p r e p a r a t i o n were a l s o phosphorylated by CAM, i n -83-FIGURE 15 ^"P-Autoradiogram of crude (C) or p u r i f i e d (P) dog c a r d i a c SR 32. phosphorylated with P-ATP f o r the v a r i o u s time i n t e r v a l s shown i n the presence of 1 juM calmodulin (CAM) and sub j e c t e d to autoradiography as d e s c r i b e d i n Methods. Molecular weights ( i n daltons) o f some prominent bands are d i s p l a y e d on the o r d i n a t e . R e s u l t shown i s a t y p i c a l experiment. - 8 4 -101,000 A 54,000 44,000 - j 39,000 24,500 A 9000 A C P 10 sec C P 15 sec C P 30 sec C P 1 min C P 2 min C P 5 min -84a-p a r t i c u l a r , 44, 39, 33, 24.5, and 14.5 Kdalton p r o t e i n s , whereas o n l y two p r o t e i n s of 101 and 54 Kdaltons were phosphorylated i n both the crude and p u r i f i e d SR. Ca , i n the absence of CAM, produced no p h o s p h o r y l a t i o n of p r o t e i n s (not shown). B. S t u d i e s on the Role of Calmodulin i n S k e l e t a l Muscle Sarcoplasmic Reticulum Previ o u s s t u d i e s i n our l a b o r a t o r y had i n d i c a t e d t h a t , u n l i k e the case i n c a r d i a c SR, exogenous calmodulin d i d not 11 s t i m u l a t e Ca t r a n s p o r t i n e i t h e r f a s t or slow SR p r e p a r a t i o n s . The f o l l o w i n g s e r i e s of experiments was t h e r e f o r e conducted i n order to determine whether CAM was i n d i g i n o u s to these p r e p a r a t i o n s . The p o s s i b l e presence of CAM was determined i n 3 ways: ( i ) by i n d i r e c t methods using red c e l l Ca -ATPase a c t i v a t i o n as an i n d i c a t o r , ( i i ) by d i r e c t means us i n g radioimmunoassay techniques and ( i i i ) use of SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . d ) I n d i r e c t measurement of calmodulin a c t i v i t y by Ca -ATPase a c t i v a t i o n . ++ 4+ The red c e l l (Ca -Mg )-ATPase i s an e x c e l l e n t enzyme f o r the d e t e r m i n a t i o n of the presence of CAM i n t i s s u e e x t r a c t s . As shown i n F i g u r e 16, pure CAM, i n a dose-dependent f a s h i o n +-•+-s t i m u l a t e d Ca -dependent ATPase a c t i v i t y , half-maximal s t i m u l a t i o n a t t a i n e d at 0.5 >ig/ml. Subsequent work was performed at a c o n c e n t r a t i o n of 1.0 ^ug/ml (60 nM) . -85-FIGURE 16 E f f e c t of c a l m o d u l i n on red c e l l calcium-dependent ATPase a c t i v i t y . Ca -dependent ATPase a c t i v i t y and f r e e Ca c o n c e n t r a t i o n was determined as d e s c r i b e d i n Methods. R e s u l t shown i s a t y p i c a l experiment. -86--86a-F o l l o w i n g the suggestion of C h i e s i and C a r a f o l i (1982) th a t b o i l i n g was an e f f e c t i v e way of d i s l o d g i n g CAM from SR v e s i c l e s , we proceeded to b o i l both f a s t (fSR) and slow (sSR) s k e l e t a l SR. In a d d i t i o n , we attempted to remove more CAM by ++ both b o i l i n g and treatment with 0.2 mM EDTA to c h e l a t e any Ca •H-away from Ca -CAM complexes. The supernatants thus obtained were then incubated with CAM-depleted red c e l l ghosts and the e f f e c t s on the (Ca -Mg )-ATPase a c t i v i t y noted. As shown i n F i g u r e 17, both b o i l e d and b o i l e d + EDTA-treated supernatants obtained from r a b b i t fSR and sSR s t i m u l a t e d (Ca -Mg )-ATPase a c t i v i t y , the. EDTA treatment appearing to enhance the s t i m u l a t i o n observed over t h a t o b tained with b o i l i n g alone. The s t i m u l a t i o n , n e v e r t h e l e s s , d i d not approach the marked (5.5-fold) enhancement t h a t pure CAM (60 nM) was able to e f f e c t on the system. To determine whether the noted s t i m u l a t i o n of (Ca* 4 -Mg + +)-ATPase a c t i v i t y by the supernatants of b o i l e d s k e l e t a l muscle SR p r e p a r a t i o n s was a t t r i b u t a b l e to CAM, we i n v e s t i g a t e d whether the anti-CAM agent, t r i f l u o p e r a z i n e (TFP) c o u l d i n h i b i t the ATPase s t i m u l a t i o n . As shown i n Table 4, +f +f s t i m u l a t i o n of red c e l l (Ca -Mg )-ATPase a c t i v i t y by CAM was i n h i b i t e d approximately 25% by 60 ^M TFP but t h i s i n h i b i t i o n was not CAM-specific s i n c e marked TFP i n h i b i t i o n of b a s a l ( i . e . no added CAM) ATPase a c t i v i t y was a l s o observed. Table 4 f u r t h e r shows t h a t the b o i l e d + EDTA-treated supernatant +4- ++ obtained from fSR s t i m u l a t e d (Ca -Mg )-ATPase a c t i v i t y i n a dose-dependent manner; TFP (60 pVl) was able to p a r t i a l l y -87-FIGURE 17 E f f e c t of b o i l e d or b o i l e d + 0.2 mM EDTA-treated supernatants (75 pi) d e r i v e d from f a s t (fSR) or slow (sSR) r a b b i t s k e l e t a l f+ 4+ X 4 4 SR. (Ca -Mg )-ATPase a c t i v i t y and f r e e Ca c o n c e n t r a t i o n (10 ;uM) was determined as d e s c r i b e d i n Methods. R e s u l t shown i s the mean ± S.D. of three experiments. - 8 8 -- 8 8 a -T a b l e 4 L e v e l s o f ( C a 2 + - M g 2 + ) - A T P a s e a c t i v i t y o b t a i n e d f o l l o w i n g i n c u b a t i o n o f b o i l e d E D T A - t r e a t e d f a s t s k e l e t a l SR e x t r a c t s i n the p r e s e n c e o r absence o f TFP (60 uM) . A d d i t i o n none c a l m o d u l i n (60 nM) b o i l e d + E D T A - t r e a t e d fSR s u p e r n a t a n t : 10 i l l 75 ul ( C a 2 + + - M g 2 + ) - A T P a s e a c t i v i t y (nmoles/mg/min) -TFP +TFP 8 .24 20 .81 9.25 1 7 . 6 5 7.07 1 5 . 4 2 7 .83 1 4 . 6 0 ( C a ^ - M g ^ + ) - A T P a s e a c t i v i t y and p r e p a r a t i o n o f s u p e r n a t a n t s were as d e s c r i b e d i n M e t h o d s . R e s u l t i s t y p i c a l o f f i v e e x p e r i m e n t s . - 8 9 -FIGURE 18 E f f e c t of 60 nM calmodulin (CAM) and 60 pM. t r i f l u o p e r a z i n e (TFP) i n the presence of CAM on calcium-dependent ATPase a c t i v i t y o f red c e l l membranes prepared a c c o r d i n g to the method 44- 4 4 of N i g g l i et a l (1981) . Ca -ATPase a c t i v i t y and f r e e Ca c o n c e n t r a t i o n (10 /iM) were determined as d e s c r i b e d i n Methods. R e s u l t shown i s the mean + S.D. of three experiments. -90-25 20 A E E CD .2 o E 15-az > o CD 0 CO CO CL r-< • CM CD o 10H Control Calmodulin TFP Calmodulin -90a-r e v e r s e t h i s s t i m u l a t i o n . T h i s r e s u l t was a l s o observed i n b o i l e d + EDTA-treated e x t r a c t s of sSR (not shown). The l e v e l o f i n h i b i t i o n of CAM s t i m u l a t i o n of red c e l l +t Ca -ATPase by 60 ;uM TFP was ra t h e r low i n comparison to other r e s u l t s found i n the l i t e r a t u r e (e.g., Raess and V i n c e n z i , 1980). I t was t h e r e f o r e decided to switch to a red c e l l membrane p r e p a r a t i o n t h a t had been shown p r e v i o u s l y to c o n t a i n ++ ++-a CAM-dependent (Ca -Mg )-ATPase a c t i v i t y t h a t was more markedly i n h i b i t e d by TFP ( N i g g l i et a l , 1981) . As shown i n F i g u r e 18, the Ca -dependent ATPase a c t i v i t y of EDTA-washed membranes prepared a c c o r d i n g to t h i s method was s t i m u l a t e d 4 - 6 - f o l d by CAM; 60 /iM TFP produced a 50% i n h i b i t i o n of t h i s CAM-stimulated Ca -dependent ATPase a c t i v i t y . Not shown i n F i g u r e 18 however, i s the marked i n h i b i t i o n (25-40%) by t h i s c o n c e n t r a t i o n of TFP of Ca -ATPase a c t i v i t y i n the absence of added CAM. In a d d i t i o n , s t i m u l a t i o n by b o i l e d + EDTA-treated e x t r a c t s was s t i l l o n l y i n h i b i t e d by 30-45% (not shown). I t was c l e a r , t h e r e f o r e , t h a t b e f o r e any c o n c l u s i o n s c o u l d be drawn concerning the presence or absence of CAM i n s k e l e t a l SR e x t r a c t s , a more s p e c i f i c i n h i b i t o r of CAM s t i m u l a t i o n was r e q u i r e d . Compound 48/80, an agent t h a t causes d e g r a n u l a t i o n of mast c e l l s (Goth, 1973) was r e c e n t l