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Role of superficial calcium binding sites in the inotropic response of isoproterenol and ouabain Fawzi, Ahmad B. 1984

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ROLE OF SUPERFICIAL' CALCIUM BINDING SITES IN THE INOTROPIC RESPONSE OF ISOPROTERENOL AND OUABAIN by AHMAD B. FAWZI Pharm.D., Tehran University, 1975 M.Sc, Dalhousie University, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Division of Pharmacology and Toxicology Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1984 © Ahmad B. Fawzi, 1984 r*4 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. , _ ^ A / W R M A C e c i T i C A L ScrewC&S Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date fz®' ist' 7 E-6 (3/81) i i A B S T R A C T Mammalian myocardial c o n t r a c t i l i t y i s b e l i e v e d to be re g u l a t e d by the amount of calcium contained i n a h i g h l y l a b i l e s u p e r f i c i a l calcium pool. The purpose of the f i r s t p a r t of t h i s study was to determine the r o l e of such s i t e s i n the po s i t i v e inotropic e f f e c t of isoproterenol. Lantha-num, an i o n known to be r e s t r i c t e d to the e x t r a c e l l u l a r space and which d i s p l a c e s the s u p e r f i c i a l l y - b o u n d calcium, was selected as a t o o l for t h i s investigation.In Langendorff prepa r a t i o n s of the guinea p i g heart, lanthanum decreased the basal c o n t r a c t i l i t y index (+dP/dt ) i n a concentra-Jr ' m a x tion-dependent fashion (0.05-3 uM) and blocked the inotropic response of isoproterenol i n a non-competitive manner (0.25-3uM). Three uM lanthanum: 1) reduced basal c o n t r a c t i l i t y and the maximum response to isoproterenol by 97 and 95%, respec-t i v e l y ; 2) had no s i g n i f i c a n t e f f e c t (p>0.05) on basal and i s o p r o t e r e n o l - i n d u c e d c y c l i c AMP l e v e l s ; and 3) had no 3 e f f e c t on the of [ H ] n i t r e n d i p i n e b i n d i n g , but r e -duced the B by 31%. While 1 u-M lanthanum reduced basal max J ^ c o n t r a c t i l i t y and the maximum response to i s o p r o t e r e n o l by 3 90 and 70%, r e s p e c t i v e l y , i t had no e f f e c t on [ H ] n i t r e n -d i p i n e b i n d i n g . These r e s u l t s suggest that the e f f e c t s of such low c o n c e n t r a t i o n s of lanthanum { <3 p.M) are not r e l a t e d to a d i r e c t a c t i o n on the calcium channels and are not mediated by an i n h i b i t i o n of i s o p r o t e r e n o l s t i m u l a t i o n of the enzyme adenylate c y c l a s e . Therefore, these r e s u l t s i i i suggest that superficially-bound calcium i s required f o r the inotropic response of isoproterenol. The purpose of the second p a r t of t h i s study was to el u c i d a t e the biochemical nature of the s u p e r f i c i a l calcium binding s i t e s , the s i a l i c acids i n p a r t i c u l a r , i n the ino-t r o p i c response of cardiotonic agents. To determine the role of the glyc o c a l y x residues of s i a l i c a c i d s i n e x c i t a t i o n -c o n t r a c t i o n coupling and the i n o t r o p i c response to card i o -t o n i c agents, I studied the e f f e c t of removal of the s i a l i c a c i d s following neuraminidase treatment on the response to ouabain, i s o p r o t e r e n o l , calcium and reduced e x t r a c e l l u l a r sodium i n Lange n d o r f f p r e p a r a t i o n s of a d u l t guinea p i g hearts. Neuraminidase treatment (0.01 U/ml, 1 h) reduced the _7 magnitude of the p o s i t i v e i n o t r o p i c response to 2.5x10 M -7 ouabain and the maximum response to 5x10 M ouabain by about 46 and 30%, r e s p e c t i v e l y , but d i d not prevent ouabain t o x i c i t y . Neuraminidase treatment d i d not a f f e c t the con-t r a c t i l i t y produced by calcium concentration a l t e r a t i o n s up to 5 mM calcium or the p o s i t i v e i n o t r o p i c e f f e c t produced by lowering external sodium to as low as 80 mM. The i n o t r o p i c -8 response to as h i g h as 10 M i s o p r o t e r e n o l was a l s o not a f f e c t e d . The c o n t r a c t i l i t y response developed to calcium — 3 c o n c e n t r a t i o n s g r e a t e r than 5 mM and to 5x10 M i s o p r o -terenol were s i g n i f i c a n t l y reduced (p<0.05) by neuraminidase treatment. The content of s i a l i c a c i d s i n neuraminidase-t r e a t e d h e a r t s used i n the above c o n c e n t r a t i o n - r e s p o n s e studies of ouabain, i s o p r o t e r e n o l , calcium, and sodium was reduced by 70.7, 66.1, 65.6 and 66.2%, r e s p e c t i v e l y . Neura-minidase treatment had no e f f e c t on b a s a l (Na +-K +)ATPase 2+ + + and Mg -ATPase a c t i v i t i e s of (Na -K ) A T P a s e - c o n t a i n -ing membrane preparations of the guinea p i g l e f t v e n t r i c l e . Neuraminidase treatment neither influenced the s e n s i t i v i t y of the enzyme (Na +-K +)ATPase t o ouabain i n h i b i t i o n nor 3 d i d i t a f f e c t the c h a r a c t e r i s t i c s of [ H]ouabain b i n d i n g to the p r e p a r a t i o n . These r e s u l t s suggest that the s i a l i c a c i d s of the g l y c o c a l y x i n the guinea p i g l e f t v e n t r i c l e play an important r o l e i n part of the i n o t r o p i c response to subtoxic concentrations of ouabain. JbAn H. McNeill, Ph.D. Thejsis Supervisor V TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i i i LIST OF FIGURES ix LIST OF ABBREVIATIONS x i ACKNOWLEDGEMENTS xiv SECTION I. INTRODUCTION 1 A. Calcium and Muscle C o n t r a c t i l i t y . 2 B. S u p e r f i c i a l Calcium and Cardiac C o n t r a c t i l i t y . 7 C. E f f e c t of Lanthanum on Cardiac Calcium Metabolism and Function. 15 D. U l t r a s t r u c t u r a l L o c a l i z a t i o n of Lanthanum i n the Heart. 22 E. Cardiac Sarcolemmal Calcium Binding. 28 F. Biochemical Nature of Cardiac Sarcolemmal Calcium Binding Sites. 37 G. Role of Sarcolemmal S i a l i c Acids i n Cardiac Calcium Metabolism and Function. 41 H. Effe c t of Cardiac Glycosides on Superficially-Bound Calcium. 46 I. Purpose of the Study and Approach to the Problem. 50 SECTION II. METHODS 53 A. Langendorff Preparations. 54 B. C y c l i c AMP Determination. 55 3 C. [ H]Nitrendipine Binding. 56 1. Membrane preparation. 56 v i PAGE 3 2. Determination of [ H]nitrendipine binding. 57 D. S i a l i c Acid Assay. 58 E. Determination of Content of S i a l i c Acids i n the Guinea Pig Heart. 60 F. Neuraminidase Assay. 61 G. Preparation of (Na +-K +)ATPase-containing Cardiac C e l l Membranes. 62 H. Assay of (Na +-K +)ATPase. 63 I. Determination of Inorganic Phosphate. 64 3 J. Determination of [ H]0uabain Binding. 66 K. Determination of Protease A c t i v i t y . 67 L. The Enzyme Neuraminidase. 68 M. Protein Assay. 69 N. S t a t i s t i c a l Analysis. 70 SECTION I I I . RESULTS 71 A. Ef f e c t of Lanthanum on Control C o n t r a c t i l i t y and Isoproterenol Inotropy. 72 B. Ef f e c t of Lanthanum on Isoproterenol-Induced C y c l i c AMP Level. 81 3 C. [ H]Nitrendipine Binding i n the Guinea Pig Left V e n t r i c l e . 84 3 D. Effects of Calcium and Lanthanum on [ H]Nit-rendipine Binding. 87 3 E. Eff e c t of Lanthanum on [ H]Nitrendipine Binding and Myocardial C o n t r a c t i l i t y . 94 F. S t a b i l i t y and A c t i v i t y of Neuraminidase. 98 G. Protease A c t i v i t y i n the Neuraminidase Product. 98 H. Neuraminidase-Releasable S i a l i c Acids. 101 V X 1 PAGE I. Ef f e c t of Neuraminidase Treatment on the Response to Ouabain. 104 J. E f f e c t of Neuraminidase Treatment on Isoproterenol Inotropy. 109 K. Eff e c t of Neuraminidase Treatment on the Response to Calcium. 109 L. E f f e c t of Neuraminidase Treatment on the Inotropic Response of Reduced E x t r a c e l l u l a r Sodium. 112 M. Eff e c t of Neuraminidase Treatment on Basal C o n t r a c t i l i t y . 115 N. Effect of Neuraminidase Treatment on Ouabain Binding and I n h i b i t i o n of the Enzyme (Na +-K +)ATPase. 121 SECTION IV. DISCUSSION , A. E f f e c t of Lanthanum on the Inotropic Response of Isoproterenol: Role of the Superficially-Bound Calcium. 128 3 B. Effects of Calcium and Lanthanum on [ H]-Nitrendipine Binding i n the Guinea Pig Left V e n t r i c l e . 134 C. S t a b i l i t y and Effectiveness of Neuraminidase. 137 D. Eff e c t of Neuraminidase Treatment on Ouabain Inotropy. 139 E. E f f e c t of Neuraminidase Treatment on the Response to Isoproterenol and Calcium. 144 SUMMARY AND CONCLUSIONS 148 SECTION V. BIBLIOGRAPHY 150 v i i i LIST OF TABLES TABLE PAGE 1. Effect.of Lanthanum on the EC^Q of Isoproterenol i n Langendorff Preparations of the Guinea Pig Heart. 7 8 2. Effects of Calcium and Lanthanum on q the Characteristics of [ H]Nitrendipine Binding i n the Guinea Pig Left V e n t r i c l e . 9 1 3. E f f e c t of 3 uM Lanthanum on the q Characteristics of [ H]Nitrendipine Binding i n the Guinea Pig Heart. 9 7 4. Left Ventricular Content of S i a l i c Acids i n Control and Neuraminidase-Treated Guinea Pig Hearts. 1 1 8 5. E f f e c t of BSA and Neuraminidase Treatments on Basal +dP/dt m a x and End D i a s t o l i c Pressure i n the Guinea Pig Left V e n t r i c l e . 1 2 0 6. E f f e c t of Neuraminidase Treatment on Basal (Na +-K +)ATPase and Mg 2 +-ATPase A c t i v i t i e s . 1 2 4 LIST OF FIGURES FIGURE 1. The concentration-response curve of the negative inotropic e f f e c t of lanthanum i n Langendorff preparations of adult guinea pig hearts. 2. E f f e c t of d i f f e r e n t concentrations of lanthanum on the p o s i t i v e inotropic e f f e c t o isoproterenol i n Langendorff preparations of adult guinea pig hearts. 3. E f f e c t of 5 \M lanthanum on the inotropic response to 5x10 M isoproterenol. 4. E f f e c t of lanthanum on basal and isoproterenol-induced c y c l i c AMP l e v e l s . 5. Typical saturation experiment of •3 [ H]nitrendipine binding i n a membrane preparation of the guinea pig l e f t v e n t r i c l e 6. Eff e c t of calcium on [ H]nitrendipine binding i n membrane preparations of the guinea pig l e f t v e n t r i c l e . 7. Concentration-dependent i n h i b i t i o n of [ H]nitrendipine binding by lanthanum i n membrane preparations of the guinea pig l e f t v e n t r i c l e . X FIGURE PAGE 8. Effe c t of lanthanum on myocardial c o n t r a c t i l i t y and [ H]nitrendipine binding i n the guinea pig heart. 95 9. Ef f e c t of bovine serum albumin (BSA) and temperature on neuraminidase s t a b i l i t y . 99 10. Neuraminidase-releasable s i a l i c acids of the guinea pig heart. 102 11. Positive inotropic e f f e c t of ouabain i n neuraminidase-treated (A) and control (B) Langendorff preparations of guinea pig hearts. 106 12. Isoproterenol concentration-response i n control and neuraminidase-treated Langendorff preparations of guinea pig hearts. 110 13. Eff e c t of neuraminidase treatment on the response of guinea pig hearts to calcium. 113 14. Eff e c t of neuraminidase treatment on the po s i t i v e inotropic e f f e c t of diminished e x t r a c e l l u l a r sodium concentration. 116 15. Eff e c t of neuraminidase treatment on ouabain binding. 122 16. Eff e c t of neuraminidase treatment on ouabain i n h i b i t i o n of the enzyme (Na +-K +)ATPase. 125 LIST OF ABBREVIATIONS adenosine 5'-triphosphate adenosine 5'-triphosphatase maximal number of binding s i t e s bovine serum albumin degree centigrade Curie c y c l i c adenosine 3',5'-monophosphate the calculated concentration of an agonist that produces 50% of the maximum e f f e c t , dose of an agonist that produces 50% of the maximum e f f e c t , ethylenediaminetetraacetic acid ethylene glycol-bis(beta-aminoethyl ether)N,N' t e t r a a c e t i c acid for example and others figure femtomole(s) gram(s) acceleration of gravity hour(s) N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid concentration of an antagonist that i n h i b i t s the response by 50%. X l l i . p . i n t r a p e r i t o n i a l i n j e c t i o n K, d i s s o c i a t i o n constant d kg kilogram(s) i n h i b i t o r y constant K Michaelis-Menten constant m 1 l i t e r ( s ) M molar concentration m m i l l i mg milligram(s) min minute(s) ml m i l l i l i t e r ( s ) mm millimeter(s) mM millimolar concentration mol mole(s) n number of observations N normal concentration ng nanogram(s) nm nanometer(s) nM nanomolar concentration nmol nanomole(s) p s t a t i s t i c a l p r o b a b i l i t y value inorganic phosphate pK negative logarithm of i o n i z a t i o n constant a pM picomolar concentration pmol picomole(s) r c o r r e l a t i o n c o e f f i c i e n t rpm revolutions per minute x i i i s second(s) S.E.M. standard error of the mean T r i s tris(hydroxymethyl)aminomethane U unit(s) a c t i v i t y u. micro ng microgram(s) |il m i c r o l i t e r (s ) uM micromolar concentration u.mol micromole(s) v/v volume by volume % percentage _< less than or equal to >^  greater than or equal to xiv ACKNOWLEDGEMENTS I am very grateful to Dr. John H. McNeill, my supervisor, for h i s patient guidance and continuous support. I wish to thank a l l members of my research committee (Drs. Jack Diamond, David V. Godin, Vladimir Palaty, B a s i l D. Roufogalis and Michael J.A. Walker) for t h e i r i n t e r e s t , constructive c r i t i c i s m s , and valuable suggestions through-out t h i s study. I g r a t e f u l l y acknowledge the f i n a n c i a l support of the Canadian Heart Foundation which made t h i s work possible. I t r u l y appreciate Mrs. Judy Wyne1s e f f o r t i n typing the thesis and preparing the figures. S E C T I O N I N T R O D U C T I O N 2 A. Calcium and Muscle C o n t r a c t i l i t y ; In 1883, Sydney Ringer demonstrated that the frog heart f a i l e d to c o n t r a c t and remained r e l a x e d when calcium was absent from the perfusion f l u i d . Ringer's observation marked the beginning of a new era i n muscle physiology. Thereafter, the task has been to determine the mechanism(s) of calcium regulation of muscle contraction. Locke (Locke and Rosenheim, 1907) reported that follow-i n g removal of perfused calcium, the spontaneous c a r d i a c a c t i o n - c u r r e n t ( e l e c t r i c a l a c t i v i t y ) remains strong long a f t e r the mechanical beat has become minimal. Later, Mines (1913) made simultaneous records of contraction and e l e c t r i -c a l a c t i v i t y i n the frog heart and reported that p e r f u s i o n of the frog heart with a calcium-free s o l u t i o n abolished the mechanical response but had no e f f e c t on the e l e c t r i c a l response. Daly and Clark (1920/1921) confirmed these obser-v a t i o n s and reported that reduction of calcium content i n the p e r f u s i n g medium has l i t t l e e f f e c t upon" the conduction of e l e c t r i c a l impulse i n the v e n t r i c l e (frog h e a r t ) . Later, Weidmann (1955) r e p o r t e d t h a t a f o u r - f o l d decrease or a f o u r - f o l d i n c r e a s e of the c a l c i u m c o n c e n t r a t i o n i n the e x t r a c e l l u l a r f l u i d (Tyrode s o l u t i o n containing 2.6 mM c a l -cium) had no marked e f f e c t on (i ) the siz e and shape of the a c t i o n p o t e n t i a l , ( i i ) the value of the maximal membrane p o t e n t i a l , and ( i i i ) the membrane r e s i s t a n c e i n s i n g l e P urkinje f i b e r s ( C a l f and Sheep), measured by means of an 3 i n t r a c e l l u l a r microelectrode. Ware et a l . (1955) a l s o re-ported that perfusion of i s o l a t e d frog hearts with calcium-f r e e Ringer s o l u t i o n had no s i g n i f i c a n t e f f e c t on the magnitude of r e s t i n g p o t e n t i a l or overshoot, the duration of a c t i o n p o t e n t i a l , or the heart rate, recorded with i n t r a c e l -l u l a r m i c r o e l e c t r o d e s . These obse r v a t i o n s c l e a r l y demon-st r a t e d that the a c t i o n of calcium on cardiac c o n t r a c t i l i t y was not related to an a l t e r a t i o n of i t s e l e c t r i c a l a c t i v i t y . I n h i b i t i o n of c a r d i a c c o n t r a c t i l i t y i n the absence of c a l c i u m w i t h o u t an e f f e c t on e l e c t r i c a l a c t i v i t y a l s o suggested that calcium may be the l i n k between e l e c t r i c a l a c t i v i t y and the mechanical response ( c o n t r a c t i l i t y ) . Over the years, evidence s u p p o r t i n g t h i s h y p o t h e s i s has been obtained from a number of laboratories. Heilbrunn (1940) showed that exposure of cut ends of i s o l a t e d frog s k e l e t a l muscle f i b e r s to c a l c i u m c h l o r i d e s o l u t i o n caused r a p i d shortening of the muscle. Later, Heilbrunn and Wiercinski (1947) showed that d i r e c t i n j e c t i o n , using pressure induced micropipettes, of rather high d i l u t i o n s of calcium ions into the i n t e r i o r of i s o l a t e d muscle f i b e r s of the frog caused an immediate and pronounced shortening of the muscle f i b e r s , and that t h i s e f f e c t was not shared by any one of the other cations normally present i n any quantity i n muscle. In 1955, Niedergerke showed that d i r e c t i n t r a c e l l u l a r i n j e c t i o n of c a l c i u m , from a m i c r o p i p e t t e by e l e c t r o l y t i c t r a n s p o r t , i n t o muscle f i b e r s caused a l o c a l shortening of the muscle. Caldwell and Walster (1963) a l s o reported that m i c r o i n j e c -4 t i o n of calcium into crab muscle f i b e r s caused marked con-t r a c t i o n of the muscle. Calcium i n very low concentrations has a l s o been shown to cause r a p i d c o n t r a c t i o n of muscle f i b e r s washed with g l y c e r o l that made the membrane h i g h l y permeable (Bozler, 1952). These experiments demonstrated that calcium inside the c e l l causes d i r e c t a c t i v a t i o n of the c o n t r a c t i l e elements. In 1952, Sandow showed t h a t f o l l o w i n g e x c i t a t i o n of the f r o g s a r t o r i o u s muscle the a c t i o n p o t e n t i a l preceded the mechanical event. Sandow (1952) introduced the term " 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 " to d e s c r i b e the e n t i r e sequence of r e a c t i o n s b e g i n n i n g w i t h e x c i t a t i o n followed by inward acting l i n k and leading to a c t i v a t i o n of contraction. Niedergerke (1956a) showed that calcium increased the amplitude and rate of r i s e of contracture i n potassium-depolarized s t r i p s of the frog v e n t r i c l e and had no s i g n i f i c a n t e f f e c t on the time course or amount of the depolarization. Frank (1958) showed that the contracture response to d e p o l a r i z a t i o n induced by potassium i n the frog toe muscle was i n h i b i t e d i n a calcium-free solution. These r e s u l t s indicated that the e x t r a c e l l u l a r calcium regulation o f muscle c o n t r a c t i l i t y was beyond the step of membrane depolarization. One obvious explanation of the above findings was that the a c t i o n p o t e n t i a l or membrane d e p o l a r i z a t i o n permits or promotes the entrance of calcium ions from the surface to 5 the i n t e r i o r of the muscle f i b e r and that these ions then i n i t i a t e the mechanical response. Indeed, Heilbrunn (1943) b e l i e v e d that e s s e n t i a l l y a l l types of s t i m u l a t i o n cause a release of calcium ions from the surface or outer region of the c e l l and t h a t t h i s c a l c i u m then enters the c e l l and produces the response (the c a l c i u m - r e l e a s e theory or the c o l l o i d chemical theory). Sandow (1952) proposed that the i n t e n s i t y of a c t i v a t i o n of the muscle would be determined at l e a s t by the amount of c a l c i u m r e l e a s e d by the t r i g g e r a c t i o n of the spike. In an e a r l i e r study, however, Fenn et a l . (1938) observed no s i g n i f i c a n t i n c r e a s e i n c a l c i u m content of the cat l e g muscle f o l l o w i n g a 30 min p e r i o d of stimulation. F a i l u r e of these investigators to detect c a l c i -um mobilization was mainly due to the i n s e n s i t i v e techniques 4 5 used. A p p l i c a t i o n of r a d i o l a b e l l e d calcium ( Ca) i n the l a t e 50's r e s u l t e d i n new developments i n t h i s area. In 1957, Niedergerke and H a r r i s showed t h a t a decrease of sodium ions or omission of potassium ions of Ringer's f l u i d 45 caused an increase i n Ca accumulation by the frog heart. Meanwhile both a decrease i n the buffer's sodium concentra-t i o n (Niedergerke and Luttgau, 1957) and an omission of the b u f f e r ' s potassium (Niedergerke, 1956b) were reported to produce an increase i n the force of contraction developed by the frog heart. Niedergerke (1959) also showed that i n 45 p o t a s s i u m - d e p o l a r i z e d f r o g v e n t r i c l e s Ca uptake was i n c r e a s e d upon r e d u c t i o n or removal of the e x t r a c e l l u l a r sodium. Si m i l a r l y , such alt e r a t i o n s of the e x t r a c e l l u l a r 6 sodium concentration were e a r l i e r found to cause an increase i n the force of contracture developed by the potassium-depo-l a r i z e d muscle (Niedergerke and Luttgau, 1957). Thus, these data i n d i c a t e d that an increased c o n t r a c t i l i t y i s accompa-nied by an increase i n calcium uptake. Similar r e s u l t s were obtained i n s k e l e t a l muscles. B i a n c h i and Shanes (1958 & 45 1959) reported that while p a s s i v e Ca i n f l u x at r e s t i n the s a r t o r i o u s muscle of the f r o g was about equal to what had been reported f o r squid axons, the i n f l u x per impulse was i n c r e a s e d to at l e a s t 30 times gre a t e r than i n nerve f i b e r s . B ianchi and Shanes (1958 & 1959) a l s o showed that the enhanced t w i t c h when n i t r a t e r e p l a c e s c h l o r i d e i n Ringer's f l u i d was associated with at l e a s t a 60% increase 4 5 i n Ca i n f l u x d u r i n g a c t i v i t y , and t h a t c a l c i u m e n t r y during potassium contracture was even more markedly augmen-ted than during e l e c t r i c a l s t i m u l a t i o n and was c o n s i s t e n t with the time course of the contractures. These observations i n d i c a t e d that calcium i n f l u x i s increased during membrane d e p o l a r i z a t i o n and suggested that the strength of contrac-t i o n i s a d i r e c t f u n c t i o n of the calcium entering the c e l l during the impulse. In an i n t e r e s t i n g study, Weidmann (1959) showed that a rapid r i s e of the calcium concentration a f t e r the beginning of an a c t i o n p o t e n t i a l i n p e r f u s e d t u r t l e v e n t r i c l e s caused an immediate increase of the c o n t r a c t i l e strength of the muscle within the period of a s i n g l e beat. Later studies confirmed the above fi n d i n g s . In a comprehen-s i v e study, Winegrad and Shanes (1962) showed a l i n e a r r e l a t i o