y shown by G i e t z e n et a l (1983) to antagonize the CAM-induced s t i m u l a t i o n of red c e l l Ca -ATPase without suppressing the " b a s a l " a c t i v i t y (that Ca + +-ATPase a c t i v i t y present i n the absence of added CAM). In our hands, compound 48/80 i n h i b i t e d CAM-induced s t i m u l a t i o n of the EDTA-washed red blood c e l l Ca -ATPase a c t i v i t y (IC50 = 3.0 -91-pq/ml) by 60-70% but, u n l i k e the f i n d i n g s of G i e t z e n et a l (1983), we observed t h a t the a n t i - c a l m o d u l i n agent a l s o i n h i b i t e d b a s a l ATPase a c t i v i t y to the same degree ( F i g 19). Compound 48/80 (10 /ig/ml) i n h i b i t e d the s t i m u l a t i o n of red c e l l ++ Ca -ATPase a c t i v i t y induced by both b o i l e d + EDTA-treated fSR and sSR e x t r a c t s by 45% and 74%, r e s p e c t i v e l y (Table 5 ) . As mentioned i n the O b j e c t i v e s , the work of MacLennan (Campbell and MacLennan, 1982) had i m p l i e d t h a t harsh treatment such as b o i l i n g of the SR membranes was not necessary i n order to l i b e r a t e bound CAM. Rather, i n c u b a t i o n of SR with 1 mM EGTA was a l l t h a t was r e q u i r e d . Our s t u d i e s , t h e r e f o r e , attempted to r e p l i c a t e the work of MacLennan (1972) who demonstrated t h a t EGTA treatment of the SR membranes r e s u l t e d i n a decreased 44 Ca -uptake a c t i v i t y which c o u l d be r e s t o r e d by r e - a d d i t i o n of the d i a l y z e d supernatant ( i . e . c a l m o d u l i n ) . As shown i n F i g u r e +t 20, Ca -uptake a c t i v i t y i n slow s k e l e t a l SR was s i g n i f i c a n t l y lower than t h a t found i n f a s t SR; the a d d i t i o n of CAM was unable to s t i m u l a t e uptake a c t i v i t y i n e i t h e r fSR or sSR. S i m i l a r to the f i n d i n g s of MacLennan (1972) , SR which had been p r e v i o u s l y washed i n 1 mM EGTA demonstrated a reduced c a p a c i t y •H-to t r a n s p o r t Ca compared to c o n t r o l . The exogenous a d d i t i o n of c a l m o d u l i n (thought by Campbell and MacLennan (1982) to be d e p l e t e d i n t h i s type of SR p r e p a r a t i o n ) d i d not r e s t o r e uptake 4 4 a c t i v i t y but, r a t h e r , appeared to s i g n i f i c a n t l y decrease Ca t r a n s p o r t i n both EGTA-washed fSR and sSR. To f u r t h e r determine whether CAM was present i n the supernatant d e r i v e d from 1 mM EGTA-washed SR, we incubated t h i s -92-FIGURE 19 C o n c e n t r a t i o n dependence of compound 48/80 on calcium-dependent ATPase a c t i v i t y i n red c e l l membranes (0.1 mg) prepared a c c o r d i n g to the method of N i g g l i et a l (1981). Calmodulin (60 nM)-stimulated (• •) , b a s a l ( ° o) Ca -ATPase a c t i v i t y , and f r e e Ca c o n c e n t r a t i o n s (10 /iM) were determined as d e s c r i b e d i n Methods. R e s u l t shown i s a t y p i c a l experiment. -93-100 Compound 48/80 (ug/mO T a b l e 5 Red C e l l membrane Ca -ATPase a c t i v i t y i n the p r e s e n c e of b o i l e d + EDTA-t r e a t e d s u p e r n a t a n t s d e r i v e d f rom f a s t and s l ow s k e l e t a l SR and the e f f e c t o f compound 48/80 (10 ug/ml) ( C a 2 + ) - A T P a s e a c t i v i t y (nmoles/mg/min) A d d i t i o n -48/80 +48/80 % i n h i b i t i o n none 3.34 1.29 61 c a l m o d u l i n (1 uM) 2 6 . 5 4 8 .77 67 b o i l e d + E D T A - t r e a t e d fSR s u p e r n a t a n t 7 .48 4 . 1 3 45 b o i l e d + E D T A - t r e a t e d sSR s u p e r n a t a n t 9 . 8 0 2 .58 74 Measurement o f C a 2 + - A T P a s e a c t i v i t y and p r e p a r a t i o n o f s u p e r n a t a n t s were p e r f o r m e d as d e s c r i b e d i n me thods . R e s u l t s r e p r e s e n t a t y p i c a l e x p e r i m e n t . -94-FIGURE 20 Calcium-uptake a c t i v i t y of s k e l e t a l f a s t SR (fSR), slow SR (sSR), or 1 mM EGTA-washed fSR and sSR i n the presence of calmodulin (0.24/iM). C a + + - u p t a k e a c t i v i t y , p r e p a r a t i o n of SR, and f r e e C a + + c o n c e n t r a t i o n s were as d e s c r i b e d i n Methods. R e s u l t shown i s the mean + S.D. of three experiments. -95-- 9 5 a -e x t r a c t with red c e l l (Ca''-Mg" )-ATPase i n the presence and absence of TFP. In a d d i t i o n , the SR p e l l e t d e r i v e d f o l l o w i n g washing with 1 mM EGTA was subjected to b o i l i n g i n the presence (and absence) of 0.2 mM EDTA to determine whether f u r t h e r amounts of CAM c o u l d be l i b e r a t e d . As shown i n Table 6 (b), the b o i l e d + EDTA t r e a t e d supernatants of 1 mM EGTA-washed fSR and +t sSR were able to s t i m u l a t e Ca -ATPase a c t i v i t y , and t h i s s t i m u l a t i o n c o u l d be i n h i b i t e d by the a d d i t i o n o f TFP. In a d d i t i o n , supernatants d e r i v e d from 1 mM EGTA-washed SR, p a r t i c u l a r l y those from fSR , were able to s t i m u l a t e Ca -ATPase a c t i v i t y with the s t i m u l a t i o n i n h i b i t e d by TFP. ( i i ) D i r e c t measurements of cal m o d u l i n by radioimmunoassay In order to d i r e c t l y determine the presence of CAM i n the s k e l e t a l SR e x t r a c t s we undertook an e s t i m a t i o n of CAM content using an I-radioimmunoassay. As shown i n Table 7, untreated fSR v e s i c l e s c o n t a i n low l e v e l s of CAM. B o i l i n g , and p a r t i c u l a r l y b o i l i n g i n the presence of EDTA, r e l e a s e d more CAM i n t o the supernatant. We found t h a t b o i l e d + EDTA-treated sSR y i e l d e d more CAM/mg s t a r t i n g p r o t e i n than s i m i l a r treatment of fSR (Table 7b) although the CAM content of both fSR and sSR ranged b r o a d l y from p r e p a r a t i o n to p r e p a r a t i o n . When we examined the supernatants of b o i l e d + EDTA-treated SR t h a t had p r e v i o u s l y been washed i n 1 mM EGTA, i t was observed t h a t at l e a s t t w o - t h i r d s of the l e v e l s of CAM were l i b e r a t e d as i n SR t h a t d i d not r e c e i v e the EGTA treatment. Supernatants from EGTA-washed fSR c o n s i s t e n t l y showed 0 or t r a c e l e v e l s of CAM -96-T a b l e 6 Measu red l e v e l s o f ( C a 2 + ) - A T P a s e i n the p r e s e n c e o f TFP (60 uM) and (a ) s u p e r n a t a n t s o f 1 mM EGTA-washed f a s t and s l ow SR v e s i c l e s b o i l e d i n the p r e s e n c e o f 0 . 2 mM EDTA, o r (b) EGTA-washed f a s t and s low SR s u p e r n a -t a n t s . A d d i t i o n -TFP (Ca^ + ) -ATPase a c t i v i t y (nmoles/mg/min)  +TFP (a ) none 2 0 . 8 9 15 .93 b o i l e d + E D T A - t r e a t e d s u p e r n a t a n t o f 1 mM EGTA-washed fSR 3 4 . 3 4 2 4 . 0 7 b o i l e d + E D T A - t r e a t e d s u p e r n a t a n t o f 1 mM EGTA-washed sSR 2 7 . 2 6 17 .35 Cb) 1 mM EGTA-washed fSR s u p e r n a t a n t 7 0 . 1 0 58 .06 1 mM EGTA-washed sSR s u p e r n a t a n t 2 7 . 9 7 19 .11 C a 2 + -ATPase a c t i v i t y and p r e p a r a t i o n o f s u p e r n a t a n t s were p e r f o r m e d as d e s c r i b e d i n M e t h o d s . R e s u l t s r e p r e s e n t a t y p i c a l e x p e r i m e n t . T a b l e 7 Rad io immunoassay f o r c a l m o d u l i n i n v a r i o u s p r e p a r a t i o n s o f f a s t and s l ow r a b b i t s k e l e t a l SR. C o n d i t i o n ng c a l m o d u l i n / m g SR p r o t e i n u n t r e a t e d fSR m i c rosomes 2.1 (a ) fSR s u p e r n a t a n t d e r i v e d f rom mic rosomes w h i c h w e r e : i ) b o i l e d 1 8 . 0 i i ) b o i l e d f o l l o w i n g 1 mM EGTA wash 12 .2 i i i ) b o i l e d + E D T A - t r e a t e d 2 8 . 5 i v ) b o i l e d + E D T A - t r e a t e d f o l l o w i n g 1 mM EGTA-wash 1 9 . 0 v ) washed i n 1 mM EGTA 0 (b) sSR s u p e r n a t a n t s d e r i v e d f rom mic rosomes w h i c h w e r e : i ) b o i l e d + E D T A - t r e a t e d 6 4 . 9 i i ) b o i l e d + E D T A - t r e a t e d f o l l o w i n g 1 mM EGTA wash 20-75 i i i ) washed i n 1 mM EGTA 0-8 S u p e r n a t a n t s d e r i v e d f rom SR and c a l m o d u l i n l e v e l s were d e t e r m i n e d as d e s -c r i b e d i n M e t h o d s . R e s u l t s a re t y p i c a l o f a t l e a s t two e x p e r i m e n t s . - 9 8 - ' while those d e r i v e d from sSR o f t e n showed moderate l e v e l s of CAM. R e c e n t l y , i t has been found t h a t radioimmunoassay-derived l e v e l s of CAM vary d r a m a t i c a l l y depending on whether or not the samples are heated (NEN t e c h n i c a l b u l l e t i n , 1983) . We t h e r e f o r e compared the u n b o i l e d supernatants from 1 mM EGTA-washed SR to an u n b o i l e d "standard" CAM and found 0 l e v e l s of CAM i n fSR supernatants whereas sSR supernatants showed ra t h e r high l e v e l s (data not shown). ( i i i ) SDS-PAGE Another method employed to determine whether CAM was pre s e n t i n e x t r a c t s was the a n a l y s i s of samples by SDS-PAGE. As shown i n F i g u r e 21, a standard CAM sample (80 ng) t y p i c a l l y ran as a doublet of 19-21 Kdaltons and maintained t h i s molecular weight even i f s u b j e c t e d to b o i l i n g i n the presence or absence of EDTA. A l l of the b o i l e d + EDTA-treated supernatants r e v e a l e d p r o t e i n bands t h a t might be i n d i c a t i v e of CAM, s i n c e CAM has been r e p o r t e d i n the l i t e r a t u r e to v a r i a b l y migrate between 15.5 and 21.5 Kdaltons on SDS-PAGE (Table 8 ) . The o n l y p r o t e i n band t h a t c o n s i s t e n t l y ran with our standard CAM sample was the 19.5 Kdalton p r o t e i n from b o i l e d + EDTA-treated, 1 mM EGTA-washed fSR supernatant. I t was of i n t e r e s t t h a t the supernatants d e r i v e d from 1 mM EGTA-washed fSR showed bands of 16.5 and 21.5 Kdaltons on SDS-PAGE, suggesting the presence of CAM, whereas the EGTA-washed sSR supernatant d i d not show any bands below 35 Kdaltons (Figure 21). The multitude of bands i n the EGTA-washed -99-FIGURE 21 Sodium-dodecyl s u l f a t e p o l y a c r y l a m i d e (12.5%) g e l e l e c t r o p h o r e t i c p r o t e i n bands of v a r i o u s supernatants d e r i v e d from e i t h e r b o i l e d + EDTA-treated or 1 mM EGTA-washed s k e l e t a l SR. Samples shown are: (a) c a l m o d u l i n standard (b) b o i l e d + EDTA-treated fSR supernatant (c) b o i l e d + EDTA-treated sSR supernatant (d) b o i l e d + EDTA-treated supernatant d e r i v e d from 1 mM EGTA-washed fSR (e) b o i l e d + EDTA-treated supernatant d e r i v e d from 1 mM EGTA-washed sSR (f) 1 mM EGTA-washed fSR supernatant (g) 1 mM EGTA-washed sSR supernatant P r e p a r a t i o n of supernatants and SDS-PAGE were as d e s c r i b e d i n Methods. R e s u l t shown i s a t y p i c a l electrophoretogram. -100-MW Standards 200,000 116,250 92,500 66,200 45,000 H tx i Mitt r 1 31,000 21,500 14,400 (a) (b) (c) (d) (e) -100a-Table 8 Molecular weight of some prominent protein bands below 20 KDaltons in rabbit skeletal SR extracts as shown in Fig.21. Condition Prominent bands (MW x 1000) calmodulin standard 19.5 boiled + EDTA-treated fSR supernatant 15.8, 16.5, 18.5 boiled + EDTA-treated sSR supernatant 15.8, 16.5, 18.5 boiled + EDTA-treated fSR supernatant following 1 mM EGTA wash 15.5, 16.0, 18.0, 19.5 boiled + EDTA-treated sSR supernatant following 1 mM EGTA wash 15.5, 18.0 supernatant from 1 mM EGTA-washed fSR 16.5, 19.5 supernatant from 1 mM EGTA-washed sSR SDS-PAGE and preparation of supernatants were performed as described in Methods. Results shown are typical of at least three preparations. -101-fSR supernatant, however, made us suspect the presence of contaminating SR membrane p r o t e i n s . We t h e r e f o r e c e n t r i f u g e d a l l of the e x t r a c t s (100,000 xg f o r 30 min) and then r e -examined t h e i r a b i l i t y to s t i m u l a t e Ca^-ATPase a c t i v i t y : there was no change i n the a b i l i t y of the b o i l e d + EDTA-treated e x t r a c t s to s t i m u l a t e ATPase a c t i v i t y but the supernatants from 1 mM EGTA-washed fSR were no longer able to s t i m u l a t e t h i s a c t i v i t y . In a d d i t i o n , SDS-PAGE re v e a l e d t h a t both the 16.5 and 21.5 k d a l t o n bands present i n the l a t t e r supernatant p r i o r to u l t r a c e n t r i f u g a t i o n were mi s s i n g f o l l o w i n g t h i s procedure, suggesting t h a t the marked s t i m u l a t i o n observed with the supernatant d e r i v e d from 1 mM EGTA-washed fSR was most l i k e l y due to SR membrane contamination. C) SR Calcium Transport i n a Chronic Disease S t a t e : E x p e r i m e n t a l l y - i n d u c e d Diabetes Table 9 shows body weights, serum glucose, and serum i n s u l i n l e v e l s of s t r e p t o z o t o c i n - i n d u c e d d i a b e t i c r a t s 120 days a f t e r the i n d u c t i o n of d i a b e t e s . Body weight and serum i n s u l i n l e v e l s at time of s a c r i f i c e were s i g n i f i c a n t l y lower i n d i a b e t i c r a t s than i n c o n t r o l s . Another index of d i a b e t e s , e l e v a t e d l e v e l s o f serum gl u c o s e , were a l s o observed i n these d i a b e t i c animals. ++ ATP-dependent t r i s o x a l a t e - f a c i l i t a t e d Ca -uptake was measured i n s k e l e t a l muscle microsomal p r e p a r a t i o n s e n r i c h e d i n SR from c o n t r o l and c h r o n i c a l l y d i a b e t i c r a t s (Figure 22). At -H-a l l f r e e Ca c o n c e n t r a t i o n s t e s t e d (0.1-2.0 /iM), the r a t e of -102-Table 9 Measurement of body weight, serum glucose, and serum i n su l i n l eve ls of con-t ro l and d iabet i c r a t s . Numbers in parentheses ind ica te the number of a n i -mals. Results are expressed as the mean + S.D. Control D iabet ic A) Body weight (g) 488 + 48 (5) 339 + 25 (6) (at time of s a c r i f i c e ) B) Serum Glucose (mg %) 133 + 15 (5) 650 + 111 (6) C) Serum i n s u l i n (uU/ml) 4 0 + 7 (5) 1 7 + 3 (5) * S i g n i f i c a n t l y less than c o n t r o l , p < 0.05. -103-FIGURE 22 E f f e c t o f c h r o n i c d i a b e t e s (120 days) on s k e l e t a l SR calcium-uptake at v a r i o u s c a l c i u m c o n c e n t r a t i o n s . C a + + - u p t a k e and f r e e C a ^ c o n c e n t r a t i o n s were determined as d e s c r i b e d i n Methods i n SR p r e p a r a t i o n s d e r i v e d from c o n t r o l (Q a ) and s t r e p t o z o t o c i n - t r e a t e d (o o) r a t s . R e s u l t shown i s the mean + S.D. of 4 c o n t r o l and 5 d i a b e t i c animals. -104-Ca2+ CONCENTRATION (IJM) -104a-Ca -uptake was s i g n i f i c a n t l y reduced (p<0.05) i n the SR p r e p a r a t i o n s obtained from d i a b e t i c r a t s compared to c o n t r o l s . L e v e l s of c a r n i t i n e and long c h a i n a c y l c a r n i t i n e were measured i n microsomal p r e p a r a t i o n s e n r i c h e d i n SR prepared from c o n t r o l and c h r o n i c a l l y d i a b e t i c r a t s (Table 10). P r e p a r a t i o n s d e r i v e d from d i a b e t i c animals contained s i g n i f i c a n t l y e l e v a t e d (p< 0.05) l e v e l s of both f r e e c a r n i t i n e and long c h a i n a c y l c a r n i t i n e s compared to c o n t r o l s . -105-Table 10 Levels of carnitine and long chain acylcarnitines in cardiac microsomal preparations enriched in sarcoplasmic reticulum from control and three month diabetic rats. Condition Metaboli te Tissue levels (nmole carnitine/mg SR) control (n = 5) (i) acid-soluble carnitine (ii) fatty acylcarnitine 3.34 ± 0.55 1.01 ± 0.20 diabetic (n = 5) (i) acid-soluble carnitine (ii) fatty acylcarnitine 5.31 ± 1.92* 1.72 ± 0.35* *significantly elevated compared to control p < 0.05 Results are expressed as the mean ± S.D. Preparation and measurement of metabolites are as described in Methods. Diabetes was induced by a single i .v . injection of 50 mg/kg streptozotocin. -106-DISCUSSION Calcium i s a c r u c i a l i o n i n the modulation of e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n both s k e l e t a l and c a r d i a c muscle. The