n s h i p between r e l a t i v e Ca uptake per beat and r e l a t i v e twitch tension developed i n d i f f e r e n t calcium con-ce n t r a t i o n s and at d i f f e r e n t frequencies of s t i m u l a t i o n i n guinea p i g l e f t a t r i a . Niedergerke (1963a) a l s o showed that beating of the heart was accompanied by an a d d i t i o n a l i n f l u x of calcium ions into the muscle f i b e r s and that t h i s i n f l u x increased both when the external calcium was increased and when the e x t e r n a l sodium was reduced, two i o n i c changes which cause the beat to become st r o n g e r . In 1967, Reuter (see also Reuter and Beeler, 1969) showed that calcium ions c a r r y an a p p r e c i a b l e membrane c u r r e n t i n the inward d i r e c t i o n when the membrane of the P u r k i n j e f i b e r was depolarized. B. S u p e r f i c i a l Calcium and Cardiac C o n t r a c t i l i t y : As early as 1883, Ringer's o r i g i n a l observation c l e a r l y demonstrated that upon removal of calcium from the perfusing f l u i d , the c o n t r a c t i l i t y of the f r o g heart d e c l i n e d very r a p i d l y . Indeed, the high s e n s i t i v i t y of the frog heart to the e x 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 was the key f o r Ringer's c o n t r i b u t i o n . In 1928, De reported that p e r f u s i o n of the frog heart with calcium-free Ringer s o l u t i o n produced a very rapid reduction i n the force of the heart beat with a 50% reduction occurring i n less than 3 s . De (1928) also r e p o r t e d t h a t the rate of recovery of the h e a r t beat, i n v e n t r i c l e s nearly a r r e s t e d with calcium-free Ringer s o l u -t i o n , upon reperfusion with normal Ringer solution, was even 8 more r a p i d and h a l f recovery occured w i t h i n 2 s. De a l s o noted that changes i n the frequency of s t i m u l a t i o n d i d not a f f e c t the speed with which calcium lack depressed the ven-t r i c u l a r response. De (1928) stated, quite c o r r e c t l y , that "...the r a p i d i t y at which calcium-free Ringer produces i t s e f f e c t upon the v e n t r i c l e and the e q u a l l y r a p i d recovery when normal Ringer i s s u b s t i t u t e d for calcium-free Ringer both suggest that deprivation of calcium produces i t s chief e f f e c t upon the surface of the heart c e l l s ." In fact, Heilbrunn's c a l c i u m - r e l e a s e theory (1943) suggested that calcium bound to the c e l l membrane was released upon stimu-l a t i o n to i n i t i a t e the protoplasmic response. Even e a r l i e r , Clark (1913/1914) noted that i n c r e a s i n g e x t r a c e l l u l a r c a l -cium concentration greatly improved the force of contraction i n the hypodynamic frog heart and suggested that the func-t i o n of calcium was to cause an a l t e r a t i o n i n the c o l l o i d a l state of the l i p o i d s at the c e l l surface. Niedergerke (1957) compared the rate of incre a s e and decrease of t e n s i o n developed by the f r o g v e n t r i c l e upon a l t e r i n g e x t r a c e l l u l a r calcium concentration with the rate 45 a t which Ca was tak e n up by, or r e l e a s e d from, the t i s s u e . Niedergerke (1957) made two important observations: ( i ) changes i n ten s i o n due to an a l t e r e d e x t e r n a l calcium concentration were f a s t e r than the corresponding net move-ments of calcium between the t i s s u e and surrounding medium, and ( i i ) the time course of the tension changes could be f i t t e d by an eq u a t i o n d e s c r i b i n g the d i f f u s i o n p r o c e s s 9 through the e x t r a c e l l u l a r t i s s u e space, but the apparent d i f f u s i o n c o e f f i c i e n t o b t a i n e d i n t h i s way was 4 times s m a l l e r than what would be expected f o r the i n t e r s t i t i a l f l u i d ( i . e . the rate of tension change was slower than the expected rate of calcium d i f f u s i o n through the i n t e r s t i t i a l f l u i d ) . Niedergerke (1957) concluded that i t i s p o s s i b l e to e x p l a i n both f i n d i n g s on the assumption t h a t a c e r t a i n quantity of calcium i s taken up r e v e r s i b l y by a s u p e r f i c i a l layer of the heart tissue while the tension changes. This meant that calcium taken up by a mobile depot located at a s u p e r f i c i a l s i t e , which i s i n r a p i d e q u i l i b r i u m with the i n t e r s t i t i a l free calcium, regulates tension development i n the heart muscle. In the study of sodium and calcium antagonism, Nieder-gerke and Luttgau (1957) proposed that calcium and sodium ions compete f o r an anionic group R (assumed for s i m p l i c i t y t o be d i v a l e n t ) , l o c a t e d presumably on the c e l l s u r face, where CaR i s the compound which a c t i v a t e s c o n t r a c t i o n whereas Na£R i s i n a c t i v e (see a l s o Luttgau and Niederger-ke, 1958). In a l a t e r study, Niedergerke (1963b) presented a more complete model for calcium movement i n the heart, based on the previous model, which proposed that inward movement of calcium i n combination with the c a r r i e r molecule R was governed by a p e r m e a b i l i t y constant which i s dependent on the magnitude of the membrane p o t e n t i a l . 4 5 Winegrad and Shanes (1962) noted t h a t Ca r e l e a s e 10 from guinea p i g l e f t a t r i a p r e v i o u s l y l o a d e d w i t h the isotope can be divided i n t o three components: ( i ) a r a p i d l y exchangeable f r a c t i o n with a h a l f - t i m e of 4.5 min; ( i i ) a slowly exchangeable f r a c t i o n with a half-time of 86 min; and ( i i i ) an inexchangeable f r a c t i o n . They a l s o found t h a t 14 [ C]-sucrose r e l e a s e from the a t r i a had two e x p o n e n t i a l components with half-times of 2.9 and 54 min, and that the f a s t e r component i n c l u d e d approximately 80% o f the t o t a l t i s s u e sucrose. Considering the d i f f e r e n c e i n the d i f f u s i o n c o e f f i c i e n t s of calcium and sucrose i n water, the calculated half-time for washout of free calcium from the e x t r a c e l l u l a r f l u i d was approximately 31% of the a c t u a l h a l f - t i m e of the 4 5 f a s t component of the Ca washout (4.5 min). Winegrad and Shanes (1962) concluded that the s u b s t a n t i a l l y longer h a l f -45 time a c t u a l l y obtained f o r Ca suggests that an a d d i t i o n -a l step besides d i f f u s i o n into the e x t r a c e l l u l a r space, such as binding to surface s i t e s , was involved. In a b r i e f review of the l i t e r a t u r e at the time, Winegrad (1961) proposed that the calcium that enters the c e l l with contraction comes from s u p e r f i c i a l s i t e s and from the e x t r a c e l l u l a r f l u i d , and the calcium that enters the c e l l from the e x t r a c e l l u l a r f l u i d d u r ing d e p o l a r i z a t i o n passes through the same s u p e r f i c i a l s i t e s to which calcium was bound i n the r e s t i n g state. 45 Grossman and Furchgott (1964a, b) reported that Ca uptake i n t o i s o l a t e d guinea p i g l e f t a u r i c l e s took place i n at l e a s t two phases: an i n i t i a l rapid phase of exchange that appeared to be complete i n about 5 min and a slower phase of Ca exchange. The uptake of Ca d u r i n g the i n i t i a l phase of exchange was dependent on the frequency of contrac-t i o n . Grossman and Furchgott (1964b) proposed that the rapid phase of calcium exchange was associated with c o n t r a c t i o n . 45 In a l a t e r study, T e i g e r and Farah (1967) s t u d i e d Ca uptake and o u t f l o w and t o t a l t i s s u e c a l c i u m c o n t e n t i n i s o l a t e d rabbit l e f t a t r i a under varying conditions of stim-u l a t i o n and varying external calcium, sodium and potassium concentrations. Teiger and Farah (1967) also found three t i s s u e - c a l c i u m compartments i n both r e s t i n g and stimulated rabbit l e f t a t r i a : (i) an unexchangeable tissue-calcium compartment; ( i i ) a rapidly exchangeable tissue-calcium compartment; and ( i i i ) a slowly exchangeable tissue-calcium compartment. The rate of exchange of the rapidly exchange-able compartment was increased by stimulation of the atrium at various r a t e s . The h a l f - t i m e s of calcium movements int o and out of the r a p i d l y exchangeable compartment were s u f f i -c i e n t l y rapid to account for the speed with which changes i n the e x t e r n a l c a l c i u m c o n c e n t r a t i o n a l t e r c o n t r a c t i l i t y . Teiger and Farah (1967) proposed that the concentration of calcium i n the r a p i d l y exchangeable compartment i s a deter-mining factor of c o n t r a c t i l e force. In an i n t e r e s t i n g study, DeCaro (1967) noted two phases of twitch-tension decline i n r a b b i t l e f t a r i a bathed i n a low c a l c i u m s o l u t i o n t h a t 45 c o r r e l a t e d with two components of Ca r e l e a s e from the t i s s u e . DeCaro (1967) suggested that the i n i t i a l rapid loss of c o n t r a c t i o n was probably caused by a release of calcium 12 from s u p e r f i c i a l b i n d i n g s i t e s , whereas the slow l i n e a r decrease i n t e n s i o n was an e f f e c t of the slow r e l e a s e of calcium from the i n t r a c e l l u l a r compartments. In a comprehensive study, B a i l e y and D r e s e l (1968) i n v e s t i g a t e d both the k i n e t i c s of calcium r e l e a s e and the de c l i n e of isometric c o n t r a c t i l e force (simultaneously) i n i s o l a t e d cat hearts perfused with zero-calcium Krebs s o l u -t i o n . Compartmental a n a l y s i s of the calcium washout curves was c h a r a c t e r i s t i c of a three compartment system. The f i r s t compartment (Ca^. ) had a s h o r t h a l f - t i m e (<5 s) t h a t was 125 c l o s e to the mean ha l f - t i m e for I-serum albumin washout (6.2 s ) . The means of the half - t i m e s for decay of contrac-t i l i t y and washout of calcium from the second compartment (Ca^) were 45.4 s and 43.2 s, respectively. The rate of calcium washout from C a - j - j w a s l i n e a r l y r e l a t e d to the rate of decay of c o n t r a c t i l e force (r = 0.79; p<0.001). Further-more, c o n t r a c t i l e f o r c e developed by hearts a t d i f f e r e n t calcium concentrations was l i n e a r l y related to the logarithm of calcium content i n C a i : i c a l c u l a t e d from the r e s u l t s of compartmental analysis (r = 0.77; p<0.001). Bailey and D r e s e l (1968) concluded that calcium content i n C a ^ was d i r e c t l y associated with cardiac muscle contraction. Bailey and Downie (1970) showed that an increase i n heart rate from 60 to 120/min caused a s i g n i f i c a n t increase i n the uptake of calcium into Ca^* They also showed that the half-times f o r the washout of Ca-j-j were r e l a t e d d i r e c t l y to the heart rate. Bailey et a l . (1972) proposed that calcium taken up 13 from the v a s c u l a r space (Ca^) was t e m p o r a r i l y s t o r e d i n Ca^j and released by depolarization of the c e l l membrane. 45 Langer and c o l l a b o r a t o r s s t u d i e d Ca exchange i n a r t e r i a l l y p e r f u s e d dog p a p i l l a r y muscle and a r t e r i a l l y perfused r a b b i t i n t e r v e n t r i c u l a r septum using a s e l e c t i v e l y developed technique that enabled them to make a sequential and simultaneous c o r r e l a t i o n of i o n i c f l u x with muscle function. Langer and Brady (1963) reported that calcium exchange i n dog p a p i l l a r y muscles demonstrated a r a p i d l y e q u i l i b r a t i n g phase (half-time = 4-6 min) and a more slowly 45 e q u i l i b r a t i n g phase. Later, Langer (1964) divided Ca exchange i n dog p a p i l l a r y muscles i n t o f i v e k i n e t i c a l l y defined phases (0 to 4). Shelburne, Serena, and Langer 4 5 (1967) found f o u r k i n e t i c a l l y d e f i n e d phases o f Ca exchange (0-3) i n a r t e r i a l l y perfused r a b b i t i n t e r v e n t r i c -45 u l a r septum. Shine, Serena and Langer (1971) studied Ca exchange i n i s o l a t e d blood-perfused i n t e r v e n t r i c u l a r septum. 4 5 They r e p o r t e d t h a t phase 1 o f Ca washout had a mean half-time of 1.98 min and the calcium content of phase 1 was l i n e a r l y r e l a t e d to calcium concentration i n the perfusate (r = 0.99). Therefore k i n e t i c considerations suggested that phase 1 was predominantly representative of calcium i n the i n t e r s t i t i u m . P e r f u s i o n of the septum with c a l c i u m - f r e e perfusate caused a rapid decline of maximal dP/dt as fa s t or 45 f a s t e r than the steady s t a t e l o s s of Ca from phase 1, and mechanical function was returned at a s i m i l a r rate when calcium was restored (Shine et a l . , 1971). Shine et a l . 14 (1971) concluded t h a t c o n t r a c t i l e c a l c i u m o r i g i n a t e s i n phase 1 and suggested that calcium released to the myofila-ments was derived from s i t e s i n r a p i d e q u i l i b r i u m with the i n t e r s t i t i u m . On the other hand, phase 2 had a half-time of about 5-6 min and was considered to represent predominantly the sarcoplasmic reticulum calcium. An increase i n force of c o n t r a c t i o n produced by a decrease i n e x t r a c e l l u l a r sodium c o n c e n t r a t i o n (Langer, 1965), and a f a l l i n temperature (Shelburne et a l . , 1967) were found to be accompanied with an increase i n phase 2 calcium content. Shine et a l . (1971) proposed that phase 2 calcium content was an index of the amount of c o n t r a c t i l e calcium which has reached the myofila-ments fom phase 1 and has been subsequently sequestered. B r i e f l y , studies from d i f f e r e n t l a b o r a t o r i e s c i t e d i n t h i s section show that k i n e t i c a n a l y s i s of calcium exchange r e v e a l s a r a p i d l y exchanging c a l c i u m p o o l whose r a t e of d e p l e t i o n i n a calcium-free medium i s the same as the rate of d e c l i n e of c o n t r a c t i l e force, and whose calcium content i s related to the c o n t r a c t i l e force developed by the cardiac muscle. This pool of calcium i s neither the free calcium i n the e x t r a c e l l u l a r space nor c a l c i u m of an i n t r a c e l l u l a r o r i g i n . K i n e t i c a l l y t h i s pool i s very l i k e l y to be located at a s u p e r f i c i a l s i t e . 15 C. E f f e c t of Lanthanum on Cardiac Calcium Metabolism and  Function; In 1910, Mines reported that a Ringer s o l u t i o n contain-ing 10 (iM lanthanum, yttrium or cerium produced an immediate e f f e c t on the f r o g h e a r t , r e d u c i n g s y s t o l e and u s u a l l y stopping the heart i n d i a s t o l e within a few minutes. He also reported that i n one experiment the heart beat was i n s t a n t l y reduced by a concentration of only 0.5 uM lanthanum. Mines (1910) a l s o s t u d i e d the e f f e c t of the lanthanides on the permeability of a r t i f i c i a l membranes to sodium c h l o r i d e . He reported that the behaviour of an a r t i f i c i a l membrane was p r o f o u n d l y m o d i f i e d by treatment with t r i v a l e n t metals. Mines (1910) suggested that the explanation of the action of the t r i v a l e n t p o s i t i v e ions i s to be sought i n the fact that these ions have i n a pre-eminent degree the power of reduc-ing or reversing the negative charges on surfaces. Lanthanum i s the most e l e c t r o p o s i t i v e element of the rare earth group, or lanthanide s e r i e s of elements, and has chemical p r o p e r t i e s most s i m i l a r t o the a l k a l i n e earths (Levy, 1924). In 1964, L e t t v i n and co-workers predicted that lanthanum, by v i r t u e of having an i o n i c radius s i m i l a r to calcium and having a higher valence than calcium, would bind to the membrane calcium binding s i t e s and would bind more s t r o n g l y than calcium. They a l s o p r e d i c t e d that lanthanum would act s i m i l a r l y to calcium i n a f f e c t i n g c a t i o n i c conduc-tance through nerve membranes and t h a t lanthanum should function as a nerve-blocking agent. Takata, Pickard, L e t t v i n 16 and Moore (1966) tested t h i s hypothesis and found that 11 mM lanthanum markedly reduced both sodium and potassium con-ductance i n l o b s t e r axon bathed i n calcium-free sea water. Since as high as 210 mM calcium would have been required to d u p l i c a t e the e f f e c t s of lanthanum, they concluded t h a t lanthanum acted l i k e a "supercalcium". On the other hand, Takenaka and Yumoto (1968) suggested that a small amount of t r i v a l e n t ions would replace divalent cations i n maintaining membrane e x c i t a b i l i t y . They showed that lanthanum concentra-t i o n s between 0.5 and 3 mM maintained the e x c i t a b i l i t y of c r a y f i s h giant axons perfused with calcium-free sea water. Lanthanum was l a t e r shown to i n h i b i t c a l c i u m f l u x e s across membranes. Van Breemen and van Breemen (1969) showed 45 t h a t 0.5 mM lanthanum completely b l o c k e d Ca t r a n s p o r t a c r o s s p h o s p h o l i p i d - c h o l e s t e r o l a r t i f i c i a l membranes. Lanthanum was a l s o shown to block c a l c i u m f l u x e s across m i t o c h o n d r i a l membranes (Mela, 1968), i n t e s t i n a l smooth muscles (Weiss and Goodman, 1969), s q u i d axolemma (van Breemen and de Weer, 1970), v a s c u l a r smooth muscles (van Breemen, 1969; van Breemen and McNaughton, 1970), and heart (Palmer and van Breemen, 1970). Sanborn and Langer (1970) reported t h a t p e r f u s i o n of r a b b i t i n t e r v e n t r i c u l a r septal t i s s u e with 5-40 nM lanthanum i n 2.5 mM c a l c i u m p e r f u s a t e produced a r a p i d d e c l i n e of a c t i v e tension to a l e s s e r steady-state value. They showed t h a t p e r f u s i o n of r a b b i t p a p i l l a r y muscles w i t h 10 M^ 17 lanthanum reduced s y s t o l i c tension by 38%, had no e f f e c t on d i a s t o l i c t ension and time to peak tensi o n , increased the duration of a c t i o n p o t e n t i a l by 15.9%, and had no e f f e c t on the amplitude of membrane depolarization. They also reported t h a t c e l l u l a r a c t i o n p o t e n t i a l s r e c o r d e d d u r i n g b r i e f p e r f u s i o n with lanthanum (5-40 uM) demonstrated e s s e n t i a l l y 45 normal regenerative d e p o l a r i z a t i o n . Perfusion of Ca-pre-l a b e l l e d i n t e r v e n t r i c u l a r septa w i t h 5-20 uM lanthanum during the early phase of washout (5-10 min) with unlabelled calcium caused a rapid concentration-dependent displacement 45 of Ca from the t i s s u e and a concomitant d e c r e a s e of developed tension. I f the time of exposure to lanthanum was 4 5 d e l a y e d , the amount of Ca d i s p l a c e d by lanthanum was 45 reduced. When septa p r e l a b e l l e d with Ca were p e r f u s e d w i t h a z e r o - c a l c i u m s o l u t i o n f o r 7 min, which caused a decline of tension to zero, lanthanum (20 uM) had no e f f e c t 45 on Ca r e l e a s e d from the t i s s u e . Furthermore, c a l c i u m caused an increase i n c o n t r a c t i l e force and released lantha-140 num from t i s s u e p r e l a b e l l e d with La. Sanborn and Langer (1970) concluded t h a t c o n t r a c t i l e dependent c a l c i u m was d e r i v e d p r i m a r i l y from " s u p e r f i c i a l l y " l o c a t e d s i t e s and that lanthanum affected the release of c o n t r a c t i l e dependent c a l c i u m by modifying the normal p e r m s e l e c t i v i t y of t h i s " s u p e r f i c i a l " membrane for activator calcium. Ong and B a i l e y (1972) studied the e f f e c t of lanthanum on calcium uptake i n calcium-depleted k i t t e n hearts.Compart-mental analysis of calcium uptake by calcium-depleted k i t t e n 18 h e a r t s upon r e p e r f u s i o n with a 2.5 mM c a l c i u m s o l u t i o n y i e l d e d two compartments of calcium uptake, Ca^ and Ca2« The f i r s t compartment (Ca^) had a mean h a l f - t i m e of 5.6 s, was f i l l e d to capacity early i n reperfusion, and the amount of calcium accumulation by t h i s compartment was not rela t e d to the return of c o n t r a c t i l e force. The second compartment (Ca.