sarcoplasmic r e t i c u l u m membrane p l a y s a c r i t i c a l r o l e s i n c e i t i s the p r i n c i p a l c a l c i u m - s e q u e s t e r i n g membrane system r e s p o n s i b l e f o r muscle r e l a x a t i o n . In s k e l e t a l muscle i t probably f u n c t i o n s as w e l l as the s t o r e of " a c t i v a t o r c a l c i u m " ; whether the SR a l s o f u n c t i o n s as the s o l e s i t e of a c t i v a t o r c a l c i u m i n mediating c a r d i a c c o n t r a c t i l i t y remains c o n t r o v e r s i a l . The s i g n i f i c a n c e of p r e c i s e r e g u l a t i o n of c a l c i u m f l u x e s i s perhaps b e s t e x e m p l i f i e d by a v a r i e t y of d i s e a s e s t a t e s , such as Duchenne Muscular Dystrophy and 4-4-Malignant Hyperthermia, i n which Ca r e g u l a t i o n , perhaps at the l e v e l of the SR, i s thought to be d e f i c i e n t . T h i s study has been an attempt to examine the f u n c t i o n and r e g u l a t i o n o f SR i n normal and d i s e a s e d t i s s u e , i n the hope of e l u c i d a t i n g the unique p o s i t i o n t h a t the SR occupies i n each case. A c c o r d i n g l y , I s h a l l d i s c u s s each aspect i n t u r n . I R e g u l a t i o n of a P u r i f i e d C a r d i a c Sarcoplasmic Reticulum P r e p a r a t i o n In c o n t r a s t to the e x t e n s i v e system present i n s k e l e t a l muscle, SR i s more scarce i n c a r d i a c muscle, with s u r f a c e membranes (SL and t r a n s v e r s e tubule) and mitochondria c o n s t i t u t i n g a higher percentage of the t o t a l membrane mass - 107 -(Fawcett and McNutt, 1969) . The v a s t m a j o r i t y of s t u d i e s on c a r d i a c SR s t r u c t u r e and f u n c t i o n have been performed on crude microsomal p r e p a r a t i o n s which have been shown to be contaminated by sarcolemmal and m i t o c h o n d r i a l membranes (Besch et a l , 1976) . A c c o r d i n g l y , we attempted to p u r i f y dog c a r d i a c SR by Ca-oxalate l o a d i n g f o l l o w e d by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n as d e s c r i b e d by Jones et a l (1979). The Ca-oxalate p r e c i p i t a t e t h a t forms i n s i d e the SR v e s i c l e s i n c r e a s e s the v e s i c l e d e n s i t y r e l a t i v e to other v e s i c l e s that e i t h e r do not t r a n s p o r t C a + + or do so at a slower r a t e . Subjected to sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , the loaded v e s i c l e s are d r i v e n to the bottom of the c e n t r i f u g a t i o n tube where they can be c o l l e c t e d . Two markers o f enzyme a c t i v i t y expressed e x c l u s i v e l y by membranes other than SR were used to determine the degree of SR v e s i c l e contamination: cytochrome c oxidase a c t i v i t y f o r +• + m i t o c h o n d r i a l membranes and Na ,K -ATPase a c t i v i t y f o r sarcolemmal membranes. M i t o c h o n d r i a l membrane content, as measured by cytochrome c oxidase a c t i v i t y , was found to be decreased t h r e e - f o l d o n l y i n those p r e p a r a t i o n s i n which Ca-oxalate l o a d i n g was performed ++-i n the presence of the m i t o c h o n d r i a l Ca -uptake i n h i b i t o r , sodium a z i d e . In t h e i r o r i g i n a l procedure, Jones et a l (1979) ne g l e c t e d the i n c l u s i o n o f t h i s i n h i b i t o r and d i d not r e p o r t the degree of m i t o c h o n d r i a l membrane contamination of t h e i r p u r i f i e d SR p r e p a r a t i o n . L e v i t z k i et a l (1976), however, d i d i n c l u d e sodium a z i d e i n t h e i r Ca-oxalate l o a d i n g procedure f o r - 108 -the p u r i f i c a t i o n of pigeon heart SR and noted a 3 . 7 - f o l d decrease i n cytochrome (a + a^) a c t i v i t y compared to crude SR. The extent of sarcolemmal membrane contamination was determined by measurement of "patent" and " l a t e n t " o u a b a i n - i n h i b i t a b l e Na ,K -ATPase a c t i v i t y measured i n the absence and presence of detergent, r e s p e c t i v e l y . ATP and ouabain are thought to b i n d to o p p o s i t e s i d e s of the transmembrane enzyme (Perrone and B l o s t e i n , 1973), such that s e a l e d r i g h t - s i d e out or i n s i d e - o u t v e s i c l e s would o n l y p r e s e n t one face f o r b i n d i n g . The presence of detergent (e.g. T r i t o n X-100) allows the membrane to become "leaky" and t h e r e f o r e p o t e n t i a l l y a v a i l a b l e to both ATP and ouabain, such that the " l a t e n t " enzymic a c t i v i t y i s now expressed. As shown i n Table + + 2, the values of "patent" Na ,K -ATPase a c t i v i t y were s i m i l a r i n both crude and p u r i f i e d SR p r e p a r a t i o n s . Incubation of both types of v e s i c l e s with 0.05% T r i t o n X-100 r e s u l t e d i n a marked e l e v a t i o n of " l a t e n t " a c t i v i t y i n the crude p r e p a r a t i o n o n l y , suggesting a d i s c r i m i n a t i o n of the two types of p r e p a r a t i o n s with r e s p e c t to content of sarcolemmal Na ,K -ATPase enzyme s i t e s . P u r i f i e d sarcolemmal v e s i c l e s have Na ,K -ATPase a c t i v i t i e s estimated between 2000 and 3000 nmoles/mg/min (Jones and Besch, 1979; Chamberlain et a l , 1983) suggesting t h a t a value of 88 nmoles/mg/min i n the p u r i f i e d p r e p a r a t i o n represented an SL contamination of 3-5%, s l i g h t l y higher than the upper l i m i t of 2% i n d i c a t e d by Jones and Besch (1979), but s t i l l below the 9% SL contamination r e p o r t e d i n the p u r i f i e d SR p r e p a r a t i o n of Chamberlain et a l (1983). - 109 -A comparison of p r o t e i n composition between crude and p u r i f i e d c a r d i a c SR microsomes u s i n g SDS-PAGE re v e a l e d s i m i l a r p o l y p e p t i d e p r o f i l e s as has been noted by Jones et a l (1979). Since d i f f e r e n c e s i n p r o t e i n banding p a t t e r n s have been found between SR and SL (Jones et a l , 1979), i t would seem t h a t the degree of SL contamination i n our crude SR p r e p a r a t i o n was not s i g n i f i c a n t enough to a l t e r the p r o t e i n p a t t e r n s observed i n the p u r i f i e d p r e p a r a t i o n . The marked exc e p t i o n between crude and p u r i f i e d SR was the absence i n the p u r i f i e d p r e p a r a t i o n of a 95,000 MW p r o t e i n , of unknown i d e n t i t y . We have sp e c u l a t e d t h a t the m i s s i n g p r o t e i n may be phosphorylase b or phosphorylase b k i n a s e . R e c e n t l y , Narahara and Green (1983) have confirmed the s e l e c t i v e l o s s of a 96,000 MW p r o t e i n i n f r o g s a r t o r i u s muscle induced to c o n t r a c t upon e l e c t r i c a l s t i m u l a t i o n or Ca i n f u s i o n , and have suggested a C a + * - a c t i v a t e d n e u t r a l protease d i r e c t e d towards phosphorylase kinase may be r e s p o n s i b l e . The l a r g e i n c r e a s e i n i n t r a c e l l u l a r Ca c o n c e n t r a t i o n o c c u r r i n g d u r i n g Ca-oxalate l o a d i n g may indeed p r e c i p i t a t e a c t i v a t i o n of such a protease and so be r e s p o n s i b l e f o r the noted disappearance of the 95 k d a l t o n p r o t e i n . •H- ++ Calcium uptake and (Ca -Mg )-ATPase a c t i v i t y was i n c r e a s e d 3 - 5 - f o l d i n the p u r i f i e d p r e p a r a t i o n compared to crude. The Ca -uptake e f f e c t appears to be one on Vmax, as the Km f o r Ca was s i m i l a r i n both p r e p a r a t i o n s . These enhancements as a r e s u l t of p u r i f i c a t i o n have p r e v i o u s l y been re p o r t e d i n SR d e r i v e d from pigeon heart ( L e v i t z k i et a l , 1976) - 110 -and dog heart (Jones et a l , 1979). The de c r e a s i n g r a t e of ca l c i u m uptake with time seen with p u r i f i e d SR (Figure 9) i s probably a form of " b a c k - i n h i b i t i o n " (Weber et a l , 1966); i n h i b i t i o n of C a + + uptake and ATPase a c t i v i t y t h a t occurs as i n c r e a s i n g l y e l e v a t e d l e v e l s of Ca i n the v e s i c l e lumen begin to ex e r t negative feedback on f u r t h e r C a + + 4-4-t r a n s p o r t . Normally, o x a l a t e a c t s as a Ca " s i n k " or b u f f e r such t h a t a Ca-oxalate s a l t of low s o l u b i l i t y forms i n the 4-t-v e s i c l e . As Ca e n t e r s , i t forms a Ca-oxalate p r e c i p i t a t e +4-which ensures a low f r e e Ca c o n c e n t r a t i o n , thus p r e v e n t i n g " b a c k - i n h i b i t i o n " from o c c u r r i n g . Loading the v e s i c l e s with Ca-oxalate, however, ay have exceede  the c a p a c i t y of the 4 + anion to maintain a low and constant f r e e Ca c o n c e n t r a t i o n . 