^) had a mean h a l f - t i m e of 46.2 s and the r e s t o r a t i o n of c o n t r a c t i l e force was d i r e c t l y r e l a t e d to the quantity of calcium accumulated by t h i s compartment (r = 0.93; p<0.01). Hence, Ong and B a i l e y (1972) suggested t h a t the ca l c i u m contained i n C a 2 was d i r e c t l y r e l a t e d to the maintenance of c o n t r a c t i l e force i n the heart. In sharp contrast, when the reperfusion f l u i d contained 5 u-M lanthanum i n a d d i t i o n to the normal c o n c e n t r a t i o n of calcium, c o n t r a c t i l e force was not restored and the calcium uptake curve could only be resolved i n t o a s i n g l e compartment. The mean ha l f - t i m e for calcium uptake (33.4 s) i n lanthanum-treated hearts did not d i f f e r s i g n i f i c a n t l y (p<0.05) from the mean h a l f - t i m e f o r the uptake of c a l c i u m by Caj i n the h e a r t s not t r e a t e d with lanthanum. The q u a n t i t y of c a l c i u m taken up by Ca^ d u r i n g r e p e r f u s i o n i n the presence of 5 u-M lanthanum was s i g n i f i c a n t l y greater (p<0.05) than the quantity of calcium taken up by C a 2 i n c o n t r o l h e a r t s , but was not s i g n i f i -c a n t l y d i f f e r e n t (p>0.05) from the t o t a l quantity of calcium t a k e n up by the c o n t r o l h e a r t . Ong and B a i l e y (1972) concluded that 5 uM lanthanum blocked the uptake of calcium by Ca, b u t n o t by C a . and t h a t Ca, was a b s o l u t e l y 19 es s e n t i a l to the coupling process i n cardiac muscle. In a l a t e r study, V i l l a n i et a l . (1976) s t u d i e d the 45 e f f e c t of 50 |j.M lanthanum on Ca exchange i n guinea p i g r i g h t a t r i a . They r e p o r t e d t h a t 50 \iM lanthanum had no e f f e c t on t o t a l t i s s u e calcium content and the slowly ex-45 changing phase of Ca, but s i g n i f i c a n t l y decreased the rate of the e a r l y phase of f a s t exchange. They a l s o showed that 5 and 50 M^ lanthanum had no e f f e c t on oxygen consump-t i o n by the muscle. V i l l a n i et a l . (1976) concluded that the f a s t exchanging compartment of calcium i s c o r r e l a t e d to the c o n t r a c t i l e force developed by the heart muscle. In c u l t u r e d monolayers of neonatal r a t h e a r t c e l l s , 0.5 mM lanthanum abo l i s h e d both calcium i n f l u x and e f f l u x (Langer and Frank, 1972). Exchangeable c a l c i u m i n these c e l l s accounted f o r 75% of t o t a l t i s s u e c a l c i u m and was k i n e t i c a l l y divided i n t o two defined phases with half-times of 1.15 min and 19.2 min. Lanthanum (0.5 mM) displaced about 50% of the r a p i d l y exchangeable phase. In the presence of 45 lanthanum, the washout of Ca from p r e l a b e l l e d t i s s u e was converted to a s i n g l e phase system with a h a l f - t i m e of 124 min. These e f f e c t s upon calcium exchange were c o i n c i d e n t w i t h a b o l i t i o n of c o n t r a c t i l e t e n s i o n and l o s s of the p l a t e a u phase of a c t i o n p o t e n t i a l , but regenerative depo-l a r i z a t i o n of tissue was maintained. Lanthanum has a l s o been shown t o i n h i b i t the slow inward calcium c u r r e n t . Katzung, Reuter and Por z i g (1973) 20 reported that p e r f u s i o n of pig v e n t r i c u l a r t r a b e c u l a with 0.4 mM lanthanum i n h i b i t e d the plateau phase of the a c t i o n p o t e n t i a l and diminished the amplitude of the slow inward calcium current. They also reported that lanthanum i n h i b i t e d the calcium-dependent regenerative depolarization i n sodium-45 f r e e s o l u t i o n and i n h i b i t e d Ca uptake by the t i s s u e . Shigenobu and Sperelakis (1972) reported that 1 mM lanthanum i n h i b i t e d the catecholamine-induced slow e l e c t r i c a l respon-ses i n i s o l a t e d v e n t r i c l e s of 9-19 day o l d chick embryonic h e a r t s made i n e x c i t a b l e by i n a c t i v a t i n g the f a s t sodium channels with e i t h e r t e t r o d o t o x i n or e l e v a t e d potassium. Kass and Tsien (1975) reported that 0.5 mM lanthanum abol-ished the slow responses i n Purkinje f i b e r s of c a l f hearts d e p o l a r i z e d t o about -50 mV by a p p l y i n g outward c u r r e n t through a microelectrode bar. They also reported that 0.5 mM lanthanum lowered the plateau phase of the action p o t e n t i a l , d e creased a c t i o n p o t e n t i a l d u r a t i o n , and steepened the pacemaker d e p o l a r i z a t i o n . S i m i l a r treatment with lanthanum completely suppressed the slow inward current i n i t i a t e d by applying standard depolarizing clamp pulses. B e l a r d i n e l l i et a l . (1979) r e p o r t e d t h a t 0.4 mM lanthanum reduced the — 6 a m p l i t u d e of slow a c t i o n p o t e n t i a l s i n d u c e d by 10 M i s o p r o t e r e n o l i n i s o l a t e d r a t a t r i a d e p o l a r i z e d by 25 mM potassium. They a l s o showed that the i n h i b i t o r y e f f e c t of lanthanum was enhanced by i n c r e a s i n g the f r e q u e n c y of s t i m u l a t i o n and was reduced by i n c r e a s i n g the calcium con-cent r a t i o n up to 5 mM. B e l a r d i n e l l i et a l . (1979) concluded 21 t h a t lanthanum may a c t by competing w i t h c a l c i u m f o r membrane binding s i t e s and that these membrane binding s i t e s appear to be characterized by frequency dependence. There i s , however, l i t t l e agreement regarding the e f f e c t + 2 + of lanthanum on Na -Ca exchange. Katzung, Reuter and P o r z i g (1973) r e p o r t e d t h a t lanthanum had no e f f e c t on + 2 + Na -Ca exchange, but i n h i b i t e d the slow inward calcium current i n cardiac muscle. They showed that 0.2 mM lanthanum had no e f f e c t on the sodium and calcium s e n s i t i v e f r a c t i o n 45 of Ca e f f l u x from pr e l o a d e d guinea p i g l e f t a t r i a . In l o n g i t u d i n a l smooth muscles of the guinea p i g ileum, Burton and Godfraind (1974) also reported that 10 mM lanthanum had 4 5 no e f f e c t on the f r a c t i o n of Ca uptake induced by an i n c r e a s e d i n t r a c e l l u l a r sodium. Coraboeuf et a l . (1981) reported that i n i s o l a t e d dog Purkinje f i b e r s bathed i n a -4 potassium-free medium or i n the presence of 10 M ouabain t o depress the e l e c t r o g e n i c sodium pump a c t i v i t y , 0.4 mM lanthanum d i d not suppress the h y p e r p o l a r i z a t i o n and the c o n t r a c t u r e developed i n a so d i u m - f r e e medium. On the contrary, the sodium-free contracture, which was re l a t e d to + 2 + th e f u n c t i o n i n g of an e l e c t r o g e n i c Na -Ca exchange mechanism, was generally increased i n the presence of la n -thanum. In contrast, Horackova and Vassort (1979) reported that 3 mM lanthanum i n h i b i t e d the development of the sodium-fre e contracture and the r e l a t e d part of h y p e r p o l a r i z a t i o n or the outward current i n frog a t r i a l muscle under voltage-clamp c o n d i t i o n s . On the other hand, Hatae (1982) showed 22 t h a t 0.2 mM lanthanum abolished t w i t c h t e n s i o n but had no e f f e c t on potassium-contracture ( 1 0 0 mM) i n frog v e n t r i c u l a r muscle. In membrane v e s i c l e s i s o l a t e d from ra b b i t v e n t r i c u -l a r t i s s u e , lanthanum i n h i b i t e d both sodium-dependent uptake and e f f l u x of calcium (Reeves and Sutko, 1 9 7 9 ) . About 0.1 mM lanthanum produced a 5 0 % i n h i b i t i o n ( I C ^ Q ) o f sodium-stimulated calcium uptake. Trosper and Ph i l i p s o n ( 1 9 8 3 ) also r e p o r t e d t h a t lanthanum i n h i b i t e d both sodium-dependent cal c i u m uptake and r e l e a s e i n canine c a r d i a c sarcolemmal v e s i c l e s . The I C C . f o r lanthanum i n h i b i t i o n of the sodium-dependent calcium uptake was about 1 |j.M, and 20 \M lanthanum c o m p l e t e l y b l o c k e d the sodium-induced c a l c i u m r e l e a s e . However, very low concentrations of lanthanum ( 0 . 1 p.M) and o t h e r t r i v a l e n t c a t i o n s ( 0 . 1 - 1 \iM) produced up to 2 0 % stimulation of the sodium-dependent calcium uptake. D. U l t r a s t r u c t u r a l L o c a l i z a t i o n of Lanthanum i n the Heart: The e l e c t r o n dense n a t u r e of lanthanum made i t s detection by electron microscopic examination possible. Such studies revealed the u l t r a s t r u c t u r a l l o c a l i z a t i o n of lantha-num i n the t i s s u e . Revel and Karnovsky ( 1 9 6 7 ) reported that sections cut from t i s s u e blocks of mouse heart treated with c o l l o i d a l lanthanum before dehydration and embedding showed lanthanum deposition, a material of high e l e c t r o n opacity, i n the e x t r a c e l l u l a r space, the basement lamina surrounding the muscle c e l l s , p i n o c y t o t i c v e s i c l e s opening onto the sur-face of the muscle or i n c a p i l l a r i e s , the large transverse 23 tubules c h a r a c t e r i s t i c of heart muscle, the i n t e r c e l l u l a r space, and i n t e r c a l a t e d d i s c s . They a l s o r e p o r t e d t h a t lanthanum did not penetrate i n t o c e l l s except i n rare cases where there was a rupture of the plasma membrane. Martinez-Palomo et a l . (1973) studied the u l t r a s t r u c t u -r a l l o c a l i z a t i o n of io n i c lanthanum i n l i v i n g cardiac c e l l s . They reported that perfusion of beating fa l s e tendons of the dog heart with 5 mM lanthanum for 1 h re s u l t e d i n lanthanum deposits only i n the surface membrane of myocardial c e l l s and i n the e x t r a c e l l u l a r space. No p r e c i p i t a t e s were found at the plasma membrane of f i b r o b l a s t s , e n d o t h e l i a l c e l l s , Schwann c e l l s , unmyelinated axons, or a r t e r i o l a r smooth muscle c e l l s found i n the same specimens. Lanthanum deposits were r e s t r i c t e d to the cytoplasmic l e a f l e t s of the sarcolem-ma. Lanthanum deposits were found i n gap junctions and i n p i n o c y t o t i c v e s i c l e s . A s e l e c t i v e d e p o s i t i o n of lanthanum was a l s o observed i n the sarcolemma of a t r i a l and v e n t r i c -u l a r c e l l s of dog, r a b b i t and cat h e a r t s . In v e n t r i c u l a r c a r d i a c c e l l s lanthanum deposits o u t l i n e d , i n a d d i t i o n to the sarcolemma, the membrane of the t r a n s v e r s e t u b u l a r system (T system), but no dep o s i t s were found w i t h i n the terminal c i s t e r n a e or i n the membranes of the sarcoplasmic reticulum. The u l t r a s t r u c t u r a l membrane deposits produced by lanthanum disappeared when the specimens were subsequently perfused with phosphate-containing Tyrode s o l u t i o n . They a l s o reported that i n t r a c e l l u l a r lanthanum d e p o s i t s were pr e s e n t only i n blocks from specimens p e r f u s e d f o r 9 h, 24 which, i n a d d i t i o n , showed morphological evidence of c e l l damage. Martinez-Palomo et a l . (1973) concluded that t h e i r r e s u l t s tend to demonstrate that a d i s t i n c t i v e feature of the sarcolemma of mammalian cardiac c e l l s i s the presence of regions with a high s u r f a c e d e n s i t y of b i n d i n g s i t e s f o r polyvalent cations. In adult r a b b i t septum perfused with 1-5 mM lanthanum f o r 20 min to 2 h, Frank et a l . (1977) r e p o r t e d t h a t an electron dense p r e c i p i t a t e was found covering the myocardial membrane complex which included the outermost l e a f l e t of the l i p i d b i l a y e r plus the surface coat and the external lamina. Lanthanum was r o u t i n e l y found l i n i n g the p i n o c y t o t i c v e s i -c l e s and the transverse tubules. However, lanthanum was not found within the cytoplasm of i n t a c t t i s s u e even when c e l l s were exposed to t h i s c a t i o n f o r 2 h (Frank et a l . , 1977). The surfaces of endothelial c e l l s were also stained intense-l y with lanthanum. In k i t t e n hearts perfused with Hepes b u f f e r containing 500 \xM lanthanum f o r 30 s, B a i l e y and Ong (1974) and Ong (1972) reported that the e l e c t r o n dense granules of lantha-num were d i s t r i b u t e d along the inner surface of the c a p i l -l a r y endothelium, along the outer s u r f a c e of the muscle membrane, and the i n t e r c e l l u l a r spaces of the c a p i l l a r y endothelium. Lanthanum granules appeared to be l i n i n g the basement membranes. They also reported that lanthanum was r e s t r i c t e d to the e x t r a c e l l u l a r space. 25 In monolayer c e l l c u l t u r e s d e r i v e d from neonatal r a t heart t r e a t e d with 1 mM LaCl^ f o r 5 min, Langer and Frank (1972) r e p o r t e d t h a t lanthanum was found to bind to the surface of the developing myocardial c e l l s and also to pene-t r a t e the interspace of the i n t e r c a l a t e d d i s c . In addition, lanthanum was present on the s u r f a c e of the f i b r o b l a s t s present i n the t i s s u e c u l t u r e . Lanthanum was e x c l u s i v e l y l o c a l i z e d i n the external lamina or basement membrane of the c e l l s . Langer and Frank (1972) a l s o reported that i n over 500 c e l l s examined, lanthanum d e n s i t i e s were never found i n the i n t e r i o r of the myoblast or f i b r o b l a s t c e l l s . Langer and Frank (1972) concluded that negatively charged s i t e s i n the basement membrane play a c r u c i a l r o l e i n the e x c i t a t i o n -contraction coupling process i n heart muscle. S i n c e lanthanum was found t o be r e s t r i c t e d to the e x t r a c e l l u l a r space and was not v i s u a l i z e d u l t r a s t r u c t u r a l l y w i t h i n i n t a c t c a r d i a c muscle c e l l s , p a t h o l o g i s t s u t i l i z e d lanthanum as a cytochemical marker of p a t h o p h y s i o l o g i c a l a l t e r a t i o n s i n i n j u r e d myocardium. Thus, i n t r a c e l l u l a r lanthanum was used as a marker f o r plasma membrane i n j u r y . H o f f s t e i n et a l . (1975) studied c o l l o i d a l lanthanum staining biopsies obtained from i n f a r c t e d , marginal and normal areas of hearts 3.5 h a f t e r ischemia was produced i n anesthetized c l o s e d - c h e s t dogs. Ischemia was induced by e l e c t r i c a l l y -induced thrombosis of the l e f t a n t e r i o r descending coronary a r t e r y . In normal c o n t r o l t i s s u e lanthanum was confined to the e x t r a c e l l u l a r spaces, i n c l u d i n g basement membranes, 26 gap junctions and portions, of the i n t e r c a l a t e d d i s c s . A l l specimens taken from near the center of frank i n f a r c t i o n s contained i n t r a c e l l u l a r as well as e x t r a c e l l u l a r lanthanum. I n t r a c e l l u l a r lanthanum was seen evenly d i s t r i b u t e d around l i p i d d r o p l e t s and i n f o c a l deposits around mitochondria. Only when mitochondria were d i s r u p t e d d i d lanthanum gain access to i n t e r n a l s i t e s on mitochondrial membranes. Few of the marginal c e l l s t h a t appeared m o r p h o l o g i c a l l y normal contained i n t r a c e l l u l a r lanthanum. Thus, H o f f s t e i n et a l . (1975) concluded that ischemic i n j u r y a f f e c t s the permeabil-i t y properties of the plasma membrane independently of other i n t r a c e l l u l a r morphologic changes and that lanthanum can be a s e n s i t i v e i n d i c a t o r of such an a l t e r a t i o n i n membrane permeability. Burton et a l . (1977) studied the r e l a t i o n s h i p between the evolution of i r r e v e r s i b l e myocardial i n j u r y induced by hypoxia i n an i s o l a t e d p a p i l l a r y muscle preparation and the development of p a t h o p h y s i o l o g i c a l a l t e r a t i o n s r e l a t e d to s e v e r e l y impaired membrane f u n c t i o n . They employed i o n i c lanthanum as a cytochemical marker to monitor the progres-s i o n of c e l l u l a r i n j u r y and c o r r e l a t e d the da t a w i t h u l t r a s t r u c t u r a l changes and measurements of c o n t r a c t i l e parameters. Examination by t r a n s m i s s i o n and a n a l y t i c a l e l e c t r o n microscopy (energy d i s p e r s i v e X-ray microanalysis) revealed lanthanum deposition only i n e x t r a c e l l u l a r regions of c o n t r o l muscles and muscles subjected to 30 min hypoxia. A f t e r 1-2 h of h y p o x i a , most muscle c e l l s had a w e l l 27 preserved u l t r a s t r u c t u r e and e x h i b i t e d minimal u l t r a s t r u c -t u r a l evidence of i n j u r y . These muscle c e l l s , however, t y p i c a l l y showed variable degrees of cytoplasmic and i n t r a -mitochondria1 lanthanum d e p o s i t i o n , but had s t r u c t u r a l l y i n t a c t plasma and m i t o c h o n d r i a l membranes. Muscles which were reoxygenated a f t e r 1 h and 15 min of hypoxia showed improved u l t r a s t r u c t u r e and d i d not e x h i b i t i n t r a c e l l u l a r lanthanum d e p o s i t s upon exposure t o lanthanum during the r e o x y g e n a t i o n p e r i o d . A f t e r 2-3 h of hypoxia, abnormal i n t r a c e l l u l a r lanthanum accumulation was a s s o c i a t e d with u 1 t r a s t r u c t u r a 1 evidence of severe muscle i n j u r y which pe r s i s t e d a f t e r reoxygenation. Burton et al.(1977) concluded t h a t the c e l l u l a r membrane a l t e r a t i o n s r e s p o n s i b l e f o r abnormal i n t r a c e l l u l a r lanthanum d e p o s i t i o n preceded the development of i r r e v e r s i b l e i n j u r y but evolved at a t r a n s i -t i o n a l stage i n the progression from r e v e r s i b l e to i r r e v e r s -i b l e i n j u r y induced by hypoxia i n i s o l a t e d f e l i n e p a p i l l a r y muscles. Thus, these data c l e a r l y i n d i c a t e that i n i n t a c t myo-c a r d i a l t i s s u e lanthanum i s r e s t r i c t e d to the e x t r a c e l l u l a r space. Furthermore, these data s t r o n g l y argue against the p r o p o s a l t h a t lanthanum i s taken up i n t r a c e 1 1 u l a r l y by i n t a c t m y o c a r d i a l c e l l s and suggest t h a t i n t r a c e l l u l a r l o c a l i z a t i o n of lanthanum reported by some i n v e s t i g a t o r s (see e.g. Weihe et a l . , 1977) may have been due to p a r t i a l hypoxia developed during tissue preparation. 28 E. Cardiac Sarcolemmal Calcium Binding: Although i t has long been b e l i e v e d that calcium binds to c e l l membranes (see Heilbrunn, 1943), d i r e c t evidence for such a calcium binding i n s k e l e t a l muscle c e l l membrane was f i r s t reported by Koketsu et a l . i n 1964. These i n v e s t i -45 g a t o r s showed t h a t Ca was bound t o i s o l a t e d membrane fragments of b u l l f r o g s k e l e t a l muscle f i b e r s , and that the b i n d i n g o f c a l c i u m ions was impeded by both sodium and potassium i o n s . They a l s o showed t h a t when the membrane fragments were extracted with a chloroform-methanol mixture (2:1 v / v ) , the e x t r a c t a b l e p o r t i o n bound c a l c i u m i o n s whereas no appreciable binding of calcium ions was observed i n the extr a c t e d residue. Koketsu et a l . (1964) concluded t h a t the r e s u l t s suggest that the binding of calcium ions takes place on the l i p i d or l i p o p r o t e i n of the s o - c a l l e d cytoplasmic membrane. In the heart, the s e n s i t i v i t y of myocardial contrac-t i l i t y to e x t r a c e l l u l a r calcium c o n c e n t r a t i o n and k i n e t i c a n a l y s i s of calcium exchange suggested that a s u p e r f i c i a l calcium pool was involved i n the r e g u l a t i o n of myocardial c o n t r a c t i l i t y . Later studies with lanthanum showed that lan-thanum s p e c i f i c a l l y uncoupled e x c i t a t i o n from co n t r a c t i o n , d i s p l a c e d calcium from a s u p e r f i c i a l calcium pool, and was bound to sarcolemmal s t r u c t u r e s . Thus, lanthanum s t u d i e s provided evidence for a sarcolemmal lo c a l e of a c o n t r a c t i l e -dependent calcium binding s i t e . Subsequent studies showed 29 that calcium does indeed bind to the cardiac sarcolemma (ATP 45 independent b i n d i n g ) . Williamson et al.(1975) studied Ca binding to a p u r i f i e d sarcolemmal preparation of the guinea p i g heart. They reported that calcium binding revealed two c l a s s e s of b i n d i n g s i t e s : a high a f f i n i t y s i t e (apparent = 16 p.M; capacity = 11.4 nmol/mg pro t e i n ) and a 10-fold g r e a t e r number of low a f f i n i t y s i t e s (apparent K m = 800 u.M; c a p a c i t y = 122.7 nmol/mg p r o t e i n ) . Ruthenium red (50 |iM), a n o n s p e c i f i c i n h i b i t o r of calcium binding to various membranes, and decreasing the pH from 7.4 to 6.6 i n h i b i t e d both the high and the low a f f i n i t y calcium b i n d i n g , while verapamil (1 \iM) s p e c i f i c a l l y i n h i b i t e d the low a f f i n i t y c a l c i u m b i n d i n g . Since the low a f f i n i t y c a l c i u m b i n d i n g s i t e s had an apparent K s i m i l a r to the o v e r a l l K f o r m m the e x t r a c e l l u l a r calcium r e q u i r e d to increase myocardial c o n t r a c t i l i t y i n i n t a c t h e a r t p r e p a r a t i o n s and were s p e c i f i c a l l y i n h i b i t e d by v e r a p a m i l , W i l l i a m s o n et a l . (1975) concluded that the low a f f i n i t y calcium binding s i t e s were located on the external surface of the sarcolemma and may r e p r e s e n t s p e c i f i c s i t e s f o r c a l c i u m e n t r y . Limas (1977) s t u d i e d the calcium b i n d i n g c h a r a c t e r i s t i c s of an i s o l a t e d sarcolemmal preparation of the rat heart. S i m i l a r -l y , he i d e n t i f i e d a high (K^ = 5 uM; capacity = 17 nmol/mg p r o t e i n ) and a low a f f i n i t y c a l c i u m b i n d i n g s i t e ( = 1.8 mM; capacity = 680 nmol/mg p r o t e i n ) . Calcium binding to both c l a s s e s of b i n d i n g s i t e s was i n h i b i t e d by ruthenium red, whereas only the low a f f i n i t y s i t e s were a f f e c t e d by 30 lanthanum. Gel e l e c t r o p h o r e s i s of the sarcolemmal proteins suggested that the high a f f i n i t y s i t e s were associated with a p r o t e i n peak of molecular weight of about 100,000, and 2 + that Ca -ATPase might be responsible f o r most of the high a f f i n i t y s i t e s . A number of reports from d i f f e r e n t labora-t o r i e s have a l s o shown ATP-independent calcium b i n d i n g i n p u r i f i e d sarcolemmal preparations from the guinea p i g heart (Hui et a l . , 1976; Morcos and Jacobson, 1979) and the rat heart (Dhalla et a l . , 1976; Takeo et a l . , 1979). Bers and Langer (1979) studied the e f f e c t of uncoupling cations on calcium binding to a p u r i f i e d sarcolemmal prepa-r a t i o n i s o l a t e d from neonatal rat hearts and c o r r e l a t e d the r e s u l t s to the negative i n o t r o p i c a c t i o n of the cations i n i s o l a t e d p a p i l l a r y muscle of the r a t h e a r t . S c a t c h a r d a n a l y s i s of calcium binding to the sarcolemma indicated two c l a s s e s of c a l c i u m b i n d i n g s i t e s ; a h i g h a f f i n i t y (K^ = 21.7 p.M; c a p a c i t y = 54 nmol/mg) and a low a f f i n i t y binding s i t e (K^ = 1.2 mM; c a p a c i t y = 216 nmol/mg). Uncoupling t r i v a l e n t and d i v a l e n t cations i n h i b i t e d calcium binding to the sarcolemma and the s e n s i t i v i t y sequence of i n h i b i t i n g 3 + 3 + 3 + c a l c i u m b i n d i n g by t h e c a t i o n s (Y > Nd > La , 2 + 2 + 2 + Cd > Co > Mg ) was t h e same as t h e e f f e c t i v e uncoupling sequence i n the i s o l a t e d p a p i l l a r y muscle. Bers and Langer (1979) also showed that the c a l c u l a t e d amount of calcium bound to the low a f f i n i t y binding s i t e s at d i f f e r e n t calcium concentrations c o r r e l a t e d very c l o s e l y to the ten-sion developed by an i n t a c t cardiac muscle. The c a l c u l a t e d 31 amount of calcium bound to the sarcolemma at 1.5 mM calcium concentration was equal to 84 nmol/mg sarcolemmal p r o t e i n , an amount representing about 700 jimol of sarcolemmal-bound calcium/kg t i s s u e wet weight (calculated by extrapolation to the i n t a c t t i s s u e ) . Bers and Langer (1979) proposed that the amount of calcium bound to the low a f f i n i t y calcium binding s i t e s of the sarcolemma was more than adequate to support t e n s i o n development and t h a t the c a l c i u m bound to these sarcolemmal s i t e s plays an important r o l e i n c o n t r o l l i n g the amount of calcium a v a i l a b l e to the myofilaments and thus myocardial c o n t r a c t i l i t y . S i m i l a r l y , P h i l i p s o n et al.