11 Although the v e l o c i t y of both the Ca -uptake and Ca -ATPase r e a c t i o n s was i n c r e a s e d i n the p u r i f i e d p r e p a r a t i o n of SR, the degree of s t i m u l a t i o n by cAMP-PK and CAM was s i m i l a r to t h a t noted i n crude SR. I t i s w e l l e s t a b l i s h e d , f o r example, 44* 44-t h a t cAMP-PK s t i m u l a t e s Ca -uptake and Ca -ATPase i n crude c a r d i a c SR 2 - 3 - f o l d (Kirchberger et a l , 1972; Tada et a l , 1975). S t i m u l a t i o n by cAMP-PK of the p u r i f i e d SR p r e p a r a t i o n 4 4 r e s u l t e d i n a 2 and 2 . 5 - f o l d s t i m u l a t i o n of Ca -ATPase a c t i v i t y a t 0.2 and 1.0 /iM f r e e Ca c o n c e n t r a t i o n s , r e s p e c t i v e l y (Figure 12) while C a + + - u p t a k e was s t i m u l a t e d by 100% a t 0.2 /iM f r e e C a + f and 22% at 1.0 ^M f r e e Ca (Figure 44-11). The low l e v e l s of Ca -uptake s t i m u l a t i o n by cAMP-PK, 44-p a r t i c u l a r l y at the higher f r e e Ca c o n c e n t r a t i o n s , are c o n t r a r y to the f i n d i n g s i n the l i t e r a t u r e i n crude SR, and may - I l l -be due to the a l r e a d y maximal l e v e l s of Ca -uptake a c t i v i t y achieved (350 nmoles/mg/min) at 1.0 /iM f r e e Ca F i g u r e s 11 and 12 show t h a t , s i m i l a r to the f i n d i n g s r e p o r t e d with crude SR (Kirchberger and Antonetz, 1982b; Plank et a l , 1983) , s t i m u l a t i o n by CAM of Ca -uptake and Ca -ATPase +•+• a c t i v i t y was g r e a t e r at low f r e e Ca (0.2 ^iM f r e e : 70-80% ++ s t i m u l a t i o n ) than at the higher f r e e Ca c o n c e n t r a t i o n s t u d i e d (1.0 /iM f r e e : 40-45% s t i m u l a t i o n ) . I t was o f t e n noted, however, t h a t 1 /iM CAM was unable to markedly s t i m u l a t e Ca -uptake a c t i v i t y at e i t h e r f r e e Ca c o n c e n t r a t i o n . Although p r e p a r a t i o n l a b i l i t y was i n i t i a l l y suspected, the r e s u l t s o b t ained f o r CAM-dependent p h o s p h o r y l a t i o n (see below) made us suspect a more s u b t l e cause. The e f f e c t of both cAMP-PK and CAM present together was ++ examined on l y with r e s p e c t to Ca -ATPase a c t i v i t y . S i m i l a r to the r e s u l t s of Lopaschuk et a l (1980) obtained i n crude SR, the presence of both r e g u l a t o r s r e s u l t e d i n an a d d i t i v e s t i m u l a t i o n compared to each r e g u l a t o r alone, at 0.2 juM f r e e C a 4 * c o n c e n t r a t i o n (Figure 12). We observed, though, a lack of +4-a d d i t i v e s t i m u l a t i o n at 1.0 ;uM f r e e Ca , which, as noted I [-above, may have been due to maximal a c t i v a t i o n of the Ca pump at t h i s higher f r e e Ca c o n c e n t r a t i o n . A comparison of the two p r e p a r a t i o n s of SR with r e s p e c t to membrane p h o s p h o r y l a t i o n r e v e a l e d t h a t i n c u b a t i o n of e i t h e r p u r i f i e d or crude SR v e s i c l e s with cAMP-PK or the c a t a l y t i c (C) subunit of PK r e s u l t e d i n s i m i l a r p r o f i l e s . T y p i c a l l y , the h i g h e s t l e v e l s of p h o s p h o r y l a t i o n were a t t a i n e d i n the presence - 112 -of cAMP-PK, both i n crude and p u r i f i e d SR. The autoradiograms f o r both p r e p a r a t i o n s were a l s o i d e n t i c a l (Figure 14): p r o t e i n s of 98, 57, 51, and 44 Kdaltons i n c r e a s e d i n c o r p o r a t i o n of phosphate with time, a 68 Kdalton p r o t e i n became dephosphorylated by two minutes, whereas a 9-11 Kdalton p r o t e i n , assumed to be phospholamban (LePeuch et a l , 1979), became maximally phosphorylated by two minutes; the l a t t e r time-course was i n agreement with the f i n d i n g s of LaRaia and Morkin (1974) and Manalan and Jones (1982) . P h o s p h o r y l a t i o n by C subunit f o l l o w e d a s i m i l a r time-course to that observed with cAMP-PK ( t 1/2 = 20-30 sec) but demonstrated a higher degree of t o t a l p h o s p h o r y l a t i o n i n the p u r i f i e d p r e p a r a t i o n compared to crude ( F i g u r e s 13a, 13b). Two marked d i f f e r e n c e s , however, were apparent when an autoradiogram comparing the time-course of p h o s p h o r y l a t i o n by cAMP-PK or C subunit were examined (Figure 14). F i r s t , many of the p r o t e i n s phosphorylated e i t h e r i n crude or p u r i f i e d SR by cAMP-PK (e.g. 98K, 68K, 51K) were not phosphorylated i n the presence of C subunit , suggesting the impuri t y of the cAMP-PK p r e p a r a t i o n , and recommending the use of C subunit i n f u r t h e r work. A 56 k d a l t o n p r o t e i n was phosphorylated by both cAMP-PK and C su b u n i t , p a r t i c u l a r l y markedly i n the former case, and may repr e s e n t a u t o p h o s p h o r y l a t i o n of the r e g u l a t o r y subunit of cAMP-PK (Erlichman et a l , 1974) . Since the C subunit i s p u r p o r t e d l y p u r i f i e d from r e g u l a t o r y s u b u n i t , i t i s l i k e l y t h a t the 56 Kdalton p h o s p h o r y l a t i o n i n p u r i f i e d SR repr e s e n t s a u t o p h o s p h o r y l a t i o n of endogenous cAMP-PK. - 113 -The second d i f f e r e n c e noted between cAMP-PK and C subunit p h o s p h o r y l a t i o n i n p u r i f i e d and crude SR was the v a r i a b i l i t y i n phospholamban p h o s p h o r y l a t i o n l a t e n c y (Figure 14). Phospholamban was phosphorylated i n t e n s e l y almost immediately ( 10 sec ) by C subunit and by 30 sec achieved maximal l e v e l s whereas p h o s p h o r y l a t i o n by cAMP-PK was r e l a t i v e l y slow i n dev e l o p i n g , a t t a i n i n g maximal l e v e l s of p h o s p h o r y l a t i o n by 2 minutes. The d i f f e r e n c e i n phospholamban p h o s p h o r y l a t i o n v e l o c i t y between these two e s s e n t i a l l y i d e n t i c a l r e g u l a t o r s may, i n p a r t , be due to the presence of i m p u r i t i e s i n the cAMP-PK p r e p a r a t i o n which may e i t h e r p h y s i c a l l y or temporally impede access o f the c a t a l y t i c subunit to phospholamban k i n a s e . D i f f e r e n c e s between the crude and p u r i f i e d p r e p a r a t i o n s of SR became apparent when we i n v e s t i g a t e d the response to calmodulin-dependent p h o s p h o r y l a t i o n . In the p u r i f i e d p r e p a r a t i o n calmodulin-dependent p h o s p h o r y l a t i o n was i n h i b i t e d by 50% compared to crude, d e s p i t e the f i n d i n g s of enhanced cAMP-dependent p h o s p h o r y l a t i o n i n the former p r e p a r a t i o n (Figures 13a, 13b). These d i f f e r e n c e s were mirrored i n autoradiograms of crude and p u r i f i e d p r e p a r a t i o n s of SR phosphorylated i n the presence of CAM (Figure 15). Both the i n t e n s i t y and time to complete p h o s p h o r y l a t i o n of phospholamban were markedly dim i n i s h e d i n p u r i f i e d SR. Many of the extraneous p r o t e i n bands phosphorylated i n the presence of CAM i n crude SR (Katz e t a l , 1983) were a l s o missing i n p u r i f i e d SR. Ne v e r t h e l e s s , the two p r e p a r a t i o n s were a l i k e i n t h a t CAM-dependent phosphate i n c o r p o r a t i o n was both slower and lower - 114 -than t h a t observed with cAMP-PK or C subunit i n c u b a t i o n . The somewhat lower l e v e l s of CAM-dependent t o t a l p h o s p h o r y l a t i o n irk may r e f l e c t the sub-obtimal Ca c o n c e n t r a t i o n s ( 2 ;uM free) used. Tada and Katz (1982) r e p o r t t h a t maximal phospholamban p h o s p h o r y l a t i o n (comparable to t h a t observed with cAMP-PK) was achieved with a f r e e Ca c o n c e n t r a t i o n between 5 and 10 ^uM. Taken to g e t h e r , these r e s u l t s suggest t h a t a p u r i f i c a t i o n o f c a r d i a c SR by Ca-oxalate l o a d i n g was s u c c e s s f u l i n t h a t Ca -dependent processes (e.g. Ca t r a n s p o r t and ATPase a c t i v i t i e s ) a s s o c i a t e d with SR were enhanced while contamination by o r g a n e l l e s other than SR was decreased. N e v e r t h e l e s s , a number of f i n d i n g s of t h i s work le a d to some doubt r e g a r d i n g the u t i l i t y of t h i s method of p u r i f i c a t i o n . F i r s t , the i d e n t i t y and f u n c t i o n of the m i s s i n g 95,000 MW band i n the p u r i f i e d p r e p a r a t i o n remains unaccounted f o r . Second, the noted r e d u c t i o n i n CAM-dependent p h o s p h o r y l a t i o n and i n c o n s i s t e n c y of CAM-stimulated Ca -uptake a c t i v i t y i n p u r i f i e d SR suggests a d i s t u r b a n c e of CAM-mediated r e g u l a t i o n i n t h i s p r e p a r a t i o n . The p o s s i b i l i t y e x i s t s t h a t l o a d i n g of the v e s i c l e s with Ca-oxalate may a c t i v a t e Ca -dependent p r o t e o l y t i c a c t i v i t y which c o u l d be r e p o n s i b l e f o r both the degradation of the 95 Kdalton p r o t e i n and the i n a c t i v a t i o n of the a c t i v e s i t e or other a c c e s s o r y p r o t e i n s c r u c i a l f o r the proper f u n c t i o n i n g of CAM-mediated events i n SR. Another p o s s i b i l i t y may be the s e l e c t i v e l o s s through " p u r i f i c a t i o n " of the CAM-dependent kinase* which to date remains u n i d e n t i f i e d ( C h i e s i and C a r a f o l i , 1983) . Regardless of the mechanism, - 115 -p u r i f i c a t i o n by Ca-oxalate l o a d i n g has r e c e n t l y come under sharp c r i t i c i s m as a means of SR v e s i c l e s e p a r a t i o n . The o b j e c t i o n s i n c l u d e : (1) Ca-oxalate l o a d i n g a l t e r i n g the p r o p e r t y of enzymes l o c a t e d i n the v e s i c l e membrane (Velema and Zaagsma, 1981) , (2) o n l y 7-10% of c a r d i a c v e s i c l e s a c t u a l l y becoming loaded (Van Winkle and Entman, 1979) , (3) such a procedure appearing to s e l e c t a s p e c i f i c subpopulation of c a r d i a c SR v e s i c l e s (Jones and C a l a , 1981), and, (4) contamination by T-tubules not being e l i m i n a t e d (Kawamoto and Baskin, 1983). An aspect of c a r d i a c SR p u r i f i c a t i o n t h a t has not been addressed i s an e x p l a n a t i o n as to why Ca -dependent a c t i v i t i e s are enhanced compared to crude SR. Kawamoto and Baskin (1983) loaded c h i c k e n s k e l e t a l muscle SR with Ca-phosphate and found an enrichment of a 100 k d a l t o n p r o t e i n which they assumed to be the (Ca -Mg )-ATPase. A s e l e c t i v e enrichment of ATPase p r o t e i n was not observed i n t h i s study (Figure 8) and i s i n *tt* agreement with the lack of Ca pump p r o t e i n enrichment observed i n SR p u r i f i e d e i t h e r i n the presence ( L e v i t z k i et a l , 1976; Jones and Besch, 1979) or absence (Chamberlain et a l , 1983) of Ca-oxalate. The enhanced Ca -uptake a c t i v i t y , then, i s p r obably due to the i s o l a t i o n of more s e a l e d , r i g h t - s i d e out v e s i c l e s which are capable of Ca t r a n s p o r t , as opposed to the somewhat haphazard c o l l e c t i o n of s e a l e d and unbroken v e s i c l e s p r e s e n t i n cruder p r e p a r a t i o n s . II S t u d i e s examining the presence of c a l m o d u l i n i n f a s t and - 116 -slow-twitch s k e l e t a l muscle SR U n l i k e the calmodulin-dependent system r e g u l a t i n g the c a r d i a c SR Ca pump, the r o l e t h a t CAM p l a y s i n the r e g u l a t i o n of s k e l e t a l SR C a + + t r a n s p o r t remains to be e l u c i d a t e d . There i s l i t t l e doubt t h a t CAM i s present i n s k e l e t a l muscle although compared with other v e r t e b r a t e t i s s u e s , i t s c o n c e n t r a t i o n i s extremely low (Klee and Vanaman, 1982). In f a s t s k e l e t a l muscle, the CAM c o n c e n t r a t i o n has been estimated as 10-20 /iM (Grand et a l , 1979) , 40% of which i s thought to be the $ subunit of phosphorylase k i n a s e . The amount of CAM i n slow s k e l e t a l muscle has not been r e p o r t e d , but the c o n t r i b u t i o n by phosphorylase kinase i s l i k e l y n e g l i g i b l e s i n c e the l e v e l of t h i s g l y c o l y t i c enzyme i s 20-50-fold lower than t h a t found i n f a s t s k e l e t a l muscle. For comparison, i t i s estimated t h a t the c o n c e n t r a t i o n o f calmodulin i n c a r d i a c muscle i s 10 ;uM, and between 3-5 >uM i n the c y t o s o l of red blood c e l l s ( S c h a r f f and Foder, 1981). Grand and P e r r y (1979) have subsequently m o d i f i e d t h e i r estimate of CAM content i n s k e l e t a l muscle by s t a t i n g t h a t the v a s t m a j o r i t y of CAM-like a c t i v i t y i s , i n r e a l i t y , t r o p o n i n C. These determinations of t i s s u e CAM content are i n d i r e c t s i n c e they are t y p i c a l l y made by b o i l i n g t i s s u e f r a c t i o n s , and then comparing the s t i m u l a t o r y e f f e c t obtained on red blood c e l l phosphodiesterase a c t i v i t y with the s t i m u l a t i o n produced by a known weight of pure CAM ( C h i e s i and C a r a f o l i , 1982). Our work has examined the c l a i m t h a t CAM i s e i t h e r t i g h t l y bound to the SR membrane and o n l y removable by such harsh - 117 -treatment as b o i l i n g ( C h i e s i and C a r a f o l i , 1982) or t h a t i t i s somewhat l e s s t i g h t l y bound and e x t r a c t a b l e by C a * + c h e l a t i o n (Campbell and MacLennan, 1982). The dichotomy of " t i g h t " and " l o o s e " b i n d i n g of CAM to c e l l u l a r c o n s t i t u e n t s i s not new. Although CAM i s g e n e r a l l y regarded as a s o l u b l e p r o t e i n , a c o n s i d e r a b l e amount of CAM a c t i v i t y has been found to be a s s o c i a t e d with p a r t i c u l a t e f r a c t i o n s of mammalian t i s s u e s even a f t e r e x t e n s i v e washing o f the p a r t i c u l a t e f r a c t i o n with EGTA (Sobue et a l , 1981; K a k i u c h i et a l , 1982). The terms "EGTA-extractable" and "EGTA-nonextractable" (or " p a r t i c l e - a s s o c i a t e d " ) have t h e r e f o r e been i n t r o d u c e d . K a k i u c h i et a l (1982), i n a d d i t i o n , have found t h a t , s i m i l a r to the " l a t e n c y " of Na +,K +-ATPase a c t i v i t y expressed f o l l o w i n g treatment o f SR v e s i c l e s with detergent (Besch e t a l , 1976), p a r t i c l e - a s s o c i a t e d CAM a c t i v i t y i s a l s o " l a t e n t " to some degree and can be unmasked by the presence of n o n i o n i c d e t e r g e n t s . Our s t u d i e s have found t h a t , u n l i k e the c l a i m s of Campbell and MacLennan (1982), a c o n s i d e r a b l e amount of CAM remains bound to both fSR and sSR even a f t e r e x t e n s i v e washing o f the SR p e l l e t with 1 mM EGTA. T h i s d e t e r m i n a t i o n was made us i n g three independent l i n e s of i n v e s t i g a t i o n . F i r s t , we examined the a b i l i t y o f the supernatants d e r i v e d from EGTA-washed f a s t and slow SR to s t i m u l a t e CAM-depleted red c e l l (Ca -Mg )-ATPase a c t i v i t y . I t was found t h a t such supernatants were