(1980b) observed two calcium binding s i t e s i n a sarcolemmal membrane preparation i s o l a t e d from the rabbit heart. Sodium i n h i b i t e d sarcolemmal calcium b i n d i n g , but potassium was much l e s s e f f e c t i v e i n i n h i b i t i n g calcium binding. Scatchard analysis 2 2 of Na binding to the sarcolemmal p r e p a r a t i o n revealed a s i n g l e c l a s s of b i n d i n g s i t e s w i t h a o f 9.1 mM and calcium appeared to behave as a competitive i n h i b i t o r of sodium binding. The e f f e c t of d i f f e r e n t sodium concentra-t i o n s on the amount of c a l c i u m bound t o the sarcolemma c o r r e l a t e d very c l o s e l y with the magnitude of the tra n s i e n t c o n t r a c t i l i t y changes produced upon a l t e r i n g the e x t r a c e l -l u l a r sodium concentration as seen i n the r a b b i t p a p i l l a r y muscle by T i l l i s c h et a l . (1979). The amount of c a l c i u m bound to the sarcolemma at d i f f e r e n t calcium concentrations i n the presence of 140 mM sodium was l i n e a r l y related to the c o n t r a c t i l e strength developed by r a b b i t i n t e r v e n t r i c u l a r 32 s e p t a p e r f u s e d w i t h d i f f e r e n t c a l c i u m c o n c e n t r a t i o n s . F urthermore, when the t o t a l number of the sarcolemmal calcium binding s i t e s (270 nmol/mg sarcolemmal protein) was extrapolated to the i n t a c t t i s s u e i t was converted to 2160 (imol of bound c a l c i u m / k g t i s s u e wet weight, an amount several f o l d higher than the amount of calcium that must be d e l i v e r e d to the myofilaments to develop maximal t e n s i o n (92.8 nmol/kg wet weight; Solaro et a l . , 1974). P h i l i p s o n et a l . (1980b) concluded that these r e s u l t s strongly implied a q u a n t i t a t i v e r e l a t i o n s h i p between sarcolemmal c a l c i u m binding and myocardial c o n t r a c t i l i t y . Such a s t r i k i n g corre-l a t i o n between cardiac c o n t r a c t i l i t y and calcium binding to i s o l a t e d cardiac sarcolemma was also found i n rabbit, neona-t a l r a t , and frog v e n t r i c u l a r t i s s u e (Bers et a l . , 1981). Reducing the sodium concentration from 140 to 20 mM d i d not a l t e r the l i n e a r r e l a t i o n s h i p between cardiac c o n t r a c t i l i t y and sarcolemmal calcium binding i n the rabbit v e n t r i c l e . In contrast, i n adult rat v e n t r i c l e and r a b b i t a t r i a , contrac-t i l i t y reached i t s maximum l e v e l at a much lower calcium c o n c e n t r a t i o n than did calcium binding to the sarcolemma. Hence, Bers et a l . (1981) suggested that frog, neonatal rat, and r a b b i t v e n t r i c l e depend more d i r e c t l y on the entry of c a l c i u m from the sarcolemmal s i t e s f o r the c o n t r o l of t e n s i o n development, whereas the a d u l t r a t v e n t r i c l e and r a b b i t atrium depend to a greater extent on calcium released from the sarcoplasmic reticulum. S i m i l a r l y , Pang (1980) observed two classes of calcium 33 binding s i t e s with d i s s o c i a t i o n constants of 20 uM (capacity = 15 nmol/mg) and 1.2 mM (capacity = 452 nmol/mg) i n a p u r i -f i e d sarcolemmal preparation i s o l a t e d from canine heart. A l l cations tested i n h i b i t e d calcium binding with the following order of potency: t r i v a l e n t > divalent > monovalent cations. The order of potency f o r the monovalent ions was: Na + > K + > L i + >^  C s + and f o r t h e d i v a l e n t and t r i v a l e n t 3+ v ... 2+ _ _ 2+ ^ _ 2+ v 2 + i o n s : L a >^Mn > S r > B a > M g — 8 C a f f e i n e (1 mM) and low concentrations of ouabain (10 & _ 7 10 M) i n c r e a s e d the c a p a c i t y of the low a f f i n i t y s i t e s , - 7 — 3 2+ w h i l e v e r a p a m i l (10 -10 M), a c i d o s i s (pH 6.4), Mn —6 and high concentrations of ouabain (> 10 M) depressed the capacity of the low a f f i n i t y s i t e s . The d i s s o c i a t i o n cons-tants of the high and low a f f i n i t y s i t e s and the capacity of the high a f f i n i t y s i t e s were not a f f e c t e d by these agents. U s i n g the a c t i v i t y of the enzyme (Na -K )ATPase as an index of the f r a c t i o n of t o t a l t i s s u e sarcolemma present i n the membrane preparation, the c a l c u l a t e d amount of calcium bound to the high and low a f f i n i t y binding s i t e s was equal t o 303 and 9151 u.mol calcium/kg wet weight, r e s p e c t i v e l y . Thus, compared with the t o t a l c o n c e n t r a t i o n of c a l c i u m necessary f o r the generation of maximum ten s i o n i n canine c a r d i a c muscle (92.8 umol calcium/kg wet weight; Solaro et a l . , 1974), the amount of calcium i n the high a f f i n i t y s i t e s alone was more than enough t o accomplish the t a s k . In a p u r i f i e d sarcolemmal preparation from the guinea p i g heart, Pang and S p e r e l a k i s (1981) a l s o observed a h i g h a f f i n i t y 34 ( = 15 (iM; c a p a c i t y = 0.45 nmol/mg) and a low a f f i n i t y c a l c i u m b i n d i n g s i t e (K^ =3.3 mM; c a p a c i t y 54 nmol/mg). L i k e w i s e , v e r a p a m i l (10 & 10 M), Mn (1 mM) , 3 + La (1 mM) and a c i d o s i s (pH 6.4 & 5.6) i n h i b i t e d calcium binding to the low a f f i n i t y s i t e s , while caffeine (1 mM) and a reduction i n the sodium concentration from 150 mM to 75 mM i n c r e a s e d c a l c i u m b i n d i n g t o the low a f f i n i t y s i t e s . B e p r i d i l (10 6 & 10 ^ M), a negative i n o t r o p i c agent and an i n h i b i t o r of the slow inward calcium current, produced e f f e c t s s i m i l a r to verapamil and i n h i b i t e d calcium binding to the low a f f i n i t y calcium b i n d i n g s i t e s by reducing the t o t a l number of the b i n d i n g s i t e s (Pang and S p e r e l a k i s , 1981). M a s - O l i v a and N a y l e r (1980) a l s o r e p o r t e d t h a t 45 verapamil (1 p.M) i n h i b i t e d the passive b i n d i n g of Ca to a sarcolemmal p r e p a r a t i o n of the r a b b i t heart. In a l a t e r study, Pang and Sperelakis (1982) obtained s i m i l a r r e s u l t s , — 6 — 5 but r e p o r t e d t h a t n i f e d i p i n e and d i l t i a z e m (10 &10 M) had no e f f e c t on calcium binding i n two d i f f e r e n t sarcolem-mal preparations of the guinea p i g h e a r t . Hence, Pang and S p e r e l a k i s (1982) concluded that the molecular mechanisms whereby the organic calcium antagonists block calcium i n f l u x may vary from one drug to another even though they a l l are c l a s s i f i e d i n the same group as calcium antagonists. I t i s i n t e r e s t i n g to note that taurine (2-amino-ethan-s u l f o n i c a c i d ) , a ubiquitous amino acid making up approxi-mately 50% of the free amino a c i d pool i n mammalian heart and which has been shown t o e x e r t a p o s i t i v e i n o t r o p i c 35 e f f e c t on the heart, was found to increase calcium binding to the low a f f i n i t y calcium binding s i t e s i n an i s o l a t e d rat heart sarcolemmal preparation (Chovan et a l . , 1979 & 1980). These i n v e s t i g a t o r s a l s o reported that taurine antagonized the i n h i b i t i o n of calcium binding to the sarcolemma caused by both verapamil and lanthanum. Feldman and Weinhold (1977a) i s o l a t e d a plasma membrane pr e p a r a t i o n from the r a t heart and c h a r a c t e r i z e d a s i n g l e c l a s s of calcium b i n d i n g s i t e s with a of 265 \iM and a maximum capacity of 65 nmol/mg p r o t e i n i n the preparation. Monovalent metal i o n s were 10-100 f o l d l e s s p o t e n t as i n h i b i t o r s compared to d i v a l e n t metal i o n s and the values f o r the d i v a l e n t metal ions were s i m i l a r to the K, d value for calcium. Lanthanum produced a potent non-competi-t i v e i n h i b i t i o n of calcium binding. Propranolol and a large v a r i e t y of i t s s t r u c t u r a l analogues were competitive i n h i b i -t o r s of the calcium binding a c t i v i t y . The l o c a l anesthetics dibucaine and procaine, the antiarrhythmic agent quinidine, and a number of experimental a n e s t h e t i c agents i n h i b i t e d calcium binding to the cardiac sarcolemmal preparation. In contrast, Ohnishi et al.(1980) reported that several general anesthetics (enflurane, halothane and ether), ethanol, and acetaldehyde, a l l known to cause myocardial depression, at c l i n i c a l l y u s e f u l concentrations increased calcium binding t o a calcium b i n d i n g l i p o p r o t e i n p u r i f i e d from dog h e a r t plasma membrane. Ohnishi et a l . (1980) concluded that these drugs, by increasing calcium binding to the plasma membrane, 3 6 perhaps through i n c r e a s i n g the a f f i n i t y of calcium to the s i t e s , l i m i t e d the a v a i l a b i l i t y of s u p e r f i c i a l l y - l o c a t e d calcium f o r e x c i t a t i o n - c o n t r a c t i o n coupling and r e s u l t e d i n depression of myocardial c o n t r a c t i l e force. Thus i n summary, r e s u l t s obtained from d i f f e r e n t labo-r a t o r i e s c l e a r l y i n d i c a t e that cardiac sarcolemma contains low a f f i n i t y calcium binding s i t e s with a very close to the E D 5 Q f o r c a l c i u m a c t i v a t i o n of c o n t r a c t i l e f o r c e i n the i n t a c t heart. In a number of species, the amount of c a l -cium bound to the sarcolemma i s very c l o s e l y r e l a t e d to the force of contraction developed by the i n t a c t cardiac muscle. A v a r i e t y of negative and p o s i t i v e i n o t r o p i c agents produce a p a r a l l e l e f f e c t on c a l c i u m b i n d i n g to the sarcolemma. Furthermore, the amount of calcium bound to the sarcolemma i s several f o l d greater than the amount of calcium required t o produce f u l l a c t i v a t i o n of the c o n t r a c t i l e elements. Hence i t i s b e l i e v e d that the calcium l o o s e l y bound to the sarcolemma may p a r t i c i p a t e i n the a c t i v a t i o n of the contrac-t i l e elements and that the i n o t r o p i c e f f e c t of a number of c a r d i a c agents seems to be r e l a t e d to t h e i r e f f e c t on c a l -cium binding to the sarcolemma. However, the mechanism of c a l c i u m r e l e a s e from these s i t e s and i t s t r a n s l o c a t i o n across the sarcolemma i s not yet known. 37 F. Biochemical Nature of Cardiac Sarcolemmal Calcium  Binding S i t e s : U s i n g a c h l o r o f o r m - m e t h a n o l m i x t u r e (1:1 v/v) t o e x t r a c t the l i p i d s of a rat sarcolemmal preparation, Limas 45 (1977) showed that 80% of the Ca bound to the sarcolemma was bound to the p r o t e i n f r a c t i o n while only 15.2% of the c a l c i u m was bound to the l i p i d e x t r a c t . Treatment of the sarcolemmal preparation with d i f f e r e n t enzymes showed that pronase and t r y p s i n reduced calcium binding t o the sarcolem-ma by 43.2 and 49.6%,respectively. In contrast, pretreatment with neuraminidase, phospholipase A^, phospholipase C, and phospholipase D reduced calcium binding by 8.2, 4.8, 3.4 and 6.4%, r e s p e c t i v e l y . Therefore these r e s u l t s suggested that p r o t e i n s accounted f o r most of the c a l c i u m b i n d i n g with phospholipids and s i a l i c acids residues p l a y i n g a secondary r o l e . On the other hand, i n t e r a c t i o n with d i f f e r e n t func-t i o n a l group reagents indicated that carboxyl residues were necessary f o r calcium b i n d i n g , whereas t h i o l , amino, and s u l f h y d r y l groups were unimportant. Philipson et al.(1980a), however, presented evidence i n d i c a t i n g that the majority of calcium was bound to the phospholipids of an i s o l a t e d rabbit sarcolemmal p r e p a r a t i o n . Calcium b i n d i n g to p h o s p h o l i p i d v e s i c l e s extracted from the sarcolemma accounted for 80% of the sarcolemmal calcium binding. Treatment of the sarcolem-mal v e s i c l e s with phospholipase C reduced calcium binding by 7 5-80%. Neuraminidase treatment of the sarcolemmal v e s i c l e s reduced the s i a l i c acids content by 57%, but s i g n i f i c a n t l y 38 reduced calcium binding to the sarcolemma at concentrations g r e a t e r than 0.5 mM calcium by only 13%. A n a l y s i s of the phospholipids by t h i n l a y e r chromatographic techniques re-vealed that phosphatidylcholine and phosphatidylethanolamine comprised 39.2 and 29.8% of the sarcolemmal phospholipids, r e s p e c t i v e l y . P h o s p h a t i d y l i n o s i t o l , phosphatidylserine, and c a r d i o l i p i n , the calcium binding phospholipids, were present as 7.4, 5.6, and 0.6% of the sarcolemmal phospholipids, res-p e c t i v e l y . The calcium binding curve of v e s i c l e s prepared from pure phospholipids mixed i n the r e l a t i v e proportion i n which they were present i n the sarcolemma was i d e n t i c a l i n shape and attained about 70-80% of the maximum obtained with the calcium binding of v e s i c l e s of phospholipids extracted from the sarcolemma. Hence, P h i l i p s o n et al.(1980a) specu-lated that sarcolemmal phospholipids have a role i n c o n t r o l -l i n g c a l c i u m movement during the 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 sequence i n c a r d i a c muscle. On the other hand, Matsukubo et a l . (1981) found that pretreatment of an i s o -l ated rat heart sarcolemmal preparation with eit h e r trypsin, p h o s p h o l i p a s e C or neuraminidase was a s s o c i a t e d with a r e d u c t i o n i n c o l l o i d a l i r o n s t a i n i n g as w e l l as decreased calcium binding a c t i v i t y at 1.25 mM calcium by 49.1, 32.0 and 42.7%, r e s p e c t i v e l y . Matsukubo et a l . (1981) concluded that both surface negative charge and calcium binding s i t e s associated with the i s o l a t e d r at heart sarcolemma were con-t r i b u t e d to by a mosaic of biomolecules i n c l u d i n g proteins, phospholipids and glycoproteins. 39 Feldman and Weinhold (1977b) i s o l a t e d and p u r i f i e d a l i p o p r o t e i n of rat heart plasma membrane with a high calcium b i n d i n g c a p a c i t y . The l i p o p r o t e i n complex had an apparent molecular weight of 71,400 and the apoprotein subunit had a molecular weight of 12,300. The l i p o p r o t e i n complex contain-ed 7.35 u.mol of l i p i d phosphorous per mg of p r o t e i n and each mole of the l i p o p r o t e i n contained 90 moles of phospho-l i p i d . The maximum c a p a c i t y of c a l c i u m b i n d i n g was 4.27 nmol/mg p r o t e i n with an apparent d i s s o c i a t i o n constant for c a l c i u m of 74 uM. Calcium b i n d i n g to the l i p o p r o t e i n was c o m p e t i t i v e l y i n h i b i t e d by a v a r i e t y of metal ions and experimental antiarrhythmic and anesthetic agents. In more recent reports, Langer and co-workers studied calcium b i n d i n g to 'gas d i s s e c t e d ' sarcolemmal membranes ob t a i n e d from monolayers of c u l t u r e d neonatal r a t h e a r t c e l l s . T h i s method of membrane p r e p a r a t i o n was based on applying a high v e l o c i t y stream of nitrogen gas across the s u r f a c e of monolayers of t i s s u e c u l t u r e which l e d to the opening of the upper surface of the c e l l s and removal of the c e l l u l a r m a t e r i a l , and the sarcolemma was l e f t on the s u r f a c e of c u l t u r e d i s c s . Langer and Nudd (1983) reported that treatment of such sarcolemmal preparations with phos-p h o l i p a s e A^ and phospholipase D s i g n i f i c a n t l y i n c r e a s e d calcium binding by 67 and 56%, r e s p e c t i v e l y . The increased calcium binding secondary to phospholipase A^ was e l i m i n a -ted by an albumin wash, which indicated that the f a t t y acid product was responsible for the enhanced binding and not the 40 formation of lysophosphoglyceride w i t h i n the membrane. The increase a f t e r phospholipase D treatment was a t t r i b u t e d to an increase i n phosphatidate, with an attendant increase i n net anionic charge on the membrane. However, phospholipase C treatment produced a n o n s i g n i f i c a n t decrease of c a l c i u m binding by about 17%. The ineffectiveness of phospholipase C was a t t r i b u t e d to the high s e l e c t i v i t y o f the cereus phospholipase C used i n the study against n e u t r a l phospha-t i d y l c h o l i n e and a n e g l i g i b l e e f f e c t against natural anionic p h o s p h o l i p i d s . S i m i l a r l y , neuraminidase treatment (0.25 U/ml; 30 min) produced a n o n s i g n i f i c a n t r e d u c t i o n of 10%. Hence, Langer and Nudd (1983) concluded that anionic phos-p h o l i p i d s probably account f o r a major p o r t i o n of calcium binding within the sarcolemma of cultured cardiac c e l l s . In the meantime, Burt and Langer (1983) showed that Polymixin B (5 mM), an a m p h i p h i l i c c a t i o n i c p e p t i d o l i p i d which i s thought to bind to anionic phospholipids, displaced calcium from 'gas dissected' cardiac sarcolemma i n amounts equal to those displaced by 1 mM lanthanum. Polymixin B (0.1 mM) also i n h i b i t e d a l l the phospholipid s p e c i f i c increment i n bound calcium produced by pretreatment with phospholipase D. Thus, i n general, sarcolemmal calcium binding s i t e s are composed of negatively charged s i t e s of carbohydrates, pro-t e i n s and phospholipids.However, negatively charged membrane phospholipids seem to play a predominant role as sarcolemmal c a l c i u m b i n d i n g s i t e s . O bviously, the d i f f e r e n c e s among reports fom d i f f e r e n t laboratories could be due to d i f f e r e n t 41 species used and d i f f e r e n t methods employed for the membrane preparation. G. Role of Sarcolemmal S i a l i c Acids i n Cardiac Calcium Metabolism and Function: Cardiac muscle c e l l s are covered with a coat known as the glycocalyx, which i s composed of g l y c o p r o t e i n s , glyco-l i p i d s and mucopolysaccharides (see f o r example Martinez-Palomo, 1970; Langer, 1978). The terminal end of the carbo-hydrate side chains of these molecules i s u s u a l l y occupied by a Cg-amino sugar, s i a l i c a c i d s , most commonly N-acetyl neuraminic ac i d (Winzler, 1970). The unsubstituted carboxyl group of s i a l i c acids are negatively charged at p h y s i o l o g i -c a l pH, pK =2.6 (Hughes, 1976). The b i o l o g i c a l importance of the glycocalyx residues of s i a l i c acids was recognized by use of the enzyme neurami-nidase, a s e l e c t i v e enzyme that cleaves the alpha-glycosidic bond between the t e r m i n a l s i a l i c a c i d s and t h e i r partner molecules of the carbohydrate chain (Drzeniek, 1972). In 1976, Langer and h i s co-workers showed that release of up to 61% of the s i a l i c a c i ds from c u l t u r e d r a t h e a r t c e l l s by neuraminidase led to a f i v e to six f o l d increase i n the rate 45 3 + o f Ca exchange and the entry of La , which i s normal-l y e x c l u d e d by t h e s e c e l l s , w h i l e K + exchange was not s i g n i f i c a n t l y a f f e c t e d . They suggested that the s i a l i c a c i d residues may play a c r u c i a l r o l e i n the regulation of trans-2 + membrane Ca f l u x and e x c i t a t i o n - c o n t r a c t i o n coupling i n 42 the heart. Later, Isenberg and Klockner (1980) reported that e l e c t r i c a l p r o p e r t i e s plus the c h a r a c t e r i s t i c s of the slow inward current i n i s o l a t e d adult r a t heart myocytes, whose glycocalyx was removed by the c e l l i s o l a t i o n procedure, were s i m i l a r to those displayed by i n t a c t t i s s u e . They concluded that the glycocalyx i s u n l i k e l y to be the s t r u c t u r e o r i g i -n a t i n g or c o n t r o l l i n g the slow inward c a l c i u m c u r r e n t . Nathan et a l . (1980) reported that removal of up to 50% of the s i a l i c acids from aggregates of 7-day chick embryo heart c e l l s following treatment with highly p u r i f i e d neuraminidase r e s u l t e d i n h y p e r p o l a r i z a t i o n of the maximum d i a s t o l i c po-t e n t i a l , reduction i n the slope of d i a s t o l i c d e p o l a r i z a t i o n leading to slowing of beating, negative s h i f t s of threshold p o t e n t i a l and the v o l t a g e at which upstroke v e l o c i t y was maximal, and an i n i t i a l i n c r e a s e i n the a c t i o n p o t e n t i a l overshoot. Woods et a l . (1982) i n v e s t i g a t e d the e f f e c t of neuraminidase treatment on the e l e c t r i c a l p r o p e r t i e s of d i f f e r e n t regions of the canine heart, and c o r r e l a t e d these observations with e l e c t r o n microscopic studies i n the same t i s s u e s . They reported that neuraminidase treatment a b o l i s h -ed the a u t o m a t i c i t y of sinus node, blocked conduction i n a t r i a l and v e n t r i c u l a r working muscle, but evoked spontane-ous f i r i n g i n quiescent f a l s e tendon c e l l s . They concluded that the glycocalyx (or s i a l i c a c i d removed by neuraminid-ase) has an important e l e c t r o p h y s i o l o g i c a l r o l e i n canine h e a r t c e l l s , but the nature of i t s f u n c t i o n i s dependent upon the type of the c e l l s . 