unable to s t i m u l a t e ATPase a c t i v i t y i f care was taken to ensure the removal of i n s o l u b l e m a t e r i a l ( i . e . SR - 118 -membranes). The prevalence of contaminating SR membranes was p a r t i c u l a r l y e v i d e n t when we employed the second method o f CAM i d e n t i f i c a t i o n , t h a t of SDS-PAGE. As shown i n F i g u r e 21, EGTA-washed fSR supernatant t h a t had not been subjected to high-speed (100,000 x g) c e n t r i f u g a t i o n to thoroughly remove i n s o l u b l e m a t e r i a l showed a number of p r o t e i n bands i n d i g i n o u s to the SR membrane. F o l l o w i n g the removal of such m a t e r i a l , the two p r o t e i n s which could be a t t r i b u t e d to CAM (see below), those of 16.5 and 19.5 Kdaltons, were no longer seen i n the electrophoretogram. As b r i e f l y mentioned above, s k e l e t a l muscle t i s s u e c o n t a i n s a s i g n i f i c a n t amount of t r o p o n i n C which has a molecular weight (20 Kdaltons) s i m i l a r to CAM. Thus, the presence of a p r o t e i n m i g r a t i n g i n the 16-20 Kdalton r e g i o n of e x t r a c t samples i s not a r e l i a b l e i n d i c a t i o n of the presence of CAM. In a d d i t i o n , the CAM standards used i n these s t u d i e s migrated between 19 and 21 Kdaltons r e g a r d l e s s of whether they 4-+ contained exogenously added Ca or c h e l a t o r (EGTA). I t i s w e l l known t h a t CAM e x h i b i t s marked v a r i a t i o n i n i t s SDS-PAGE 44 m o b i l i t y depending on whether i t i s run i n the presence of Ca (MW: 15,800 + 700 daltons) or EGTA (MW: 18,200 ± 200 daltons) (Klee and Vanaman, 1982) . As mentioned above, we d i d not see 44-t h i s change i n m o b i l i t y . An e x p l a n a t i o n may be t h a t the Ca or EGTA must not o n l y be present i n the sample but a l s o i n the g e l matrix and running b u f f e r b e f o r e the change i n m o b i l i t y i s observed (Dr. A. M o l l a , p e r s o n a l communication). Thus we r e l i e d h e a v i l y on the t h i r d and most d i r e c t means of CAM - 119 -i d e n t i f i c a t i o n , t h a t o f a^I-CAM radioimmunoassay ( R I A ) . As shown i n T a b l e 7, i t was found t h a t washing SR, p a r t i c u l a r l y fSR, i n the presence o f EGTA was i n c a p a b l e o f l i b e r a t i n g enough CAM t o be d e t e c t a b l e by RIA, a l t h o u g h SDS-PAGE r e v e a l e d ( F i g u r e 21) t h a t the EGTA-washed e x t r a c t s c o n t a i n e d membrane fra g m e n t s . How was i t p o s s i b l e , t h e n , t h a t the RIA f o r CAM was below t h e s e n s i t i v i t y o f t h e e l e c t r o p h o r e t o g r a m ? The answer may l i e w i t h the f a c t t h a t i n t a c t fSR membrane v e s i c l e s show v e r y l i t t l e CAM by RIA (Table 7 ) , p o s s i b l y due t o t h e i n a c c e s i b i l i t y o f the CAM p r e s e n t i n the SR v e s i c l e s t o the anti-CAM a n t i b o d y o f t h e RIA d e t e c t i o n system. The r e s u l t s c i t e d above a l l o w f o r the c o n c l u s i o n t h a t , i n d e e d , t h e CAM p r e s e n t i n s k e l e t a l muscle may be E G T A - n o n e x t r a c t a b l e . I n o r d e r , t h e n , t o demonstrate the " l a t e n t " a c t i v i t y o f CAM i n t h e s e v e s i c l e s h a r s h e r t r e a t m e n t was r e q u i r e d . We t h e r e f o r e b o i l e d the SR v e s i c l e s i n e i t h e r t h e pr e s e n c e or absence o f EDTA, e i t h e r p r i o r or f o l l o w i n g EGTA-washing, and a g a i n d e t e r m i n e d by the t h r e e independent l i n e s o f i n v e s t i g a t i o n whether or not CAM was p r e s e n t . B o i l i n g o f s k e l e t a l SR membranes, p a r t i c u l a r l y i n t h e pr e s e n c e o f 0.2 mM EDTA, and i s o l a t i o n o f s u p e r n a t a n t r e s u l t e d i n ( i ) a dose-dependent s t i m u l a t i o n o f CAM-depleted r e d b l o o d c e l l ++ +-f (Ca -Mg )-ATPase a c t i v i t y (Table 4 ) , ( i i ) the p r e s e n c e , on SDS-PAGE, o f a number o f p r o t e i n bands r a n g i n g between 15.5 and 19.5 K d a l t o n s , t h e a c c e p t a b l e m i g r a t i o n range o f CAM (Table 8 ) , U S ' and ( i i i ) , measureable l e v e l s o f CAM as de t e r m i n e d by I-CAM - 120 -RIA (Table 7 ) . A l l of the above were found r e g a r d l e s s of whether the SR v e s i c l e s were pre-washed i n 1 mM EGTA. CAM l e v e l s , as measured by RIA, were decreased by 33%, however, i n the fSR v e s i c l e s pre-washed with EGTA (Table 7 ) . T h i s l a t t e r p o i n t i s somewhat p u z z l i n g s i n c e one would expect to f i n d t h i s presumably EGTA-extractable CAM i n the supernatant and so to be RIA-detectable (which i t was n o t ) . Most of the r e s u l t s obtained with fSR mirrored those obtained with sSR with one notable e x c e p t i o n : the CAM content of sSR, as determined by RIA, was higher (per mg p r o t e i n ) than i n fSR. T h i s c o u l d r e f l e c t e i t h e r endogenous CAM c o n c e n t r a t i o n d i f f e r e n c e s or d i s s i m i l a r i t i e s i n the degree to which CAM i s bound to the r e s p e c t i v e SR membranes. Although t h i s aspect of s k e l e t a l muscle b i o c h e m i s t r y has not y e t been addressed i n the l i t e r a t u r e , our data would sugggest the l a t t e r p o s s i b i l i t y on the b a s i s of our f i n d i n g of EGTA-extractable CAM i n the supernatants of EGTA-washed sSR and not fSR (Table 7b). In a d d i t i o n , we were able to r e c o r d a value of 85 ng/ mg SR p r o t e i n i n EGTA-washed sSR supernatant when an u n b o i l e d CAM standard was used; EGTA-washed fSR supernatant had 0 l e v e l s of CAM. The use of the u n b o i l e d standard was based on the recent d i s c o v e r y (NEN t e c h n i c a l b u l l e t i n , 1983) t h a t b o i l i n g of CAM uncovered more immunoreactive s i t e s f o r the anti-CAM antibody. Our p r e v i o u s use of a " b o i l e d " standard CAM f o r an u n b o i l e d supernatant sample c o u l d r e s u l t i n a gross underestimation of the a c t u a l amount of CAM present i n the sample. The high l e v e l of EGTA-extractable CAM d e r i v e d from sSR i s f u r t h e r i n d i r e c t - 121 -evidence t h a t perhaps the p r o t e i n i s l e s s a v i d l y bound to the sSR membrane than to the fSR. T h i s study a l s o demonstrated an i n a b i l i t y of the anti-CAM agents, TFP and Compound 48/80, to r e s t r i c t t h e i r i n h i b i t o r y 44-a c t i o n to CAM-stimulated Ca -ATPase a c t i v i t y . N o n - s p e c i f i c e f f e c t s u n r e l a t e d to i n t e r a c t i o n s with CAM made c o n c l u s i o n s r e g a r d i n g the presence or absence of CAM i n s k e l e t a l muscle SR untenable. U n l i k e other r e p o r t s i n the l i t e r a t u r e (Gietzen e t a l , 1980; Raess and V i n c e n z i , 1980) documenting a 50% i n h i b i t o r y potency of l e s s than 20 uM TFP on CAM-stimulated red +4 4-4 c e l l (Ca -Mg )-ATPase a c t i v i t y , and s i m i l a r to the f i n d i n g s of L e v i n and Weiss (1980) i n r a t e r y t h r o c y t e s , we were unable to show a s i g n i f i c a n t decrease i n a c t i v i t y below 50 ;uM TFP. I n i t i a l s t u d i e s performed on red c e l l membranes hemolyzed i n the presence of d i s t i l l e d , d e - i o n i z e d water demonstrated poor TFP anti-CAM potency. Subsequent s t u d i e s on membranes prepared a c c o r d i n g to N i g g l i et a l (1981) u t i l i z e d hypotonic t r i s - C l , pH 7.4 ( i n the presence of 1 mM EDTA) as the hemolyzing agent, and were much more amenable to a T F P - i n h i b i t i o n of CAM-stimulated ATPase a c t i v i t y ( F igure 18). I t i s not c l e a r why the anti-CAM potency of TFP should vary with membrane p r e p a r a t i o n , but i t may be t h a t the poor b u f f e r i n g c a p a c i t y of d i s t i l l e d water, used i n the hemolysis step of the former p r e p a r