43 Cardiac glycosides have been the only cardiac agonists i n v e s t i g a t e d for a p o s s i b l e r o l e of s i a l i c a c i ds i n t h e i r actions on the heart. Bailey and Fawzi (1980) reported that neuraminidase treatment of La n g e n d o r f f p r e p a r a t i o n s of guinea p i g heart prevented the p o s i t i v e i n o t r o p i c e f f e c t of _7 th e r a p e u t i c concentrations of ouabain (10 M), and had no — 6 e f f e c t on ouabain t o x i c i t y (10 M). Grupp et a l . (1980), however, reported that neuraminidase treatment had no e f f e c t on the i n o t r o p i c r e s p o n s e of the g u i n e a p i g h e a r t to ouabain. Meanwhile, Harding and H a l l i d a y (1980) reported that neuraminidase treatment of the guinea p i g l e f t a t r i a released up to 79% of t o t a l t i s s u e s i a l i c a c i d , but had no - 7 e f f e c t on the response to i n o t r o p i c (2x10 M) or t o x i c —6 (10 M) concentrations of ouabain. Histochemical studies demonstrated that the surface of myocardial c e l l s contained abundant s i a l i c acids d i s t r i b u t e d i n two d i s t i n c t layers, one i n the surface coat next to the l i p i d b i l a y e r , the other i n the e x t e r n a l lamina at the i n t e r s t i t i a l i n t e r f a c e (Frank et al.,1977). Electron microg-raphs have shown that removal of up to 61% of t o t a l c e l l u l a r s i a l i c acids f o l l o w i n g treatment with neuraminidase (0.25 U/ml; 15 min) markedly decreased lanthanum and c o l l o i d a l i r o n hydroxide s t a i n i n g of the sarcolemma of neonatal r a t myoblasts i n c u l t u r e (Frank et a l . , 1977). Langer (1978) concluded t h a t the g l y c o c a l y x s i a l i c a c i ds account f o r a s i g n i f i c a n t number of s u p e r f i c i a l calcium-binding s i t e s i n c a r d i a c t i s s u e . Frank (1978) s p e c u l a t e d t h a t the s i a l i c 44 a c i d residues on the surface membrane of heart c e l l s bind c a l c i u m as a f i r s t step i n the c a l c i u m i n f l u x mechanism. Indeed, Jaques et a l . (1977) reported that calcium strongly and p r e f e r e n t i a l l y binds to free s i a l i c a c i d i n s o l u t i o n at pH 7 i n a 1:1 r a t i o and with a d i s s o c i a t i o n constant of 8.3 mM. However, there i s a c o n s i d e r a b l e c o n t r o v e r s y i n the l i t e r a t u r e r e g a r d i n g c a l c i u m b i n d i n g to the s i a l i c a c i d residues of c a r d i a c sarcolemma. Limas (1977) reported that neuraminidase treatment of a sarcolemmal preparation of the 45 r a t heart reduced Ca binding by 8.2%, and suggested that the s i a l i c a c i d residues play a secondary r o l e i n calcium b i n d i n g t o the sarcolemma. Pang (1980) r e p o r t e d t h a t treatment of an i s o l a t e d canine c a r d i a c sarcolemma with neuraminidase had no s i g n i f i c a n t e f f e c t on calcium binding t o the sarcolemma. P h i l i p s o n et a l . (1980) showed t h a t neuraminidase treatment of an i s o l a t e d sarcolemmal prepara-t i o n of the r a b b i t heart caused a 57% reduction i n s i a l i c a c i d c o n t e n t , had no e f f e c t on c a l c i u m b i n d i n g t o the sarcolemma at up to 0.5 mM calcium concentration, and only s i g n i f i c a n t l y reduced calcium binding at calcium concentra-tions higher than 0.5 mM by about 13%.Langer and Nudd (1983) showed t h a t neuraminidase treatment of ' g a s - d i s s e c t e d ' sarcolemmal membranes obtained from monolayers of c u l t u r e d n e o n a t a l r a t h e a r t c e l l s produced a n o n s i g n i f i c a n t 10% r e d u c t i o n of calcium binding to the membrane. In contrast, Taeko et a l . (1980) showed that treatment of a r a t heart sarcolemmal p r e p a r a t i o n with neuraminidase reduced s i a l i c 45 a c i d content of the pre p a r a t i o n by about 50% and s i g n i f i -c a n t l y diminished calcium binding at a concentration of 0.1 45 mM Ca by about 34%. Matsukubo et a l . (1981) showed that neuraminidase treatment of a r a t heart sarcolemmal prepara-t i o n released approximately 63% of the t o t a l sarcolemmal-bound s i a l i c acids and reduced calcium binding to the prepa-45 r a t i o n at 0.1 and 1.25 mM c o n c e n t r a t i o n of Ca by 41.2 and 42.7%, r e s p e c t i v e l y . Ma , Baker and B a i l e y (1979) reported that neuraminidase treatment of sarcolemmal ghosts i s o l a t e d from normal hamster hearts reduced the capacity of the low a f f i n i t y c alcium b i n d i n g s i t e s (K^ = 2.17 mM) by about 70% and revealed a higher a f f i n i t y c a l c i u m b i n d i n g s i t e ( K d = 0.67 mM). B a i l e y and Fawzi (1980 and 1982a) showed that neuraminidase treatment of i s o l a t e d guinea p i g c a r d i a c myocytes with an i n t a c t g l y c o c a l y x r e l e a s e d about 47% of t o t a l c e l l u l a r s i a l i c a c i d content and reduced the maximum capacity of the low a f f i n i t y and lanthanum-sensitive calcium binding s i t e s by 52.4%. In the l a t t e r study, calcium binding to the c e l l preparation was measured by a continuous flow d i a l y s i s technique i n order to avoid the washout of the l o o s e l y bound calcium t h a t may occur i n r a p i d f i l t r a t i o n techniques of binding assays. The i n a b i l i t y of some i n v e s t i -gators to detect calcium binding to the cardiac sarcolemmal s i a l i c acids i s very l i k e l y due to removal of the glycocalyx during the preparation of the sarcolemma and/or the i n s e n s i -t i v i t y of the b i n d i n g assays used to detect the very low a f f i n i t y calcium binding to the s i a l i c acids. 46 H. Ef f e c t of Cardiac Glycosides on Superficially-Bound  Calcium: Through d i f f e r e n t experimental approaches, therapeutic concentrations of cardiac glycosides have c o n s i s t e n t l y been shown to increase calcium content i n a l a b i l e s u p e r f i c i a l calcium pool i n the heart of c a r d i a c g l y c o s i d e s - s e n s i t i v e species. Lullmann and Holland (1962) reported that ouabain i n concentrations producing a p o s i t i v e i n o t r o p i c e f f e c t i n i s o l a t e d guinea p i g a t r i a , under c o n d i t i o n s o f v a r y i n g e x t r a c e l l u l a r calcium concentration and time of exposure to the agent, had l i t t l e or no e f f e c t on t o t a l t i s s u e calcium content. Such concentrations, however, caused an increase i n the exchangeable calcium f r a c t i o n which was correlated to c o n t r a c t i l e tension. B a i l e y and Harvey (1969) i n v e s t i g a t e d 45 the e f f e c t of ouabain on the k i n e t i c s of Ca f l u x i n the l e f t v e n t r i c l e of a goat heart-lung preparation. They found t h a t ouabain s i g n i f i c a n t l y i n c r e a s e d c a l c i u m i n f l u x and t o t a l calcium content i n a l a b i l e calcium pool, compartment II , with a concomitant increase i n c o n t r a c t i l i t y . They also found that increasing l e f t v e n t r i c u l a r work accelerated the d i f f u s i o n of calcium into compartment II and r e s u l t e d i n an a c c u m u l a t i o n of c a l c i u m i n compartment I I . Hence, they suggested that compartment II was the postulated c o n t r a c t i l e pool of calcium and concluded that t h e i r r e s u l t s support the hypothesis that ouabain produced a p o s i t i v e i n o t r o p i c e f f e c t i n the heart by increasing the rate of calcium i n f l u x i n t o a l a b i l e calcium pool involved i n the contraction of the heart 47 i muscle. B a i l e y and Sures (1971) s t u d i e d the e f f e c t s of a — 8 t h e r a p e u t i c concentration of ouabain (5x10 g/ml) on the k i n e t i c s of calcium washout and calcium uptake i n Langen-d o r f f preparations of cat h e a r t s . They found t h a t ouabain a f f e c t e d n e i ther the e f f l u x of calcium from Ca.^, the con-tracti l e - d e p e n d e n t calcium pool (Bailey and Dresel/ 1968), nor the decay of c o n t r a c t i l e f o r c e when the h e a r t s were washed out w i t h c a l c i u m - f r e e K r e b s - H e n s e l e i t s o l u t i o n . In c o n t r a s t , ouabain s i g n i f i c a n t l y i n c r e a s e d the r a t e of calcium accumulation and the quantity of calcium taken up by the heart with a concomitant increase i n the rate of resto-r a t i o n of c o n t r a c t i l e force to a s i g n i f i c a n t l y higher l e v e l . In a l a t e r study, Bailey (1977) showed that ouabain s p e c i f i -c a l l y increased calcium content i n Ca^, a s u p e r f i c i a l and r e l a t i v e l y l a b i l e calcium pool d i s p l a c e d s p e c i f i c a l l y by lanthanum (Ong and B a i l e y , 1972). In a more recent study, Maitland et a l . (1982) showed that i n Langendorff prepara-t i o n s of guinea pig heart a concentration of ouabain (0.15 |j.M) which enhanced the force of contraction of the heart by 60% without evidence of t o x i c i t y , i n c r e a s e d the f l u x and q u a n t i t y of exchangeable calcium i n the vascular space and i n the c e l l u l a r compartment with the h i g h e s t f r a c t i o n a l t r a n s f e r r a t e . In contrast, ouabain decreased the f l u x and f r a c t i o n a l t r a n s f e r rate of exchangeable calcium i n the two c e l l u l a r compartments with the lowest f r a c t i o n a l t r a n s f e r r a t e s . Maitland et a l . (1982) concluded that t h e i r r e s u l t s are consistent with the conclusion that one of the actions 48 o f ouabain on myocardial muscle c e l l s i s to i n c r e a s e the q u a n t i t y of r a p i d l y - e x c h a n g e a b l e c a l c i u m bound t o the e x t r a c e l l u l a r s i t e s on the sarcolemma and/or present i n the myoplasm. C a r r i e r et a l . (1974) reported that, i n i s o l a t e d guinea _ 7 p i g a t r i a , ouabain (1.5x10 M) enhanced the s i z e of a f a s t exchanging calcium f r a c t i o n that was reduced by aging and by treatment w i t h p h e n o b a r b i t a l . In an i n t e r e s t i n g study, Nayler (1973) showed that i n o t r o p i c a l l y a c t i v e concentra-45 t i o n s of ouabain increased the amount of Ca d i s p l a c e d by lanthanum, an ion r e s t r i c t e d to the e x t r a c e l l u l a r space and which displaces calcium from the s u p e r f i c i a l binding s i t e s , 45 from Ca l a b e l l e d canine v e n t r i c u l a r t r a b e c u l a r muscles. _ 7 S h e r i d a n (1978) a l s o showed t h a t ouabain (2x10 M) i n -45 creased the s u p e r f i c i a l l y - b o u n d c a l c i u m detected as Ca d i s p l a c e d by lanthanum i n r i g h t v e n t r i c u l a r p a p i l l a r y muscles of adult cat hearts. In an i s o l a t e d sarcolemmal p r e p a r a t i o n of the dog h e a r t , Pang (1980) r e p o r t e d t h a t low c o n c e n t r a t i o n s of — 8 — 7 ouabain (10 & 10 M) i n c r e a s e d the amount of c a l c i u m bound t o the low a f f i n i t y c a l c i u m b i n d i n g s i t e s of the preparation by increasing the capacity of the binding s i t e s . -5 - 3 But high c o n c e n t r a t i o n s of ouabain (10 -10 M) depress-ed the capacity of the low a f f i n i t y calcium binding s i t e s . In i s o l a t e d cardiac myocytes of the guinea p i g heart. Bailey and Fawzi (1982a & b) have shown t h a t ouabain i n c r e a s e d 49 calcium binding to a low a f f i n i t y and lanthanum-sensitive c a l c i u m b i n d i n g s i t e . Ouabain produced a c o n c e n t r a t i o n -dependent increase i n the a f f i n i t y of calcium for the s i t e s , but had no e f f e c t on the t o t a l number of b i n d i n g s i t e s . B a i l e y and Fawzi (1980) have a l s o shown that neuraminidase treatment of the c e l l s i n h i b i t e d the e f f e c t of ouabain on calcium binding. 50 I. Purpose of the Study and Approach to the Problem: I t was evident from the l i t e r a t u r e that the ro l e of the s u p e r f i c i a l l y - b o u n d calcium i n the i n o t r o p i c response of a major class of inotropic agents that produce t h e i r inotropic e f f e c t by elevating c e l l u l a r c y c l i c AMP l e v e l was not known. Hence, i n the f i r s t p a r t of t h i s study, I attempted to determine the r o l e of the superficially-bound calcium i n the p o s i t i v e i n o t r o p i c response of isoproterenol, a beta-agonist w e l l known t o in c r e a s e c e l l u l a r c y c l i c AMP l e v e l s i n the hea r t . I used lanthanum, an i o n known to be r e s t r i c t e d to the e x t r a c e l l u l a r space and which displaces calcium from the s u p e r f i c i a l binding s i t e s , as a t o o l to e l u c i d a t e the role of s u p e r f i c i a l l y - b o u n d calcium i n the i n o t r o p i c response of iso p r o t e r e n o l . I speculated that i f the su p e r f i c i a l l y - b o u n d calcium plays any ro l e i n the p o s i t i v e i n o t r o p i c response of i s o p r o t e r e n o l , then one would expect that displacement of c a l c i u m from these s i t e s by pretreatment with lanthanum would i n h i b i t , or a l t e r , the p o s i t i v e i n o t r o p i c response of iso p r o t e r e n o l . Therefore, I studied the e f f e c t of lanthanum on b a s a l c o n t r a c t i l i t y and the i n o t r o p i c r e s p o n s e o f i s o p r o t e r e n o l i n Langendorff p r e p a r a t i o n s of adult guinea p i g h e a r t s . Hearts were perfused with a Hepes b u f f e r con-t a i n i n g no carbonate or phosphate to avoid the p r e c i p i t a t i o n of lanthanum s a l t s , and the c o n t r a c t i l i t y of the l e f t ven-t r i c l e was monitored by a ba l l o o n i n s e r t e d i n s i d e the l e f t v e n t r i c l e . To examine the s e l e c t i v i t y of the lanthanum e f f e c t : 1) I determined the e f f e c t of lanthanum on bas a l 51 and isoproterenol-induced c y c l i c AMP l e v e l s i n the heart, to determine the p o s s i b l e i n t e r a c t i o n s of lanthanum with the beta-receptor and i t s e f f e c t on the coupling of the beta-r e c e p t o r w i t h the enzyme a d e n y l a t e c y c l a s e ; and 2) I 3 examined the e f f e c t of lanthanum on [ H ] n i t r e n d i p i n e b i n d i n g i n an i s o l a t e d membrane pr e p a r a t i o n of the guinea p i g l e f t v e n t r i c l e , to i n v e s t i g a t e the d i r e c t a c t i o n of lanthanum on the slow inward calcium channels. Obviously, the f o l l o w i n g step i n such a study was to determine the biochemical nature of the s u p e r f i c i a l calcium b i n d i n g s i t e s i n v o l v e d i n the i n o t r o p i c response of each a g o n i s t . T h e r e f o r e , i n the second p a r t of t h i s study, I i n v e s t i g a t e d the r o l e of s i a l i c a c i d s i n the i n o t r o p i c response of i s o p r o t e r e n o l , ouabain and calcium. Hence, I studied the e f f e c t of s e l e c t i v e removal of the s i a l i c acids, f o l l o w i n g treatment with the enzyme neuraminidase, on the i n o t r o p i c response of these agents i n Langendorff prepara-t i o n s of adult guinea p i g hearts. A l l hearts were perfused with the Hepes b u f f e r containing no carbonate or phosphate to avoid the p r e c i p i t a t i o n of calcium s a l t s at high calcium c o n c e n t r a t i o n s i n the calcium study and to be c o n s i s t e n t throughout the study. I a l s o examined the e f f e c t of neura-minidase treatment of the hearts on the t r a n s i e n t p o s i t i v e i n o t r o p i c response produced by reducing the e x t r a c e l l u l a r concentration of sodium i n order to evaluate the e f f e c t of + 2 + such a treatment on the f u n c t i o n o f Na -Ca exchange i n an i n t a c t h e a r t . In o r d e r t o e v a l u a t e the r o l e of the 52 enzyme ( N a + - K + ) ATPase i n the e f f e c t of neuraminidase treatment on the i n o t r o p i c response of ouabain, I examined th e e f f e c t of a s i m i l a r n e u r a m i n i d a s e t r e a t m e n t of a membrane preparation of the guinea pig l e f t v e n t r i c l e on the a c t i v i t y of the enzyme ( N a + - K + ) A T P a s e and i t s s e n s i -t i v i t y to i n h i b i t i o n by ouabain. The c h a r a c t e r i s t i c s of 3 [ H]ouabain binding were also studied. 53 S E C T I O N I I M E T H O D S 54 A. Langendorff Preparations: Adult guinea pigs of e i t h e r sex (300-900 g) were hepa-r i n i z e d (1,000 U/kg, i.p.) 20 min before being anesthetized with d i e t h y l e ther. Hearts were immediately removed and plac e d i n oxygenated c o l d b u f f e r on i c e (4°C). Extraneous t i s s u e was removed and h e a r t s were r a p i d l y mounted on a modified Langendorff apparatus. Hearts were perfused with Hepes buffer (pH = 7.4) containing: 140 mM NaCl, 4.5 mM KC1, 1.2 mM MgC^, 3 mM Hepes (N-2-hydroxy-ethylpiperazine-N'-2-ethansulfonic a c i d ; Sigma Chemical Company) and 11.2 mM glucose. The b u f f e r was bubbled with 100% and perfu s i o n was c a r r i e d out at 30°C and 60 mm Hg. Hearts were paced at 3.5 Hz at double t h r e s h o l d v o l t a g e . C o n t r a c t i l i t y of the hearts was monitored by a latex balloon i n s e r t e d insi d e the l e f t v e n t r i c l e . The s i z e of the b a l l o o n was g r a d u a l l y increased and adjusted to obtain a maximum recording of the c o n t r a c t i l i t y index (+dP/dt ). A l l h e a r t s were f i r s t J ' max perfused with the buffer for 45-60 min to reach equilibrium. Then hearts were perfused with Hepes buffe r containing d i f -f e r e n t agents according to a s p e c i f i c experimental design. In the lanthanum study, hearts were perfused with increasing concentrations of lanthanum, isoproterenol, or isoproterenol plus a single concentration of lanthanum (see Results). For neuraminidase treatments, hearts were perfused with Hepes b u f f e r c o n t a i n i n g 0.01 U/ml neuraminidase plus 0.3 mg/ml bovine serum albumin (BSA). A t o t a l volume of 100 ml 55 was used and the s o l u t i o n was r e c i r c u l a t e d while being con-t i n u o u s l y oxygenated f o r 1 h. Contr o l hearts were tr e a t e d s i m i l a r l y with b u f f e r c o n t a i n i n g 0.3 mg/ml BSA. Following t h i s treatment, hearts were perfused with Hepes b u f f e r for 15-20 min p r i o r to further treatment. At the end of each ex-periment, hearts were taken down and the a t r i a were removed, the v e n t r i c l e s were blotted, weighed and a piece of the l e f t v e n t r i c l e was frozen i n l i q u i d nitrogen and stored at -70°C for l a t e r assay of s i a l i c acid content. B. C y c l i c AMP Determination: Langendorff preparations of ad u l t guinea p i g hearts, prepared as described above, were used for the determination of myocardial c y c l i c AMP l e v e l s . F o l l o w i n g e q u i l i b r a t i o n with Hepes b u f f e r (45-60 min) and treatment with d i f f e r e n t agents, hearts were r a p i d l y f r o z e n at an appropriate time u s i n g tongs cooled i n l i q u i d n i t r o g e n . Frozen v e n t r i c l e s were stored at -70°C and c y c l i c AMP content was measured l a t e r using a Becton Dickinson radioimmunoassay k i t . I n i t i a l l y a group of hearts were frozen at d i f f e r e n t — 8 time points f o l l o w i n g p e r f u s i o n with 5x10 M i s o p r o t e r e n o l and c y c l i c AMP content of the hearts was measured i n order to determine the time at which isoproterenol-induced c y c l i c AMP l e v e l was at i t s maximum. A s i m i l a r study was c a r r i e d out i n hearts f i r s t perfused with 3 u.M lanthanum f o r 3 min — 8 f o l l o w e d by 3 u.M lanthanum p l u s 5x10 M i s o p r o t e r e n o l . 56 In both c o n t r o l and l a n t h a n u m - t r e a t e d h e a r t s , maximum i s o p r o t e r e n o l - i n d u c e d c y c l i c AMP l e v e l s were o b t a i n e d following 9-10 s of treatment with isoproterenol. To study the e f f e c t of lanthanum on basal and isoprote-renol-induced c y c l i c AMP l e v e l s , four groups of hearts were frozen a f t e r : a) e q u i l i b r a t i o n with Hepes b u f f e r ; b) 3 min p e r f u s i o n with 3 M^ lanthanum; c) 9-10 s p e r f u s i o n with — 8 5x10 M i s o p r o t e r e n o l ; and d) 9-10 s p e r f u s i o n w i t h — 8 5x10 M i s o p r o t e r e n o l plus 3 |i.M lanthanum f o l l o w i n g 3 min perfusion with 3 |iM lanthanum, and c y c l i c AMP content of the frozen v e n t r i c l e s was measured. 3 C. C H]Nitrendipine Binding: C - l . Membrane Preparation: Guinea pigs of eit h e r sex weighing 300-600 g were hepa-r i n i z e d (1000 U/kg, i.p.) 20 min before being anesthetized with d i e t h y l ether. Hearts were r a p i d l y removed and placed i n i c e - c o l d s a l i n e . Hearts were p e r f u s e d w i t h i c e - c o l d s a l i n e to f l u s h blood out of the muscle. L e f t v e n t r i c l e s were removed, cut i n t o small p i e c e s , b l o t t e d and weighed r a p i d l y . The t i s s u e was homogenized i n 30 volumes (based on t i s s u e weight) of a s o l u t i o n containing 10 mM Tris-EDTA and 0.25 M sucrose (pH = 8 at 25°C) using a Polytron (PT 10 20 3 500) homogenizer at speed s e t t i n g 6 f o r 4 x 15 s. Homoge-nates were l e f t on i c e f o r 1-2 min between homogenizations. The homogenates were centrifuged at 2442 x g (Sorvall RC2-B, 57 r o t o r SS-34, 4500 rpm) f o r 20 min. The supernatants were c a r e f u l l y decanted and p l a c e d on i c e . The p e l l e t s were rehomogenized i n 30 volumes of 10 mM Tris-EDTA and 0.25 M sucrose (pH = 8) and centrifuged as described i n the p r e v i -ous step. Supernatants were c a r e f u l l y decanted, mixed with previous supernatants and centrifuged at 96,000 x g (Beckman L5-50, r o t o r SW27, 27,000 rpm) f o r 1 h. The supernatants were d i s c a r d e d and the p e l l e t s were homogenized i n 10 volumes of 50 mM T r i s - H C l b u f f e r (pH = 7.4) using an a l l -g l a ss hand homogenizer, and the suspension was cen t r i f u g e d at 96,000 x g (Beckman L5-50, r o t o r SW27, 27,000 rpm) f o r 1 h. The l a t t e r step was repeated and the f i n a l p e l l e t was suspended i n 50 volumes (based on o r i g i n a l t i s s u e weight) of 50 mM Tris-HCl buffer (pH = 7.4). T r i p l i c a t e s of 0.5 ml from the f i n a l s u s p e n s i o n were taken f o r p r o t e i n assay by a mod i f i e d Lowry method (Hartree, 1972) using bovine serum albumin as a standard. 3 C-2. Determination of [ H]Nitrendipine Binding: Samples of the membrane preparation containing 0.2-0.3 mg p r o t e i n were incubated with s e l e c t e d c o n c e n t r a t i o n ( s ) 3 (see Results) of [ H] n i t r e n d i p i n e (New England Nuclear, 70 Ci/mmol). Incubation was ca r r i e d out i n the dark (since n i t -rendipine i s l i g h t s e n s i t i v e ) at room temperature (23°C) i n 50 mM Tris-HCl buffer (pH = 7.4; f i n a l volume = 1 ml) for 90 min. The reaction was terminated by rapid f i l t r a t i o n through Whatman GF/B g l a s s m i c r o f i b r e f i l t e r s . T e s t tubes and 58 f i l t e r s were washed r a p i d l y with 3 x 4 ml i c e - c o l d 50 mM T r i s - H C l b u f f e r (pH = 7.4 at 25°C). The f i l t e r papers were t r a n s f e r r e d i n t o s c i n t i l l a t i o n v i a l s , 10 ml toluene-based s c i n t i l l a t i o n f l u i d was added, the v i a l s were shaken f o r one hour at 4°C and r a d i o a c t i v i t y was counted i n a Mark III l i q u i d s c i n t i l l a t i o n system spectrometer ( S c i n t i l l a t i o n System Model 6880, S e a r l e , USA). N o n s p e c i f i c b i n d i n g was — 6 determined i n i the presence of 10 M n i f e d i p i n e . Assays were performed i n duplicate or t r i p l i c a t e . D. S i a l i c Acid Assay: S i a l i c a c i d was determined by the t h i o b a r b i t u r i c a c i d assay d e s c r i b e d by Warren (1959). The assay i s based on o x i d i z i n g s i a l i c acids with metaperiodate to formylpyruvate, followed by the formation of a reddish-pink complex with t h i o b a r b i t u r i c a c i d . Absorbance of the complex s o l u t i o n i n cyclohexanone at 549 nm (wavelength of maximum absorbance f o r the s i a l i c a c i d product) i s p r o p o r t i o n a l to the amount of s i a l i c a c i d present. To increase the s e n s i t i v i t y of the method the volume of cylohexanone used t o e x t r a c t the chromophore was reduced from 4.3 to 2 ml. This process was found to g i v e s a t i s f a c t o r y r e s u l t s . The s t a n d a r d curve o b t a i n e d u s i n g d i f f e r e n t c o n c e n t r a t i o n s of s i a l i c a c i d overlapped the standard curve obtained from Warren's (1959) r e p o r t . T his method allows d e t e r m i n a t i o n of as low as 2 nmoles s i a l i c acid. Glycosidically-bound s i a l i c acids do not i n t e r f e r e w i t h the assay, and c o u l d only be determined 59 following acid hydrolysis. B r i e f l y , 0.1 ml of sodium periodate s o l u t i o n (0.2 M i n 9 M phosphoric acid) was added to an a l i q u o t of 0.2 ml of the sample. The mixture was shaken and allowed to stand at room temperature f o r 20 min. Then 1 ml of the a r s e n i t e s o l u t i o n (10% sodium a r s e n i t e i n a s o l u t i o n of 0.5 M Na 2SO 4~0.1 N H 2 S 0 4 ) was added t o the m i x t u r e and the t e s t tube was shaken u n t i l the yellowish-brown c o l o r disap-peared. This was followed by the a d d i t i o n of 3 ml of the t h i o b a r b i t u r i c a c i d s o l u t i o n (0.6% i n 0.5 M N a 2 S 0 4 ) . The t e s t tube was shaken, capped with a gl a s s bead, and then heated i n a vi g o r o u s l y b o i l i n g water bath f o r 15 min. The tube was then removed and placed i n a cold water bath f o r 5 min. To the t o t a l s o l u t i o n , 2 ml cyclohexanone was added and the mixture was vigorously shaken to extract the co l o r . The mixture was then c e n t r i f u g e d at 1500 x g f o r 5 min to separate the two phases. The top cyclohexanone l a y e r was c a r e f u l l y i s o l a t e d and the absorbance of the s o l u t i o n was measured at 549 nm against a s i m i l a r l y prepared blank. Since the standard curve overlapped the values reported by Warren (1959), the molar e x t i n c t i o n c o e f f i c i e n t of 57,000 reported by Warren (1959) was used to c a l c u l a t e the amount of s i a l i c "acid present i n the sample. The assay was c a r r i e d out i n duplicate or t r i p l i c a t e . 60 E. Determination of Content of S i a l i c Acids i n the  Guinea Pig Heart: For the determination of t o t a l content of s i a l i c acids i n the guinea p i g heart, about 100 mg t i s s u e was accurately weighed, p l a c e d i n 1 ml 0.1 N H2SO^ and homogenized by Polytron (PT 10 20 3 500; speed 6-7 for 15-20 s ) . Homogenates were incubated i n a water bath at 80°C f o r 1 h to re l e a s e glycosidically-bound s i a l i c acids. Samples were then c e n t r i -fuged f o r 20 min (bench top c e n t r i f u g e ) . Aliquots of 200 \xl of supernate were used for the determination of content of s i a l i c acids as described before. A l l determinations were c a r r i e d out i n t r i p l i c a t e . Recovery of s i a l i c acid following acid hydrolysis was 92%. Other substances present i n the guinea p i g heart tiss u e homogenate i n t e r f e r e with t h i s assay forming a rose-colored complex that c o n t r i b u t e s to the absorbance at 549 nm. The c o n t r i b u t i o n of t h i s c o l o r to the absorbance at 549 nm i s high. The i n t e r f e r i n g substance(s) was re l e a s e d f o l l o w i n g a c i d h y d r o l y s i s , s i n c e no c o l o r was formed b e f o r e a c i d h y d r o l y s i s . Maximum absorbance of t h i s chromophore was at 532 nm, suggesting that the substance i s 2-deoxyribose, un-saturated l i p i d s , or any substance that y i e l d s malonaldehyde upon periodate oxidation. To cor r e c t f o r t h i s interference, r e a d i n g s were made r o u t i n e l y at 562, 549 and 532 nm and content of s i a l i c acids was c a l c u l a t e d using both formulas p r o v i d e d by Warren (1959). Content o f s i a l i c a c i d s was 61 c a l c u l a t e d as nmoles s i a l i c a c i d s / g t i s s u e wet weight. In t h i s study p i e c e s of the l e f t v e n t r i c l e were used, and t h e r e f o r e r e s u l t s correspond to the guinea p i g l e f t ven-t r i c u l a r content of s i a l i c a c i d s . The term s i a l i c a c i d s (plural) i s used to refer to N-acetylneuraminic ac i d and i t s d e r i v a t i v e s , s i n c e the a c t u a l nature of the a c i d i n the heart i s not known. F. Neuraminidase Assay; The a c t i v i t y of the enzyme neuraminidase was determined by the method described by Cassidy et a l . (1965). B r i e f l y , the reaction mixture (125 \il) contained 25 u.1 sodium acetate b u f f e r (pH = 5), 25 ul of 1.5 mg/ml BSA, and 50 ul N-acetyl-neuramin lactose (Sigma type I) containing 0.5 p o l e glyco-s i d i c a l l y - b o u n d s i a l i c a c i d . The r e a c t i o n was s t a r t e d by adding 25 u-1 of enzyme s o l u t i o n . The mixture was incubated at 37°C f o r 5 min and the reaction was stopped by incubating the mixture i n a b o i l i n g water bath f o r 1.5 min. C o n t r o l samples l a c k e d enzyme. The volume of the r e a c t i o n was adjusted to 200 ul with d i s t i l l e d water and the s i a l i c acid released was measured as described above. One u n i t neuraminidase i s d e f i n e d as the amount of enzyme that l i b e r a t e s 1 jimole of s i a l i c acid from N-acetyl-neuramin lactose per minute at pH 5 and 37°C. Neuraminidase used i n t h i s study was prepared from C l o s t r i d i u m p e r f r i n - gens, obtained from Sigma (Sigma type X). 62 G. Preparation of (Na +-K +)ATPase-Containing  Cardiac C e l l Membranes; The method of membrane p r e p a r a t i o n and the ( N a + - K + ) -ATPase assay are based on e a r l i e r reports (see e.g. Schwartz et a l . , 1971). Five guinea pigs of e i t h e r sex weighing 350-500 g were used f o r each membrane preparation. Animals were hep a r i n i z e d (1000 U/kg, i.p.) 20 min before being anesthe-t i z e d with d i e t h y l ether. Hearts were r a p i d l y removed and pl a c e d i n i c e - c o l d s a l i n e . Unless otherwise i n d i c a t e d a l l subsequent steps were c a r r i e d out on i c e . A l l hearts were perfused with c o l d s a l i n e to f l u s h blood out of the muscle. L e f t v e n t r i c l e s were removed, cut i n t o small pieces, b l o t -ted , r a p i d l y frozen i n l i q u i d nitrogen and kept at -70°C. About 3-4 g of the l e f t v e n t r i c u l a r muscle was a c c u r a t e l y weighed and homogenized i n 20 volumes of a s o l u t i o n contain-i n g 5 mM Tris-EGTA [ethylene g l y c o l - b i s ( b e t a - a m i n o e t h y l -ether)-N,N'-tetraacetic a c i d ] , 0.25 M sucrose and 0.1% de-oxycholate at pH 7 using a Polytron (PT 10 20 3500) at speed 5 for 10-15 s. Homogenates were kept on i c e and homogenized 5-6x over a 30 min period. The tubes were placed i n the ice between homogenizations. The homogenates were centrifuged at 2,000 x g (Sorvall RC2-B, rotor SS-34, 4000 rpm) f o r 15 min. The supernatant was c a r e f u l l y decanted and kept on i c e . The p e l l e t s were rehomogenized i n 20 volumes of 0.25 M sucrose with 1 mM Tris-EGTA and c e n t r i f u g e d as d e s c r i b e d b e f o r e (2000 x g for 15 min). Supernatants were c a r e f u l l y decanted, mixed with the p r e v i o u s supernatant, and c e n t r i f u g e d at 96,000 x g (Beckman L5-50, rotor SW27, 27,000 rpm) for 1 h. The p e l l e t was suspended i n 8 ml (2 volumes) 1 mM Tris-EGTA. Then 4 ml (1/2 the Tris-EGTA volume) of a s o l u t i o n contain-i n g 6 M Nal, 15 mM EGTA, 7.5 mM MgCl 2 and 150 mM Tris-base (pH = 8) was added drop by drop with continuous s t i r r i n g on i c e . The mixture was s t i r r e d on i c e f o r 30 min, 18 ml (1.5 x t o t a l volume) 1 mM Tris-EGTA was added and the mixture s t i r r e d for 5-10 min. The mixture was centrifuged at 96,000 x g (Beckman L5-50, ro t o r SW 27, 27,000 rpm) f o r 1 h. The p e l l e t was washed twice with 1 mM Tris-EGTA and the f i n a l p e l l e t was suspended i n 2.5 x volumes (based on t i s s u e wet weight) 1 mM Tris-EGTA. The suspension was q u i c k l y frozen i n an acetone-dry ice bath and kept at -70°C u n t i l used. The preparation was l a t e r thawed and t r i p l i c a t e s of 100 [il samples were taken for p r o t e i n assay by a modified Lowry method (Hartree, 1972) using BSA as a standard. The remain-ing suspension was di v i d e d into two equal portions and the volume was adjusted by approximately a two-fold d i l u t i o n so that i t contained ( c o n t r o l ) 0.3 mg/ml BSA and (neuramini-dase-treated) 0.3 mg/ml BSA plus 0.01 U/ml neuraminidase (Sigma type X). Both samples were incubated at 37°C for 1 h. H. Assay of (Na +-K +)ATPase: A c t i v i t y of the enzyme (Na +-K +)ATPase was assayed by measuring the amount of i n o r g a n i c phosphate l i b e r a t e d from ATP. B r i e f l y , a l i q u o t s o f the above p r e p a r a t i o n 64 c o n t a i n i n g about 200 u.g p r o t e i n (about 200 u.1) were i n c u -bated f o r 30 min at 37°C i n a medium ( f i n a l volume = 1 ml) containing 2.5 mM Tris-ATP, 3 mM MgCl 2, 1 mM Tris-EGTA, 50 mM T r i s - H C l b u f f e r (pH = 7.4), 100 mM NaCl, and 10 mM KC1. 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 ATP and was stopped by the a d d i t i o n of 1 ml i c e - c o l d 10% t r i c h l o r a c e t i c a c i d . Samples were c e n t r i f u g e d at 3,000 x g f o r 10 min and 1 ml of the supernatant was used for the determination of l i b e r a t e d i n o r g a n i c phosphate by the method of Martin and 2 + Doty (1949). Mg -dependent ATPase a c t i v i t y was determined i n the absence of NaCl and KCl.To investigate the concentra-tion-dependent i n h i b i t i o n of the enzyme (Na +-K +)ATPase by ouabain, the assay was c a r r i e d out i n the presence of — 9 -3 i n c r e a s i n g c o n c e n t r a t i o n s of ouabain (10 -10 M). The assay was car r i e d out i n duplicate. I. Determination of Inorganic Phosphate: Inorganic phosphate l i b e r a t e d by the h y d r o l y s i s of ATP was measured by the c o l o r i m e t r i c method described by Martin and Doty (1949). The method i s based on the formation of a phosphomolybdate complex and rapid extraction of the complex i n t o an isobutanol-benzene mixture. Since only i n o r g a n i c phosphate i s extracted as phosphomolybdate complex leaving ATP and other organic phosphate compounds i n the aqueous la y e r , t h i s process allows rapid separation of the enzyma-t i c a l l y released inorganic phosphate and prevents i n t e r f e r -ence from the subsequent a c i d h y d r o l y s i s o f ATP. C o l o r 65 development i s then e f f e c t e d by stannous c h l o r i d e , and the i n t e n s i t y of the blue c o l o r developed at the peak wave-lengths of maximum absorbance of 730 or 625 nm i s propor-t i o n a l to the amount of inorganic phosphate present i n the sample. B r i e f l y , 1 ml of the sample solut i o n was transferred to a t e s t tube containing 1 ml molybdate s o l u t i o n (1.25% ammon-ium molybdate i n IN H2SC<4) and 2.5 ml isobutanol-benzene mixture (1:1 v/v), and the mixture was vigorously shaken for 15 s. The t e s t tube was then c e n t r i f u g e d f o r 5 min at 1500 x g to separate the two phases. A volume of 1 ml of the upper isobutanol-benzene layer was tr a n s f e r r e d i n t o another t e s t tube c o n t a i n i n g 2 ml of e t h a n o 1 - H 2 S O 4 d i l u t i n g s o l u t i o n (mixture of 98 ml absolute ethanol and 2 ml concen-t r a t e d H 2 S 0 4 ) . Then 0.1 ml of f r e s h l y prepared reducing stannous c h l o r i d e s o l u t i o n (stock s o l u t i o n was made of 40 g stannous c h l o r i d e i n 100 ml concentrated HC1; the reducing s o l u t i o n was made by a 200 x d i l u t i o n of the stock s o l u t i o n i n 1 N H 2S0 4) was added t o the mixture and the contents were mixed. The absorbance of the blue c o l o r developed was measured at 625 nm against a s i m i l a r l y prepared blank. The absolute amount of inorganic phosphate was c a l c u l a t e d using a s i m i l a r l y prepared standard curve. 66 J. Determination of [ H]Ouabain Binding; 3 The [ H]ouabain b i n d i n g assay i s based on e a r l i e r r e p o r t s by d i f f e r e n t i n v e s t i g a t o r s (see Erdmann, 1981). ( N a + - K + ) A T P a s e - c o n t a i n i n g g u i n e a p i g l e f t v e n t r i c u l a r membrane p r e p a r a t i o n s were prepared as d e s c r i b e d before except the step of Nal treatment was e l i m i n a t e d . C o n t r o l and n e u r a m i n i d a s e - t r e a t e d p r e p a r a t i o n s were p r e p a r e d 3 s i m i l a r l y . [ H]Ouabain b i n d i n g was determined by a r a p i d f i l t r a t i o n technique. B r i e f l y , samples of about 0.2-0.5 mg p r o t e i n were incubated at room temperature (24°C) for 1 h i n a medium ( t o t a l volume =0.5 ml) containing 1 mM Tris-EGTA, 4 mM MgCl 2, 3 mM T r i s - p h o s p h a t e , 50 mM T r i s - H C l b u f f e r (pH = 7.4), and d i f f e r e n t c o n c e n t r a t i o n s (12-380 nM) of 3 [ H]ouabain (1.7 Ci/mmol.). Nonspecific binding was deter--3 mxned xn the presence of 10 M ouabain. The r e a c t i o n was terminated by r a p i d f i l t r a t i o n through Whatman GF/B glass m i c r o f i b r e f i l t e r s . T e s t tubes and f i l t e r s were washed twice with 4 ml i c e - c o l d deionized d i s t i l l e d water. Radio-a c t i v i t y trapped on the f i l t e r s was determined by l i q u i d s c i n t i l l a t i o n counting (10 ml Biofluor, New England Nuclear; Mark I I I l i q u i d s c i n t i l l a t i o n system Model 6880, Se a r l e , U.S.A.). T r i p l i c a t e s and duplicates of t o t a l and nonspecific binding, respectively, were carried out i n each assay. 67 K. Determination of Protease A c t i v i t y ; The a c t i v i t y of p r o t e o l y t i c enzymes was determined by an u l t r a s e n s i t i v e c o l o r i m e t r i c method described by Rinder-knecht et a l . (1968). The method i s based on the protease s o l u b i l i z a t i o n of an i n s o l u b l e p r o t e i n substrate complexed with a dye. In t h i s assay, the substrate was derived from hide powder l a b e l l e d covalently with the dye Remazobrilliant Blue. The s o l u b i l i z e d s u b s t r a t e c a r r i e s the dye i n t o the s o l u t i o n and the i n t e n s i t y of the color i s used as an index of the p r o t e o l y t i c a c t i v i t y of the enzyme. This method i s s e n s i t i v e to as low as 1-2 ng/ml assay mixture of t r y p s i n , f i b r i n o l y s i n and elastase. B r i e f l y , t e s t tubes (1.5 x 15 cm) were charged with 40 mg of Rem a z o b r i l l i a n t Blue-hide (Hide powder azure, Sigma Chemical Company). A measured amount of the enzyme so l u t i o n was added and the volume of the i n c u b a t i o n mixture was adjusted to 5 ml with T r i s - H C l b u f f e r (pH = 7.4 at 37°C). Test tubes were stoppered, placed i n an incubator at 37°C and agitated gently at 5 min i n t e r v a l s for a 30 min period. At the end of the incubation period, t e s t tubes were cooled i n an ice-water bath and the contents were f i l t e r e d through Whatman No. 1 f i l t e r papers. The i n t e n s i t y of the blue c o l o r of the f i l t r a t e was measured by a spectrophotometer at 595 nm, the wavelength of maximum absorbance of the dye. Blanks were prepared s i m i l a r l y , except t h a t the enzyme s o l u t i o n s were added at the end of the i n c u b a t i o n p e r i o d 68 immediately before f i l t r a t i o n of the mixture. Blank values were subtracted from readings furnished by the t e s t samples. To a c t i v a t e calcium-dependent proteases, the assay was also conducted i n the presence of a f i n a l concentration of 2 mM calcium. L. The Enzyme Neuraminidase: The enzyme neuraminidase (Sigma Chemical Company, type X) used i n t h i s study was prepared and p u r i f i e d from C l o s t r i d i u m p e r f r i n g e n s . The p r o c e s s i n c l u d e d s a l t f r a c t i o n a t i o n (Sigma type V), followed by chromatographic p u r i f i c a t i o n (Sigma type V I I I ) , and a f u r t h e r p u r i f i c a t i o n by a f f i n i t y chromatography to o b t a i n type X. The product was then d i a l y z e d and l y o p h i l i z e d . The f i n a l product con-tained 100% protein and had a s p e c i f i c a c t i v i t y of about 150 units/mg p r o t e i n . One unit neuraminidase was defined as the amount of enzyme that l i b e r a t e s 1.0 ^mole of N-acetylneura-minic a c i d ( s i a l i c acid) per min at pH 5.0 and 37°C, using N-acetylneuramin l a c t o s e as a su b s t r a t e . Each v i a l of the enzyme product was accompanied with a data sheet that i n d i -c a ted the c h a r a c t e r i s t i c s of the enzyme and the l e v e l of i m p u r i t i e s present. The data provided by the s u p p l i e r f o r an enzyme sample used i n t h i s study i n d i c a t e d t h a t the product's protease and aldolase a c t i v i t i e s were < 0.002 and 0.004 unit/mg p r o t e i n , r e s p e c t i v e l y . Hence, the protease and aldolase a c t i v i t i e s of t h i s product were equivalent to — 5 — 5 < 1.3x10 and 2.7x10 u n i t / u n i t neuraminidase, respec-69 t i v e l y . Other batches o f the enzyme provided by the same sup p l i e r contained s i m i l a r amounts of protease and aldolase i m p u r i t i e s . One u n i t protease a c t i v i t y was defined as the amount of enzyme that w i l l hydrolyze casein to produce color e q u i v a l e n t to 1.0 p o l e (181 ng) of t y r o s i n e per min at pH 7.5 and at 37°C ( c o l o r per F o l i n - C i o c a l t e u r e a g e n t ) . One u n i t of aldolase a c t i v i t y was defined as the amount of enzyme that w i l l release 1.0 umole of pyruvate from N-ace-tylneuraminic acid per min at pH 7.2 and at 37°C. M. Protein Assay; Samples of membrane p r e p a r a t i o n s (0.1-0.5 ml) were mixed with 1 ml 1 N NaOH and allowed to stand at room tem-perature for 3-4 h. These solutions were then neutralized by the a d d i t i o n of 1 ml 1 N HC1. Proper volumes of the f i n a l s o l u t i o n (0.1-0.5 ml) containing 10-100 \xg protein were used f o r the f i n a l p r o t e i n assay. The assay was c a r r i e d out according to the modified Lowry method described by Hartree (1972). B r i e f l y , samples were d i l u t e d with d i s t i l l e d water to 1 ml and 0.9 ml of sol u t i o n A was added ( s o l u t i o n A: 2 g potassium sodium t a r t r a t e and 100 g were d i s s o l -ved i n 500 ml 1 N NaOH and d i l u t e d with d i s t i l l e d water to 1 l i t e r ) . Test tubes were shaken and placed i n a water bath at 50°C f o r 10 min. Test tubes were then removed from the water bath and allowed to stand f o r 25 min to reach room temperature. This was followed by the add i t i o n of 0.1 ml of s o l u t i o n B ( s o l u t i o n B: 2 g potassium sodium t a r t r a t e and 70 1 g CuSO^.Sf^O were d i s s o l v e d i n 90 ml d i s t i l l e d water and then 10 ml 1 N NaOH was added). Samples were shaken and l e f t to stand at room temperature for at l e a s t 10 min. Then 3 ml of s o l u t i o n C ( s o l u t i o n C: 1 volume F o l i n - C i o c a l t e u reagent was d i l u t e d with 15 volumes of d i s t i l l e d water; t h i s s o l u t i o n was f r e s h l y prepared) was f o r c e d i n r a p i d l y to ensure mixing with 2-3 s. Test tubes were again heated i n a water bath at 50°C f o r 10 min and then cooled to room tem-perature. Absorbance of the solutions was measured at 6 50 nm ag a i n s t a s i m i l a r l y prepared blank. Standard curves were simultaneously prepared i n each assay using a standard solu-t i o n of bovine serum albumin (0.5 mg/ml). The amount of the sample's protein was calculated using the l i n e a r part of the standard curve (10-100 \xg) , and t h e r e f o r e the c a l c u l a t e d amount of pr o t e i n was presented as mg of p r o t e i n equivalent to mg of bovine serum albumin. The assay was c a r r i e d out i n duplicate or t r i p l i c a t e . N. S t a t i s t i c a l Analysis; A l l data were evaluated s t a t i s t i c a l l y by an appropriate t e s t u s i n g a n a l y s i s of v a r i a n c e , u n p a i r e d t - t e s t , or a paired t - t e s t . A p r o b a b i l i t y of 95% (p<0.05) was preselected as the c r i t e r i o n of s t a t i s t i c a l s i g n i f i c a n c e . A l l data are presented as mean values ± the standard e r r o r of the mean (S.E.M.). 71 S E C T I O N I I I R E S U L T S 72 A. Eff e c t of Lanthanum on Control C o n t r a c t i l i t y and  Isoproterenol Inotropy: Langendorff p r e p a r a t i o n s o f a d u l t guinea p i g hea r t s were perfused with i n c r e a s i n g concentrations of lanthanum ranging from 0.05-3 uM• P e r f u s i o n with each conc e n t r a t i o n of lanthanum was continued for 3-5 min to obtain a maximum e f f e c t . As shown i n F i g . l , lanthanum caused a concentration-dependent decrease i n b a s a l c o n t r a c t i l i t y . Concentrations as low as 0.5 u-M lanthanum produced about an 82% decrease i n the basal c o n t r a c t i l i t y index (+dP/dt ). The c a l c u l a t e d J max concentration of lanthanum that produced 50% i n h i b i t i o n of b a s a l c o n t r a c t i l i t y ( I C 5 0 ) was 0.19 ± 0.01 uM (mean ± S.E.M.; n = 5). The maximum c o n c e n t r a t i o n of lanthanum used (3 |iM) c o n s i s t e n t l y produced a 97% decrease i n the +dP/dt of the heart, max To study the e f f e c t of lanthanum on the p o s i t i v e ino-t r o p i c response of i s o p r o t e r e n o l , Langendorff preparations of adult guinea pig hearts were f i r s t perfused with a single concentration of lanthanum (0.25-3 uJM) for 3-5 min followed -9 by i n c r e a s i n g c o n c e n t r a t i o n s of i s o p r o t e r e n o l (10 — 8 5x10 M) c o n t a i n i n g the same c o n c e n t r a t i o n of lanthanum. F i g . 2 shows the concentration-response curves of i s o p r o -terenol i n the control hearts and i n the presence of 0.5, 1, and 3 uM lanthanum. The calculated concentrations of isopro-t e r e n o l that produced 50% of the maximum e f f e c t (EC^ Q) i n the presence of d i f f e r e n t concentrations of lanthanum were 73 FIG. 1. The c o n c e n t r a t i o n - r e s p o n s e curve of the n e g a t i v e i n o t r o p i c e f f e c t of lanthanum i n Langendorff preparations of a d u l t guinea p i g h e a r t s . Hearts were perfused with Hepes b u f f e r containing 1.8 mM calcium and i n c r e a s i n g concentra-t i o n s of lanthanum. Each c o n c e n t r a t i o n of lanthanum was perfused f o r 3-5 min to obta i n a maximum e f f e c t . Results are mean values ± S.E.M. (n = 5). 