a t i o n , may be p a r t l y r e s p o n s i b l e . U n l i k e the f i n d i n g s of G i e t z e n et a l (1983), Compound 48/80 e x h i b i t e d i n h i b i t i o n of CAM-independent Ca^-ATPase a c t i v i t y . The c o n c e n t r a t i o n of Compound 48/80 which e x h i b i t e d - 122 -50% i n h i b i t i o n of CAM-stimulated Ca -ATPase a c t i v i t y (IC50) was 3.0 /ig/ml, almost a 4 - f o l d higher c o n c e n t r a t i o n than the value of 0.85 yug/ml obtained by G i e t z e n et a l (1983). D i f f e r e n c e s i n membrane p r e p a r a t i v e techniques or commercial p r e p a r a t i o n s of Compound 48/80 may be r e s p o n s i b l e f o r the noted d i f f e r e n c e s . These s t u d i e s t h e r e f o r e demonstrated t h a t CAM i s bound t i g h t l y to both fSR and sSR (although perhaps more t i g h t l y to fSR) and remains l a r g e l y ( g r e a t e r than 2/3 ) EGTA-nonextractable. I t i s of i n t e r e s t t h a t Diamond et a l (1980) r e p o r t e d t h a t r a b b i t fSR v e s i c l e s possess a c l a s s of t i g h t l y - b o u n d c a l c i u m ions i n a c c e s s i b l e to EGTA l o c a t e d , presumably, i n a hydrophobic "pocket" i n , or i n c l o s e p r o x i m i t y to the ATPase. I t i s tempting to s p e c u l a t e t h a t perhaps the EGTA-nonextractable CAM may be a s s o c i a t e d with such t i g h t l y bound Ca + 4" i o n s , and perhaps e x e r t i t s r e g u l a t o r y e f f e c t i n c l o s e p r o x i m i t y to the ATPase. A major unresolved q u e s t i o n concerns the nature of the s t i m u l a t o r t h a t MacLennan (1972) observed t h a t was able to r e s t o r e the depressed Ca -uptake a c t i v i t y f o l l o w i n g EGTA-washing of fSR v e s i c l e s , and t h a t Campbell and MacLennan (1982) found able to enhance, i n a CAM-like manner, p h o s p h o r y l a t i o n i n SR v e s i c l e s p u r p o r t e d l y d e p l e t e d of CAM by treatment with 1 mM EGTA. With r e f e r e n c e to the f i r s t f i n d i n g of MacLennan (1972) , supernatant ( e i t h e r u n t reated or f o l l o w i n g b o i l i n g ) d e r i v e d from EGTA-washed fSR were able to r e s t o r e the depressed Ca t r a n s p o r t a c t i v i t y of the washed v e s i c l e s . Once - 123 -the supernatant was d i a l y z e d f o r o n l y 4 hours, however, i t s a b i l i t y to r e s t o r e t h i s a c t i v i t y was almost completely l o s t . One may t h e r e f o r e t e n t a t i v e l y suggest t h a t the r e s t o r a t i o n of a c t i v i t y was perhaps due to the presence of high EGTA c o n c e n t r a t i o n s ( i . e . Ca-EGTA complexes) which have been shown ++ ++ by Berman (1982) to markedly s t i m u l a t e the (Ca -Mg )-ATPase a c t i v i t y of r a b b i t s k e l e t a l SR. A s i m i l a r e x p l a n a t i o n f o r the enhanced CAM-like p h o s p h o r y l a t i o n of EGTA-washed SR v e s i c l e s observed by Campbell and MacLennan (1982) i s not warranted. In t h e i r study, the supernatant d e r i v e d from EGTA-washed v e s i c l e s was b o i l e d f o r 5 minutes, c e n t r i f u g e d (100,000 x g) to remove i n s o l u b l e m a t e r i a l , and d i a l y z e d . I t i s our c o n t e n t i o n , however, t h a t the i n i t i a l wash i n the high EGTA c o n c e n t r a t i o n r e s u l t s i n suspension of membrane fragments (which indeed r e q u i r e s a c e n t r i f u g a t i o n of 100,000 x g to sediment). The b o i l i n g procedure t h a t Campbell and MacLennan (1982) then undertake p r i o r to c e n t r i f u g a t i o n , may r e l e a s e bound CAM present i n the contaminating SR membranes suspended i n the supernatant. The r e s u l t i n g CAM, purported to have o r i g i n a t e d from EGTA washing of s k e l e t a l SR, i s t h e r e f o r e more l i k e l y t o have been d e r i v e d from b o i l i n g of suspended SR membranes. As t h i s study has shown, a remnant of the o r i g i n a l CAM ( l e s s than 1/3 ) may be r e l e a s e d by EGTA treatment, but, by f a r , the m a j o r i t y of CAM remains E G T A - i n a c c e s s i b l e and removable o n l y by harsher treatment. - 124 -I l l The R e g u l a t i o n of S k e l e t a l SR i n a Disease S t a t e : C h r o n i c , Experimentally-Induced D i a b e t e s . T h i s study demonstrated both a s i g n i f i c a n t decrease i n Ca -uptake a c t i v i t y at a l l f r e e c a l c i u m c o n c e n t r a t i o n s t e s t e d , and a s i g n i f i c a n t i n c r e a s e of a c i d - s o l u b l e and l o n g - c h a i n a c y l c a r n i t i n e l e v e l s i n microsomes e n r i c h e d i n SR d e r i v e d from the s k e l e t a l muscle of d i a b e t i c r a t s , as compared to c o n t r o l s . These f i n d i n g s p a r a l l e l e d the r e s u l t s obtained i n d i a b e t i c r a t h e art SR (Lopaschuk et a l , 1983) , and suggest a g e n e r a l i z e d p a t h o p h y s i o l o g i c a l mechanism a f f e c t i n g muscle t i s s u e i n the d i a b e t i c animal. The d e f e c t i n both Ca + + /-uptake a c t i v i t y and l e v e l s of l o n g - c h a i n and f r e e c a r n i t i n e i n d i a b e t i c r a t h eart SR has been l i n k e d to the noted d i m i n i s h e d a b i l i t y of i s o l a t e d working h e a r t s from d i a b e t i c r a t s to respond to i n c r e a s i n g p r e l o a d or a f t e r l o a d (Vadlamudi et a l , 1982; Ingebretson et a l . 1980) . I t i s b e l i e v e d t h a t such an a l t e r a t i o n i n h eart f u n c t i o n may be due to the accumulation of c e l l u l a r l o n g - c h a i n a c y l c a r n i t i n e s 44 which have been shown to be potent i n h i b i t o r s of SR Ca t r a n s p o r t (Adams et a l , 1979). The f i n d i n g of a decreased 44, Ca -uptake i n d i a b e t i c r a t s k e l e t a l muscle may a l s o have a c o r r e l a t e i n the noted c l i n i c a l complaint of muscle weakness i n many d i a b e t i c p a t i e n t s ( E l l e n b e r g , 1976) . Current b e l i e f suggests t h a t muscle weakness i n d i a b e t e s may be the end r e s u l t o f axonal degeneration, a l s o thought r e s p o n s i b l e f o r the c l i n i c a l f i n d i n g s of decreased conduction v e l o c i t y , d e m y e l i n a t i o n , and a l t e r e d axonal t r a n s p o r t (Thomas et a l , - 125 -1982). The decrease i n s k e l e t a l muscle SR Ca -uptake may p l a y an as yet unrecognized r o l e i n the e t i o l o g y of such neuromuscular d y s f u n c t i o n . Whether e l e v a t e d l e v e l s of c a r n i t i n e d e r i v a t i v e s are ++ p r i m a r i l y r e s p o n s i b l e f o r the decrease i n Ca -uptake a c t i v i t y i s unknown. A number of a l t e r a t i o n s i n the s k e l e t a l muscle of c h r o n i c a l l y d i a b e t i c r a t s have been noted; of p a r t i c u l a r r e l e v a n c e i s a s i g n i f i c a n t decrease i n both ATP c o n c e n t r a t i o n and pH i n d i a b e t i c r a t s oleus muscle compared to c o n t r o l s (Moore et a l , 1983) . Both decreased l e v e l s of ATP (Weber et a l , 1966) and H+ c o n c e n t r a t i o n (Mandel et a l , 1982) have been shown ++ to depress Ca -uptake a c t i v i t y . The e l e v a t e d l e v e l s of l o n g - c h a i n a c y l c a r n i t i n e s observed may be of s e r i o u s consequence to the SR membrane. The e f f e c t of such detergents range from i n c o r p o r a t i o n and consequent a l t e r a t i o n of the l i p i d b i l a y e r at low c o n c e n t r a t i o n s , to i r r e v e r s i b l e l o s s of membrane p h o s p h o l i p i d s and p h y s i c a l d i s r u p t i o n of the membrane at high c o n c e n t r a t i o n s l e a d i n g , perhaps, to u n c o n t r o l l e d Ca f l u x , and u l t i m a t e l y c e l l death (Katz, 1982). The l a t t e r sequence of events may or may not be r e s p o n s i b l e f o r the noted muscle weakness i n d i a b e t e s . 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