74 75 FIG. 2. E f f e c t of d i f f e r e n t concentrations of lanthanum on the p o s i t i v e i n o t r o p i c e f f e c t of i s o p r o t e r e n o l i n Langendorff prep a r a t i o n s of adult guinea p i g h e a r t s . Hearts were per-fu s e d w i t h Hepes b u f f e r c o n t a i n i n g 1.8 mM c a l c i u m . The curves show the response to i s o p r o t e r e n o l (+dP/dt ) i n ^ ^ ' max co n t r o l preparations ( f i l l e d t r i a n g l e s ) and i n the presence of 0.5 (open squares), 1.0 ( f i l l e d c i r c l e s ) , and 3.0 (open tr i a n g l e s ) \xM lanthanum. Lanthanum-treated hearts were f i r s t perfused with a s i n g l e concentration of lanthanum f o r 3-5 min followed by i n c r e a s i n g concentrations of i s o p r o t e r e n o l containing the same concentration of lanthanum. The response to isoproterenol i n the presence of 0.25 and 0.75 \iM lantha-num was also studied, but the data are not shown to increase the c l a r i t y of the f i g u r e . The concentration-response curve of isoproterenol i n the presence 0.25 \iM lanthanum overlap-ped the c o n t r o l curve. Note that the maximum response to i s o p r o t e r e n o l i n the presence of 3.0 |j.M lanthanum was 95% l e s s than that of c o n t r o l . Both i n c o n t r o l and i n the pre-sence of lanthanum, concentrations of i s o p r o t e r e n o l higher — 8 th a n 5x10 M d i d not cause a f u r t h e r i n c r e a s e i n the — 8 maximum +dP/dt d e v e l o p e d at 5x10 M i s o p r o t e r e n o l max c (tested i n a few control and lanthanum-treated h e a r t s ) . The data are mean values ± S.E.M. (n = 5 i n each group). (*) Control (n = 5) (•) 0.5 jjm La (n= 5) 3 0 0 0 - . (•) L O u m L a (n=5) (A) 3.0 jum La (n = 5) 2500-2000-1500-1000-5 0 0 -9 8 7 ISOPROTERENOL, - log (M) not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) from control (Table 1). In a few experiments higher concentrations of isoproterenol (>5xl0 M) were used to determine i f the i n h i b i t o r y e f f e c t of lanthanum could be reversed. Concentrations of i s o p r o -t e r e n o l h i g h e r than 5x10 M d i d not inc r e a s e the maximum i n o t r o p i c r esponse of i s o p r o t e r e n o l d e v e l o p e d at the — 8 5x10 M concentration e i t h e r i n c o n t r o l or i n the presence of the concentrations of lanthanum tested (0.25-3 yM). While 0.25 p.M lanthanum reduced basal c o n t r a c t i l i t y by 63% (Fig. 1), i t had no e f f e c t on the response to isoprote-r e n o l . The concentration-response curve of isoproterenol i n the presence of 0.25 [iM lanthanum overlapped the c o n t r o l curve (data not shown). On the other hand 3 (iM lanthanum reduced b a s a l c o n t r a c t i l i t y ( F i g . 1) and the maximum re s -— 8 ponse t o i s o p r o t e r e n o l (5x10 M; F i g . 2) by 97 and 95%, respectively. F i g . 3 shows the e f f e c t of 5 |j.M lanthanum on the — 8 response to 5x10 M i s o p r o t e r e n o l . In t h i s experiment the — 8 heart was f i r s t treated with 5x10 M i s o p r o t e r e n o l and the drug was r a p i d l y washed out. Following a 45 min p e r f u s i o n w i t h Hepes b u f f e r , b a s a l c o n t r a c t i l i t y r e t u r n e d to the i n i t i a l c o ntrol l e v e l . The heart was then treated with 5 \iM lanthanum followed by the same concen t r a t i o n of lanthanum — 8 p l u s 5x10 M i s o p r o t e r e n o l . I s o p r o t e r e n o l caused a s l i g h t i n c r e a s e i n the c o n t r a c t i l i t y . Compared to the i n i t i a l response to i s o p r o t e r e n o l i n the absence of lanthanum, the 78 TABLE 1. Ef f e c t of Lanthanum on the EC,-Q of Isoproterenol in Langendorff Preparations of the Guinea Pig Heart. Treatment E C50 (10 9M) Control 4.9 ± 0.9 Lanthanum: 0.5 uM 6.4 ± 1.0 1.0 uM 4.1 ± 0.6 3.0 [iM 5.2 ± 0.6 NOTE: E<"50 '""S t^ i e c a l c u l a t e ( ^ concentration of isoprote-renol that produces 50% of the maximum e f f e c t . Data were obtained from the concentration-response curves of isopro-terenol shown i n Fi g . 2. Note that the EC,-Q values of isoproterenol i n the presence of d i f f e r e n t concentrations of lanthanum were not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) from control. Results are mean values ± S.E.M. (n = 5 i n each group). 79 FIG. 3. E f f e c t of 5 |j.M lanthanum on the i n o t r o p i c response to — 8 5x10 M i s o p r o t e r e n o l . T h i s f i g u r e shows the o r i g i n a l t r a c i n g of a Langendorff preparation of an adult guinea pig heart perfused with Hepes buffer containing 1.8 mM calcium. The upper p a r t of the t r a c e shows the i n t r a v e n t r i c u l a r pressure of the l e f t v e n t r i c l e , and the bottom part shows the f i r s t d e r i v a t i v e of the p r e s s u r e curve (+dP/dt ) * ' max recorded simultaneously. Following 45 min e q u i l i b r a t i o n with Hepes buffer, the heart was treated with the maximum e f f e c -— 8 t i v e c o n c e n t r a t i o n of i s o p r o t e r e n o l (5x10 M). The drug was r a p i d l y washed out and p e r f u s i o n was continued with Hepes b u f f e r for 45 min. Following washout with the b u f f e r b a s a l c o n t r a c t i l i t y was r e t u r n e d t o i t s i n i t i a l c o n t r o l l e v e l . Then the h e a r t was p e r f u s e d w i t h 5 uM lanthanum f o l l o w e d by the same c o n c e n t r a t i o n of lanthanum p l u s _ g 5x10 M i s o p r o t e r e n o l . I s o p r o t e r e n o l produced a p o s i t i v e i n o t r o p i c e f f e c t i n the presence of lanthanum; however the response compared to the i n i t i a l response i n the absence of lanthanum was reduced by 94%. Note that a t e n - f o l d increase -7 i n i s o p r o t e r e n o l concentration to 5x10 M d i d not cause a further increase i n the response to isoproterenol. 80 5x10"8M 5uM 5x10 _ 8M 5x10"7M Control Isoproterenol La 3 * Isoproterenol 81 response to i s o p r o t e r e n o l was shows that a t e n - f o l d increase _7 t i o n to 5x10 M d i d not cause c o n t r a c t i l i t y . reduced by 94%. F i g . 3 a l s o i n i s o p r o t e r e n o l concentra-any f u r t h e r i n c r e a s e i n the B. E f f e c t of Lanthanum on Isoproterenol-Induced  C y c l i c AMP Level; To determine the p o s s i b l e e f f e c t o f lanthanum on i s o p r o t e r e n o l b i n d i n g to the beta-receptor and subsequent c o u p l i n g t o the enzyme adenylate c y c l a s e , the e f f e c t of lanthanum on b a s a l and i s o p r o t e r e n o l - i n d u c e d c y c l i c AMP l e v e l s was investigated. In t h i s study c y c l i c AMP content of L a n g e n d o r f f p r e p a r a t i o n s of c o n t r o l h e a r t s and h e a r t s — 8 t r e a t e d with 3 |aM lanthanum, 5x10 M i s o p r o t e r e n o l , and — 8 5x10 M i s o p r o t e r e n o l p l u s 3 |iM lanthanum was measured ( F i g . 4). Note that c y c l i c AMP content of h e a r t s t r e a t e d with 3 \iM lanthanum was not d i f f e r e n t from control untreated hearts. Although the isoproterenol-induced c y c l i c AMP l e v e l i n the presence of 3 |j.M lanthanum was lower than c o n t r o l i s o p r o t e r e n o l - t r e a t e d preparations, the two values were not s i g n i f i c a n t l y d i f f e r e n t (p>0.05). In the presence of 3 i^M — 8 lanthanum, 5x10 M i s o p r o t e r e n o l produced about a 120% increase i n c y c l i c AMP content of the hearts. 82 FIG. 4. E f f e c t of lanthanum on basal and isoproterenol-induced c y c l i c AMP l e v e l s . Langendorff preparations of adult guinea p i g hearts were frozen a f t e r : a) e q u i l i b r a t i o n with Hepes b u f f e r (U), b) 43 min treatment with 3 p.M lanthanum, c) 9-— 8 10 s treatment with 5x10 M i s o p r o t e r e n o l , and d) 9-10 s — 8 treatment with 5x10 M i s o p r o t e r e n o l plus 3 |iM lanthanum following a 3 min treatment with 3 \iM lanthanum, and c y c l i c AMP content of the v e n t r i c l e s was determined. Results shown are mean ± S.E.M. of 5 observations i n each group. Note that 3 \iM lanthanum had no e f f e c t on b a s a l and i s o p r o t e r e n o l -induced c y c l i c AMP level s (p>0.05). [La3 +] = 3 pM 84 3 C. [ H]Nitrendipine Binding i n the Guinea Pig Left V e n t r i c l e : 3 S p e c i f i c C H j n i t r e n d i p i n e b i n d i n g i n the membrane p r e p a r a t i o n of the guinea p i g l e f t v e n t r i c l e reached a maximal l e v e l f o l l o w i n g 60 min incubation at room tempera-t u r e (23°C) and remained unchanged f o r up to the 120 min inves t i g a t e d . In t h i s study a 90 min incubation was used to ensure maximal b i n d i n g . The s p e c i f i c b i n d i n g of 38 pM 3 [ H ] n i t r e n d i p i n e was l i n e a r l y p r o p o r t i o n a l to the concen-t r a t i o n of p r o t e i n i n the assay up to 0.4 mg/ml (data not shown). F i g . 5 shows a t y p i c a l s a t u r a t i o n experiment. Note 3 t h a t s p e c i f i c b i n d i n g of [ H ] n i t r e n d i p i n e ( F i g . 5A) was h y p e r b o l i c while the n o n s p e c i f i c b i n d i n g was l i n e a r and nonsaturable. Scatchard a n a l y s i s of s p e c i f i c binding (Fig. 5B) was l i n e a r (r = 0.997) su g g e s t i n g the presence of a 3 s i n g l e c l a s s of [ H ] n i t r e n d i p i n e b i n d i n g s i t e s . R e s u l t s obtained from f i v e independent experiments showed a d i s s o -c i a t i o n constant (K^) i n the c o n t r o l preparation of 90.0 ± 3.4 pM (mean ± S.E.M.) and a maximal number of binding s i t e s (B ) of 70.5 ± 6 . 3 fmol/mg protein (Table 2; page 91). 85 FIG. 5. 3 T y p i c a l s a t u r a t i o n experiment of [ H ] n i t r e n d i p i n e b i n d i n g i n a membrane p r e p a r a t i o n of the guinea p i g l e f t v e n t r i c l e . Samples of a EDTA-washed membrane p r e p a r a t i o n (see Methods) containing 0.27 mg protein were incubated with 3 varying concentrations of [ H] n i t r e n d i p i n e (24.4-517.7 pM) i n the dark for 90 min at 23°C (50 mM T r i s - H C l ; pH = 7.4; t o t a l volume = 1 ml) i n the presence and absence of 10 M^ n i f e n d i p i n e . The concentration-dependent s p e c i f i c ( f i l l e d c i r c l e s ; mean of t r i p l i c a t e ) and nonspecific (open c i r c l e s ; 3 mean of d u p l i c a t e ) [ H ] n i t r e n d i p i n e b i n d i n g i s shown i n A, and Scatchard p l o t of the s p e c i f i c binding i s shown i n B. Linear regression analysis of the Scatchard p l o t (r = 0.997) shows a K, value (-1/slope) of 84.9 pM and a B (X-d \ i t- i f max intercept) of 73.3 fmol/mg protein. 86 87 D. Effects of Calcium and Lanthanum on 3 [ H]Nitrendipine Binding: 3 To i n v e s t i g a t e the e f f e c t of c a l c i u m on [ H ] n i t -r e n d i p i n e b i n d i n g , b i n d i n g of a s i n g l e c o n c e n t r a t i o n of 3 [ H ] n i t r e n d i p i n e ( 90-120 pM) was c a r r i e d o ut i n the 2 + presence of d i f f e r e n t concentrations of Ca (0.1-10 mM), 2 + 1 mM EDTA and 1 mM EDTA plus 3 mM Ca ( F i g . 6). S p e c i f i c b i n d i n g was expressed as percentage of c o n t r o l s p e c i f i c 2 + b i n d i n g . As shown i n F i g . 6, Ca (0.1-10 mM) i n c r e a s e d 3 the s p e c i f i c b i n d i n g of [ H ] n i t r e n d i p i n e with a maximal e f f e c t at 1 mM c o n c e n t r a t i o n . A f u r t h e r i n c r e a s e i n the 2 + Ca c o n c e n t r a t i o n from 1 mM to 10 mM d i d not cause any increase i n s p e c i f i c binding. However, s p e c i f i c binding of 3 2 + [ H ] n i t r e n d i p i n e a t 10 mM Ca was s l i g h t l y l e s s than 2 + t h a t observed at 1 mM Ca , but was not s i g n i f i c a n t l y d i f f e r e n t (p>0.05). F i g . 6 a l s o shows t h a t s p e c i f i c 3 [ H ] n i t r e n d i p i n e binding i n the presence of 1 mM EDTA was reduced to 16.3% of con t r o l and t h i s e f f e c t was reversed by 2 + 3 mM Ca . However a second wash of the membrane prepara-t i o n with 10 mM EDTA had no e f f e c t on the b a s a l l e v e l of 3 [ H]nitrendipine binding (data not shown). To study the e f f e c t s of calcium and lanthanum on the 3 c h a r a c t e r i s t i c s of [ H ] n i t r e n d i p i n e b i n d i n g , s a t u r a t i o n experiments were c a r r i e d out i n the presence of e i t h e r or both cations. Results of t h i s study, summarized i n Table 2 2 + (page 91), show t h a t i n the p r e s e n c e of 2 mM Ca the 88 FIG. 6. 3 E f f e c t of c a l c i u m on [ H ] n i t r e n d i p i n e b i n d i n g i n membrane p r e p a r a t i o n s of the guinea p i g l e f t v e n t r i c l e . Membrane preparations washed with 10 mM EDTA were incubated 3 with a s i n g l e c o n c e n t r a t i o n of [ H ] n i t r e n d i p i n e (90-120 pM; see Methods) i n the presence of varying concentrations 2+ of Ca . T o t a l and n o n s p e c i f i c binding ( i n the presence of — 6 10 M n i f e d i p i n e ) were c a r r i e d out i n d u p l i c a t e . S p e c i f i c b i n d i n g was expressed as a percentage of c o n t r o l s p e c i f i c 2 + b i n d i n g (open bar) i n the absence of Ca or c h e l a t o r . Results shown are mean values of 5 independent experiments ± 2+ 3 S.E.M. Note t h a t Ca enhanced [ H ] n i t r e n d i p i n e b i n d i n g 2 + ( c r o s s hatched bars) with a maximum e f f e c t at 1 mM Ca concentration. Also note that 1 mM EDTA ( s o l i d bar) greatly 3 d i m i n i s h e d [ H ] n i t r e n d i p i n e b i n d i n g and t h i s e f f e c t was 2 + reversed with 3 mM Ca (hatched bar). 90 3 Bmax °^ **3nitrendipine b i n d i n g was i n c r e a s e d to about 154.3% of c o n t r o l and the was s l i g h t l y and s i g n i f i c a n t -l y (p<0.05) i n c r e a s e d to about 140 pM. Meanwhile 0.1 mM EDTA reduced the B to about 25.6% of c o n t r o l but had no max s i g n i f i c a n t e f f e c t on the K,. The B i n the presence d max 3 + of 10 (iM La was reduced to 63.2% of c o n t r o l . Calcium 3 + (10 mM) p a r t i a l l y r e v e r s e d the e f f e c t of 10 \iM La and s i g n i f i c a n t l y i n c r e a s e d (p<0.05) the B m a x t o 90.6% of control (Table 2). F i g . 7 shows the concentration-dependent i n h i b i t i o n of 3 3 + s p e c i f i c [ H ] n i t r e n d i p i n e b i n d i n g by La . In t h i s study 3 binding of a s i n g l e concentration of [ H] n i t r e n d i p i n e (90-120 pM) was measured i n the presence of i n c r e a s i n g concen-3 + t r a t i o n s of La , i n the absence and presence of 10 mM 2+ 3 + Ca . The c a l c u l a t e d c o n c e n t r a t i o n of La t h a t caused 3 a 50% i n h i b i t i o n ( I C 5 Q ) of s p e c i f i c [ H ] n i t r e n d i p i n e binding i n the control preparation was 10.4 ± 0.9 p.M (mean ± !50 3 + S.E.M.). The IC,.. of La was s l i g h t l y but s i g n i f i c a n t l y 2 + i n c r e a s e d i n the presence of 10 mM Ca to 22.8 ± 2.9 |j.M (mean ± S.E.M.). 91 TABLE 2. Effects of Calcium and Lanthanum on the Charac-3 t e r i s t i c s of [ H]Nitrendipine Binding i n the Guinea Pig Left V e n t r i c l e . B max Treatment K, (pM) (fmol/mg) % of controlt Control 90. 0±3.4 70. 5±6. 3 100 2 mM C a 2 + 140. 0±13.6* 107. 6±8. 3* 154. 3±8. 6 0.1 mM EDTA 119. 5±12.9 18. 6±3. 2* 25. 6±2. 7 10 uM L a 3 + 123. 1+9.5* 45. 0±5. 3* 63. 2±2. 3 10 u-M L a 3 + _ + 10 mM Ca 145. 4±13.5* 64. 3±7. 3 90. 6±2. Q* * NOTE: Saturation experiments of [ H]nitrendipine binding (17-550 pM) i n 10 mM EDTA-washed membrane preparations of the guinea pig l e f t v e n t r i c l e were ca r r i e d out (see Methods and legend of F i g . 5, page 85) i n the absence (control) and presence of the agents indicated under treatment. t Since i n each experiment a l l treatments were carried out on the same membrane preparation, the B was expressed ^ * max ^ as percentage of the control B to eliminate the e f f e c t c max of v a r i a b i l i t y among membrane preparations. * S i g n i f i c a n t l y d i f f e r e n t from control (p<0.05). 3+ 2+ ** The B i n the presence of 10 u.M La + 10 mM Ca was max r s i g n i f i c a n t l y greater (p<0.05) than the B i n the ^ J ^ max 3 + presence of 10 uM La Results are mean values of 5 independent experiments ± S.E.M. 92 F I G . 7. 3 Concentration-dependent i n h i b i t i o n of [ H ] n i t r e n d i p i n e binding by lanthanum i n membrane preparations of the guinea 3 p i g l e f t v e n t r i c l e . S p e c i f i c [ H ] n i t r e n d i p i n e binding was 3 measured ( s i n g l e c oncentration of 90-120 pM [ H ] n i t r e n d i -pine; see Methods) i n presence of increasing concentrations 3 + o f La i n c o n t r o l p r e p a r a t i o n s (n = 5; f i l l e d c i r c l e s ) 2 + and i n the presence of 10 mM Ca (n = 4; open c i r c l e s ) . 3 + The c a l c u l a t e d concentration of La that caused 50% i n h i -3 b i t i o n (ICJ-Q) of [ H] n i t r e n d i p i n e b i n d i n g was s i g n i f i -2 + c a n t l y i n c r e a s e d (p<0.05) i n the presence of 10 mM Ca (from 10.4 ± 0.9 p.M to 22.8 ± 2.9 fxM) . Resu l t s shown are mean values ± S.E.M. ( • ) C O N T R O L ( n = 5 ) COD 1 0 m M C A L C I U M (n=4D L A N T H A N U M , log (MD 94 E. E f f e c t of Lanthanum on [ H]Nitrendipine Binding  and Myocardial C o n t r a c t i l i t y ; F i g . 8 shows the conc e n t r a t i o n - d e p e n d e n t e f f e c t of 3 lanthanum on [ H ] n i t r e n d i p i n e b i n d i n g and m y o c a r d i a l c o n t r a c t i l i t y i n Langendorff preparations of the guinea pig heart (data shown i n F i g . 1). Both studies were c a r r i e d out at 30°C. Note that 1 uM lanthanum reduced basal myocardial c o n t r a c t i l i t y by about 90% but had no s i g n i f i c a n t e f f e c t on 3 [ H ] n i t r e n d i p i n e b i n d i n g . The c a l c u l a t e d c o n c e n t r a t i o n of 3 lanthanum that produced 50% i n h i b i t i o n of [ H N i t r e n d i p i n e binding was 12.4 ± 1.1 uM (mean ± S.E.M.; n = 4), while the c a l c u l a t e d concentration of lanthanum that reduced myocar-d i a l c o n t r a c t i l i t y by 50% was 0.19 ± 0.01 uM (mean ± S.E.M.; n = 5). Table 3 shows the e f f e c t of 3 |iM lanthanum on the 3 c h a r a c t e r i s t i c s of [ H ] n i t r e n d i p i n e b i n d i n g . Note t h a t 3 u-M lanthanum had no e f f e c t on the d i s s o c i a t i o n constant 3 ( ) of [ H ] n i t r e n d i p i n e b i n d i n g . However, lanthanum (3 uM) caused a s i g n i f i c a n t decrease i n the maximal number 3 ( B m a x ) of [ H ] n i t r e n d i p i n e b i n d i n g s i t e s (p<0.05). The 3 ^max °^ ^ n i t r e n d i p i n e b i n d i n g i n the presence of 3 u.M lanthanum was 69.3 ± 3.3% of cont r o l (mean ± S.E.M.; n = 5). Note that 3 |iM lanthanum c o n s i s t e n t l y reduced basal myocar-d i a l c o n t r a c t i l i t y by about 97% (Fig. 8). 95 FIG. 8. E f f e c t of lanthanum on myocardial c o n t r a c t i l i t y and 3 [ H]nitrendipine binding i n the guinea pig heart. The concentration-dependent negative i n o t r o p i c e f f e c t of lanthanum ( f i l l e d c i r c l e s ; the curve on the l e f t ) was s t u d i e d i n Langendorff p r e p a r a t i o n s of a d u l t guinea p i g hearts perfused with Hepes buffer containing 1.8 mM calcium and i n c r e a s i n g c o n c e n t r a t i o n s of lanthanum at 30°C (data shown i n F i g . 1). The cal c u l a t e d concentration of lanthanum t h a t produced 50% i n h i b i t i o n of b a s a l c o n t r a c t i l i t y was 0.19 ± 0.01 |iM (mean ± S.E.M.; n = 5). 3 The concentration-dependent i n h i b i t i o n of [ H ] n i t r e n -d i p i n e b i n d i n g by lanthanum (closed squares; the curve on the r i g h t ) was s t u d i e d i n membrane p r e p a r a t i o n s of the 3 guinea p i g l e f t v e n t r i c l e . S p e c i f i c [ H ] n i t r e n d i p i n e bind-3 i n g ( s i n g l e c o n c e n t r a t i o n of 90-120 pM [ H ] n i t r e n d i p i n e ; see Methods) was measured i n the presence of i n c r e a s i n g concentrations of lanthanum at 30°C (50 mM Tris - H C l buffer; pH = 7.4). The c a l c u l a t e d concentration of lanthanum that 3 produced 50% i n h i b i t i o n of s p e c i f i c [ H j n i t r e n d i p i n e bind-ing was 12.4 ± 1.1 \xM (mean ± S.E.M.; n = 4). Results shown are mean values ± S.E.M. (•) CONTRACTILITY (n=5) (•) |3H)NITRENDIPINE BINDING (n=4) 1 2 0 - , LANTHANUM, log CM) 97 TABLE 3. Effe c t of 3 uM Lanthanum on the Char a c t e r i s t i c s 3 of [ H]Nitrendipine Binding i n the Guinea Pig Heart. Treatment K,(pM) B (fmol/mg) d ^ max ' ^  2 mM Ca 2+ 2 mM Ca 2 ++ 3 uM L a 3 + 140.0 ± 13.6 136.1 ± 10.3 107.6 ± 8.3 74.8 ± 7.0* NOTE: Membrane preparations of the guinea pig l e f t v e n t r i c l e 3 were incubated with increasing concentrations of [ H]nitren-dipine (17-550 pM) for 90 min at room temperature (23°C; 50 mM Tris-HCl buffer; pH = 7.4) i n the presence and absence —6 of 10 M nifedipine. The K, and B values were obtained c d max from the lin e a r regression analysis of Scatchard p l o t s . Results are mean values of 5 independent experimentslS.E.M. * S i g n i f i c a n t l y d i f f e r e n t from control (p<0.05). These data are presented separately i n order to c l a r i f y the r e s u l t s . 98 F. S t a b i l i t y and A c t i v i t y of Neuraminidase: E a r l y i n t h i s study i t was n o t i c e d t h a t the enzyme neuraminidase r a p i d l y loses i t s a c t i v i t y i n s o l u t i o n . F i g . 9 shows the a c t i v i t y of neuraminidase s o l u t i o n (0.1 U/ml) i n the presence and absence of 0.3 mg/ml BSA at 30 and 37°C. Note t h a t during the 2 h i n c u b a t i o n p e r i o d s t u d i e d , BSA protected the enzyme at both temperatures. In the presence of BSA, neuraminidase a c t i v i t y was 95% of i t s maximum a c t i v i t y 2 h f o l l o w i n g i n c u b a t i o n . In the absence of BSA, the enzyme l o s t 50% of i t s a c t i v i t y w i t h i n 15 min and was almost i n a c t i v e a f t e r 2 h. Note that s i m i l a r r e s u l t s were obtained at 30 and 37°C. In a l l experiments the a c t i v i t y of the enzyme was found to be c o n s i s t e n t with the s u p p l i e r ' s report. G. Protease A c t i v i t y i n the Neuraminidase Product: Protease a c t i v i t y of the neuraminidase product used i n t h i s study (Sigma type X) was examined by the c o l o r i m e t r i c method of Remazobrilliant Blue-hide (see Methods) described by Rinderknecht et a l . (1968). No protease a c t i v i t y was detected following a 30 min incubation period using 0.1 unit neuraminidase, with or without BSA. A d d i t i o n of calcium to the incubation medium ( f i n a l concentration of 2 mM) d i d not a l t e r the r e s u l t s . When a sample of the enzyme c o n t a i n i n g 0.5 u n i t s of neuraminidase (without BSA) was incubated for 2 4 h, the absorbance of the sample containing the enzyme was 99 FIG. 9. E f f e c t of bovine serum albumin (BSA) and temperature on neuraminidase s t a b i l i t y . Neuraminidase s o l u t i o n (0.1 U/ml) prepared with ( s o l i d l i n e s ) and without (broken l i n e s ) 0.3 mg/ml BSA was incubated at 30 ( f i l l e d squares) and 37°C ( f i l l e d t r i a n g l e s ) . A l i q u o t s of the enzyme s o l u t i o n were taken at v a r i o u s time i n t e r v a l s and enzyme a c t i v i t y was measured as described i n Methods. Results are expressed as a percentage of the maximum amount of s i a l i c a c i d released during 5 min incubation with the substrate. Note that BSA pro t e c t e d the enzyme at both temperatures. A l s o note that i n i t i a l a c t i v i t y of the enzyme i n the absence of BSA was about 79% of that i n the presence of BSA i n d i c a t i n g a loss of a c t i v i t y during preparation. TIME (min) 101 0.183 (blank = 0.156). This was c a l c u l a t e d to be equivalent t o the a c t i v i t y of about 0.073 ng t r y p s i n (considering 65 ng t r y p s i n produces an absorbance of 0.5 during a 30 min incu-bation period; Rinderknecht et a l . , 1968). Therefore a unit of neuraminidase may contain an amount of protease enzyme(s) e q u i v a l e n t to the a c t i v i t y of 0.146 ng t r y p s i n . This i s , however, an a r t i f a c t u a l f i g u r e s i n c e i t i s beyond the s e n s i t i v i t y of the assay. T h e r e f o r e , a c c o r d i n g t o the Remazobrilliant Blue-hide assay f o r proteases, the neurami-nidase product used i n t h i s study (Sigma type X) does not contain any detectable amount of protease a c t i v i t y . H. Neuraminidase-Releasable S i a l i c Acids; To i n v e s t i g a t e the optimum c o n d i t i o n s f o r maximum amount of s i a l i c acids r e l e a s e d by neuraminidase, homoge-nates of the guinea p i g heart were incubated with d i f f e r e n t c o n c e n t r a t i o n s of neuraminidase f o r d i f f e r e n t periods of time and s i a l i c acids released was measured ( F i g . 10). The maximum amount of s i a l i c a c i d s r e l e a s e d f o l l o w i n g a 2 h incubation with 0.1 U/ml neuraminidase under optimal condi-t i o n s (acetate b u f f e r pH = 5 and 37°C), was about 75% of t o t a l a c i d - r e l e a s a b l e s i a l i c a c i d s ( F i g . 10A). Under ex-perimental conditions of the Langendorff preparation (Hepes b u f f e r pH = 7.4 and 30°C), the s i a l i c acids released by 0.1 U/ml neuraminidase during 1 h i n c u b a t i o n was about 70% of the t o t a l acid releasable s i a l i c acids (Fig. 10B). Note that 0.01 U/ml neuraminidase r e l e a s e d comparable amounts of 102 FIG. 10. Neuraminidase-releasable s i a l i c acids of the guinea pig h e a r t . T i s s u e homogenates were incubated with d i f f e r e n t concentrations of neuraminidase for 15 ( f i l l e d t r i a n g l e s ) , 30 ( f i l l e d double t r i a n g l e s ) , 60 ( f i l l e d squares), or 120 ( f i l l e d c i r c l e s ) min i n acetate buffer (pH = 5) at 37°C (A) and i n Hepes b u f f e r (pH = 7.4) at 30°C (B). A l l samples contained 0.3 mg/ml BSA. Reactions were stopped by p l a c i n g the samples i n a b o i l i n g water bath f o r 1.5 min. Samples were c e n t r i f u g e d f o r 15 min (bench top c e n t r i f u g e ) and content of s i a l i c acids of the supernate was measured by the t h i o b a r b i t u r i c a c i d assay (Warren, 1959; see Methods). R e s u l t s are expressed as a p e r c e n t a g e of t o t a l t i s s u e content of acid-releasable s i a l i c acids. 103 A. <pH=5, 37°C) (•<) 15 min («-") 30 min 80- ) 1 20 min £ 60 CO Q 40 o < o < 20 CO -3 -2 -1 NEURAMINIDASE, log (U/ml) B. (pH=7.4, 30°C) ( « ) 15 min (•) 60 min 80 M y *—s CO 60-o IDS 40-ACI O IALI 20-CO -3 -2 -1 NEURAMINIDASE, log (U/ml) s i a l i c a c i d s ( F i g . 10A and 10B). C o n s i d e r i n g the above f i n d i n g s and the p r i c e of neuraminidase, a 1 h p e r f u s i o n with Hepes b u f f e r c o n t a i n i n g 0.01 U/ml neuraminidase was selected for treatment of i n t a c t Langendorff preparations of the guinea pig hearts. Note that t h i s s o l u t i o n contained 0.3 mg/ml BSA to protect the enzyme. I. Ef f e c t of Neuraminidase Treatment on the Response to Ouabain: To investigate the e f f e c t of neuraminidase treatment on the p o s i t i v e i n o t r o p i c response to ouabain, f o l l o w i n g 1 h e q u i l i b r a t i o n with Hepes b u f f e r , hearts were perfused with -7 2.5x10 M ouabain f o r 30 min followed by a 30 min washout with the b u f f e r . Hearts were then t r e a t e d with 0.01 U/ml neuraminidase plus 0.3 mg/ml BSA for 1 h followed by a 15-20 _ 7 mm washout. H e a r t s were t r e a t e d a g a i n w i t h 2.5x10 M ouabain f o r 30 min, f o l l o w e d by a 30 min treatment with - 7 5x10 M ouabain, and f i n a l l y t r e a t m e n t c o n t i n u e d w i t h — 6 10 M ouabain u n t i l the hearts were a r r e s t e d i n c o n t r a c -ture (5-10 min).The maximum e f f e c t of ouabain at 2.5x10 M c o n c e n t r a t i o n was u s u a l l y reached f o l l o w i n g a 20-25 min p e r f u s i o n . To ensure a maximum e f f e c t a 30 min p e r f u s i o n was selected. Control hearts were treated s i m i l a r l y , except neuraminidase plus BSA was substituted with buffer contain-i n g 0.3 mg/ml BSA o n l y . The magnitude of the p o s i t i v e -7 i n o t r o p i c response to 2.5x10 M ouabain f o l l o w i n g neura-minidase treatment compared to the i n i t i a l magnitude of 105 response p r i o r to neuraminidase treatment ( F i g . 11A) was s i g n i f i c a n t l y lower (p = 0.003) by about 46.3%. A s i m i l a r treatment w i t h BSA ( F i g . 11B), however, d i d not have a -7 s i g n i f i c a n t e f f e c t on the i n o t r o p i c response to 2.5x10 M ouabain. This protocol was followed i n order to resolve the apparent controversy i n the l i t e r a t u r e . Nevertheless, the _7 magnitude of the i n o t r o p i c response to 2.5x10 M ouabain following neuraminidase treatment shown i n F i g . 11A was s i g -n i f i c a n t l y lower (p = 0.001) than that observed i n controls (F i g . 11B) following BSA treatment by about 42.2%. Treatment - 7 w i t h 5x10 M o u a b a i n f u r t h e r i n c r e a s e d the +dP/dt ' ma x which reached a peak wit h i n 5-15 min, followed by a steady d e c l i n e f o r up to 30 min of p e r f u s i o n when treatment was — 6 c o n t i n u e d w i t h 10 M ouabain. Comparing neuraminidase-t r e a t e d h e a r t s ( F i g . 11A) to c o n t r o l h e a r t s ( F i g . 11B), neuraminidase treatment s i g n i f i c a n t l y reduced (p = 0.023) _7 the magnitude of the maximum i n o t r o p i c response to 5x10 M ouabain by 29.7%, but there was no d i f f e r e n c e between the response of the two p r e p a r a t i o n s at 30 min of treatment. Treatment with 10 M ouabain produced a f u r t h e r d e c l i n e i n + d p / d t m a x and developed i n t r a v e n t r i c u l a r pressure accom-panied by a gradual increase i n end d i a s t o l i c pressure and a r r h y t h m i a i n both c o n t r o l s and n e u r a m i n i d a s e - t r e a t e d h e a r t s . F i n a l l y , hearts were a r r e s t e d i n contracture 2-10 — 6 min f o l l o w i n g p e r f u s i o n with 10 M ouabain. Note that the +dP/dt r e c o r d e d p r i o r t o c a r d i a c a r r e s t was not ' ma x d i f f e r e n t between the two preparations ( F i g . 11A and 11B). 106 FIG. 11. P o s i t i v e i n o t r o p i c e f f e c t of ouabain i n neuraminidase-t r e a t e d (A) and c o n t r o l (B) Langendorff p r e p a r a t i o n s of guinea p i g h e a r t s . R e s u l t s are shown as a percentage i n -crease i n +dP/dt from that p r i o r to ouabain treatment. max The number of obs e r v a t i o n s was 5 i n both A and B. At the arrow, neuraminidase-treated hearts (A) were perfused with Hepes b u f f e r c o n t a i n i n g 0.01 U/ml neuraminidase plus 0.3 mg/ml BSA f o r 1 h. C o n t r o l h e a r t s (B) were perfused with Hepes buffer containing only 0.3 mg/ml BSA. The two bars of _7 2.5x10 M ouabain show the response before (open bar) and a f t e r ( s o l i d bar) treatment with neuraminidase plus BSA (A) and BSA ( B ) , r e s p e c t i v e l y . The f o l l o w i n g two b a r s of _ 7 5x10 M show the peak response (hatched b a r ) 5-15 min f o l l o w i n g t r e a t m e n t and the response 30 min f o l l o w i n g — 6 treatment (open b a r ) , r e s p e c t i v e l y . The e f f e c t of 10 M ouabain i s the response recorded (best estimate) just before c a r d i a c a r r e s t i n c o n t r a c t u r e . Two of the c o n t r o l hearts — 6 were arrested i n contracture p r i o r to treatment with 10 M ouabain. Note that neuraminidase treatment diminished the magnitude of the i n o t r o p i c response to subtoxic concentra-t i o n s of ouabain but had no e f f e c t at t o x i c concentrations nor did i t prevent t o x i c i t y . 107 A. NEURAMINIDASE (n=5) 1 2 0 1 0 0 -0. ~o + 8 0 u. O < til cc « 4 0 ^ 2 0 N E U R A M I N I D A S E • B S A 2.5 2.5 5 5 1 0 X 10 M OUABAIN B. CONTROL (n=5) 120- , 1 0 0 0. X) + 8 0 g 6 0 < 111 CC " 4 0 2 0 -0 J B S A 'I rh 2.5 2.5 5 5 1 0 X 10"' M OUABAIN 108 The end d i a s t o l i c pressure developed p r i o r to cardiac arrest i n c o n tracture (at 10 ouabain) i n c o n t r o l and neurami-n i d a s e - t r e a t e d hearts was 143.5 ± 18.9 and 119.8 ± 14.3% (% of c o n t r o l ; mean ± S.E.M.), r e s p e c t i v e l y , and were not s i g n i f i c a n t l y d i f f e r e n t at p<0.05 l e v e l . - 7 Treatment w i t h 2.5x10 M o u a b a i n produced a m i l d arrhythmia i n only one heart used i n the BSA Study (con-t r o l s ; n = 5) both before and a f t e r treatment with BSA. In _7 the neuraminidase study, treatment with 2.5x10 M ouabain produced an arrhythmia i n two hearts (n = 5) p r i o r to t r e a t -ment with neuraminidase but an arrhythmia was not observed f o l l o w i n g treatment with neuraminidase i n these two hearts -7 and i n the remaining three. During treatment with 5x10 M ouabain, an arrhythmia was observed i n two of BSA-treated hearts and these hearts were arrested i n contracture p r i o r to the end of the 30 min pe r i o d of treatment. In three of the neuraminidase-treated hearts, an arrhythmia was observed -7 d u r i n g treatment w i t h 5x10 M ouabain, but none of the hearts were arrested i n contracture during the 30 min period _7 of treatment with 5x10 M ouabain. Content of s i a l i c acids of the l e f t v e n t r i c l e of hearts used i n t h i s study was measured i n order to quantitate the e f f e c t of neuraminidase treatment. As shown i n Table 4 (page 118), mean content of s i a l i c a c i d s of the neuraminidase-treated hearts was 70.7% lower than controls. 109 J. E f f e c t of Neuraminidase Treatment on Isoproterenol  Inotropy; Following a 1 h e q u i l i b r a t i o n , Langendorff preparations of guinea p i g hea r t s were e i t h e r perfused with 0.01 U/ml neuraminidase plus 0.3 mg/ml BSA or 0.3 mg/ml BSA f o r 1 h (see Methods). Increasing concentrations of ^ - i s o p r o t e r e -nol were then perfused through the hearts. As shown i n F i g . 12 there was no s i g n i f i c a n t d i f f e r e n c e (p<0.05) between the — 8 response of the two groups of h e a r t s to as hi g h as 10 M — 8 — 8 i s o p r o t e r e n o l . Responses to 10 M and 5x10 M i s o p r o t e -reno l i n neuraminidase-treated hearts were lower than con-t r o l by about 15.3% and 16.8%, r e s p e c t i v e l y , but were only — 8 s i g n i f i c a n t l y d i f f e r e n t a t 5x10 M c o n c e n t r a t i o n (p = 0.073 and 0.008, r e s p e c t i v e l y ) . As shown i n Table 4 (page 118), mean content of s i a l i c acids of neuraminidase-treated hearts was 66.1% lower than controls. K. Ef f e c t of Neuraminidase Treatment on the Response to Calcium: To investigate the e f f e c t of neuraminidase treatment on the response to d i f f e r e n t concentrations of calcium, neura-minidase-treated and c o n t r o l hearts (prepared as described under Methods) were perfused with Hepes b u f f e r c o n t a i n i n g 0.5 mM calcium followed by perfusion with b u f f e r containing i n c r e a s i n g c o n c e n t r a t i o n s of calciu m . No adjustment f o r changes i n osmolarity was made i n t h i s study. Results, shown 110 FIG. 12. I s o p r o t e r e n o l c o n c e n t r a t i o n - r e s p o n s e i n c o n t r o l and neuraminidase-treated Langendorff preparations of guinea pig h e a r t s . Data are mean va l u e s and bars r e p r e s e n t S.E.M. Number of c o n t r o l (open squares) and neuraminidase-treated (open t r i a n g l e s ) h e a r t s was 7 and 6, r e s p e c t i v e l y . The as t e r i s k indicates a s i g n i f i c a n t difference at p<0.05 l e v e l . I l l (•) control CrW) (<) neuraminidase Cn=6) 0 112 i n F i g . 13, show the maximum +dP/dt expressed as a per-3 ' max r centage of c o n t r o l observed at each calcium concentration. Note t h a t there was no d i f f e r e n c e between the magnitude of c o n t r a c t i l i t y developed by the two groups of hearts for up to 5 mM calcium. At calcium concentrations greater than 5 mM the +dP/dt developed by n e u r a m i n i d a s e - t r e a t e d ' max ^ 1 hearts was lower than control. The values were s i g n i f i c a n t l y d i f f e r e n t (p<0.05) at 7, 10 and 15 mM calcium (p = 0.01, 0.0005 and 0.002, r e s p e c t i v e l y ) . The average magnitude of c o n t r a c t i l i t y developed by neuraminidase-treated hearts at 7, 10, and 15 mM calcium was 13.2, 17.7 and 21.2% lower than that developed by c o n t r o l s , r e s p e c t i v e l y . The mean content of s i a l i c a c i d s of n e u r a m i n i d a s e - t r e a t e d h e a r t s i n t h i s study was 65.6% lower than corresponding controls (Table 4; page 118). L. Ef f e c t of Neuraminidase Treatment on the Inotropic  Response of Reduced E x t r a c e l l u l a r Sodium: The e f f e c t of neuraminidase treatment on the i n o t r o p i c response of reduced e x t r a c e l l u l a r sodium concentration was i n v e s t i g a t e d i n order to determine the p o s s i b l e e f f e c t of + 2 + the enzyme on the Na -Ca exchange process i n an i n t a c t preparation. Control and neuraminidase-treated (0.01 U/ml, 1 h) Langendorff p r e p a r a t i o n s of a d u l t guinea p i g hearts prepared as described under Methods were perfused with Hepes b u f f e r containing decreasing concentrations of sodium: 160, 140, 110, and 80 mM NaCl, r e s p e c t i v e l y . Osmolarity of the 113 FIG. 13. E f f e c t of neuraminidase treatment on the response of guinea p i g hearts to calcium. Langendorff preparations of neuraminidase-treated (open t r i a n g l e s , n = 5) and c o n t r o l (open squares, n = 7) h e a r t s were prepared as d e s c r i b e d under Methods and were perfused with Hepes buffer containing incre a s i n g concentrations of calcium ranging from 0.5 to 15 mM f o r 1-3 min to reach the maximum response. Results show the maximum response c a l c u l a t e d as a percentage of c o n t r o l . Values are mean ± S.E.M. Stars i n d i c a t e s i g n i f i c a n t d i f f e r -ence at p<0.05 l e v e l . 114 (•) control Cn=7D (<} neuraminidase (n=5D 300n CALCIUM (mM) 115 s o l u t i o n s was kept constant by r e p l a c i n g NaCl with choline c h l o r i d e . Each s o l u t i o n was p e r f u s e d f o r 5-6 min. By red u c i n g the p e r f u s a t e sodium c o n c e n t r a t i o n a t r a n s i e n t increase i n the c o n t r a c t i l i t y was developed and was followed by a steady d e c l i n e . The maximum +dP/dt developed at •* J ' max c each sodium concentration was determined and the data were expressed as percentage of the maximum +dP/dt m a x developed at 140 mM Na +. As shown i n F i g . 14 neuraminidase treatment had no s i g n i f i c a n t e f f e c t (p<0.05) on the magnitude of the i n o t r o p i c e f f e c t produced by reducing e x t r a c e l l u l a r Na + concentration from 140 mM to 110 mM and then to 80 mM. The maximum c o n t r a c t i l i t y index (+dP/dt ) developed at 160 J max ^ mM Na + by neuraminidase-treated and c o n t r o l p r e p a r a t i o n s was 91.1 ± 1.3% (mean ± S.E.M.) and 87.8 ± 0.07% of the +dP/dt developed at 140 mM Na +, r e s p e c t i v e l y . These ' max ^ values are s i g n i f i c a n t l y d i f f e r e n t (p = 0.037); however, the d i f f e r e n c e between the two values i s only 3.8%. Tot a l con-tent of s i a l i c acids of the neuraminidase-treated hearts was 66.2% less than that of controls (Table 4). M. Effe c t of Neuraminidase Treatment on Basal  C o n t r a c t i l i t y : The c o n t r a c t i l i t y i ndex (+dP/dt ) and t h e end J ' m a x d i a s t o l i c pressure obtained before and a f t e r treatment with BSA and neuraminidase were compared i n order to evaluate the e f f e c t of neuraminidase treatment on b a s a l c o n t r a c t i l i t y . Results obtained from a l l the Langendorff preparations used 116 FIG. 14. E f f e c t of neuraminidase treatment on the p o s i t i v e i n o t r o p i c e f f e c t of diminished e x t r a c e l l u l a r sodium concen-t r a t i o n . Control and neuraminidase-treated (0.01 U/ml, 1 h) Langendorff p r e p a r a t i o n s of a d u l t guinea p i g hea r t s (see Methods) were perfused with Hepes buffer containing decreas-ing concentrations of NaCl (160-80 mM). Osmolarity of the b u f f e r was kept constant by r e p l a c i n g NaCl with c h o l i n e c h l o r i d e . A l l s o l u t i o n s were p e r f u s e d f o r 5-6 min. The maximum + d P / d t j n a x developed at each N a + c o n c e n t r a t i o n was expressed as a percentage of the maximum + d P / d t m a x developed at 140 mM Na +. The a s t e r i s k i n d i c a t e s a s i g n i -f i c a n t d i f f e r e n c e at p<0.05 l e v e l . Note that there was a s i g n i f i c a n t d i f f e r e n c e at the p<0.05 l e v e l between the two treatments only at 160 mM Na +. 140-1 130H X CO 3 120 T3 "D + _ l o o O 110H 100 90H (x) CONTROL (n=7) ( ° ) NEURAMINIDASE (n=6) 80-+— 170 140 110 mM INa+L 8 118 TABLE 4. Left Ventricular Content of S i a l i c Acids i n Control and Neuraminidase-Treated Guinea Pig Hearts. Content of S i a l i c Acids (nmoles/g tissue wet weight) % Deer-Treatment Control Neuraminidase easet Ouabain [11] 11 393. 1±10.2(5) 115. 0±6. 4 (5) 70. 7 Isoproterenol[12] 337. 0±11.0(7) 114. 3±4. 3 (6) 66. 1 Calcium [13] 359. 5±7.8 (7) 123. 6±6. 3 (5) 65. 6 Sodium [14] 338. 2±2.8 (7) 114. 5 + 7. 2 (6) 66. 2 Content of s i a l i c acids i s the t o t a l tissue content of acid-releasable s i a l i c acids measured by the thiobarbitu-r i c acid assay (see Methods) described by Warren (1959). IT Left ventricular samples were obtained from the hearts used for the dose-response study of the agent indicated, at the end of the experiment. The numbers i n brackets indicate the figure number that the data correspond to. t Percentage decrease of mean content of s i a l i c acids com-pared to corresponding controls. Results are mean values ± S.E.M., and numbers i n parenthesis indicate the number of hearts. 119 i n the above neuraminidase s t u d i e s were pooled together (Table 5) to assess the o v e r a l l e f f e c t of neuraminidase treatment. Both the +dP/dt and the end d i a s t o l i c p r e s s u r e ' ma x developed a f t e r neuraminidase treatment compared to p r i o r treatment i n the same hearts, using a p a i r e d t - t e s t , were s i g n i f i c a n t l y increased (p<0.05). In c o n t r a s t , n e i t h e r of these parameters were s i g n i f i c a n t l y a l t e r e d (p>0.05; paired t - t e s t ) f o l l o w i n g BSA treat m e n t . However, the a b s o l u t e v a l u e s of the +dP/dt and the end d i a s t o l i c p r e s s u r e max d e v e l o p e d f o l l o w i n g n e u r a m i n i d a s e t r e a t m e n t were not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) from the values obtained following BSA treatment (unpaired t - t e s t ) . The +dP/dt and the end d i a s t o l i c p ressure d e v e l -max oped f o l l o w i n g BSA and neuraminidase treatment were a l s o expressed as a percentage of the c o n t r o l values p r i o r to t r e a t m e n t ( T a b l e 5 ) . In t h i s case o n l y the +dP/dt 1 1 max following neuraminidase treatment was s i g n i f i c a n t l y (p<0.05) h i g h e r than the BSA t r e a t e d c o n t r o l s . The +dP/dt and ' max the end d i a s t o l i c pressure following neuraminidase treatment were i n c r e a s e d by 12.4 ± 2.8% and 11.3 ± 4.2% (mean ± S.E.M.; n = 22 ), respectively. TABLE 5. E f f e c t of BSA and Neuraminidase Treatments on Basal +dP/dt max and End D i a s t o l i c Pressure i n the Guinea Pig Heart. +dP/dt (mm Hg/sec) End D i a s t o l i c Pressure (mm Hg) ' max ^ ^ Treatment Control Treated % of Cont. Control Treated % of Cont. BSA (control) 679±16 665±20 98.0±1.8 16.1±0.5 16.U0.5 101.5±3.6 n = 26 Neuraminidase 619±15* 689±12f 112.4±2.8* 15.7±0.5 17.3±0.6t 111.3±4.2 (0.01 U/ml, lh) n = 22 NOTE : Results i n t h i s table are mean ± S.E.M. of values obtained from a l l Langendorff preparations (see Methods) of the guinea pig hearts used i n the neuraminidase study. Data presented show the absolute values obtained before (control) and aft e r (treated) treatment with BSA or neuraminidase, and the r e l a t i v e e f f e c t of treatment presented as the percentage of control. * Neuraminidase-treated are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from BSA-treated controls (unpaired t - t e s t ) . t Values obtained a f t e r neuraminidase treatment are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from p r i o r to treatment controls (paired t - t e s t ) . 121 N. Eff e c t of Neuraminidase Treatment on Ouabain Binding  and In h i b i t i o n of the Enzyme (Na +-K +)ATPase; Since neuraminidase treatment was found to reduce the magnitude of the p o s i t i v e i n o t r o p i c response of ouabain, I examined the e f f e c t of neuraminidase treatment on ouabain b i n d i n g and i n h i b i t i o n of the enzyme ( N a + - K + ) A T P a s e . Membrane p r e p a r a t i o n s c o n t a i n i n g the enzyme ( N a + - K + ) -ATPase were prepared from guinea p i g l e f t v e n t r i c l e s and ouabain b i n d i n g was c a r r i e d out under optimal c o n d i t i o n s 2 + ( i n t h e p r e s e n c e o f Mg and P^, see M e t h o d s ) f o r o u a b a i n b i n d i n g t o the enzyme ( N a + - K + ) A T P a s e (Erdman, 1981). Under these c o n d i t i o n s Scatchard p l o t s of s p e c i f i c 3 [ H]ouabain b i n d i n g were l i n e a r s u g g e s t i n g the presence of a s i n g l e c l a s s of b i n d i n g s i t e s . As shown i n F i g . 15, neuraminidase treatment (0.01 U/ml, 1 h at 37°C) of the p r e p a r a t i o n had no e f f e c t on the a f f i n i t y and the binding capacity of ouabain binding. S i m i l a r l y neuraminidase treatment had no e f f e c t on + + 2 + b a s a l (Na -K )ATPase and Mg -ATPase a c t i v i t i e s of the preparation (Table 6). As shown i n F i g . 16, the concentra-t i o n - e f f e c t curves f o r ouabain i n h i b i t i o n o f the enzyme ( N a + - K + ) A T P a s e of n e u r a m i n i d a s e - t r e a t e d p r e p a r a t i o n s overlapped t h a t of the c o n t r o l s . The c a l c u l a t e d ouabain c o n c e n t r a t i o n t h a t p r o d u c e d 50% i n h i b i t i o n of t o t a l (Na + - K + ) A T Pase a c t i v i t y ( I C ^ ) o f n e u r a m i n i d a s e -t r e a t e d p reparations was not s i g n i f i c a n t l y d i f f e r e n t from control (Fig. 16). 122 FIG. 15. E f f e c t of neuraminidase treatment on ouabain binding. This figure shows Scatchard plots of a t y p i c a l experiment of 3 s p e c i f i c [ H]ouabain binding to c o n t r o l (open squares) and neuraminidase-treated (0.01 U/ml, 1 h and 37°C; open t r i a n -g l e s ) ( N a + - K + ) A T P a s e - c o n t a i n i n g g u i n e a p i g l e f t ven-3 t r i c u l a r membrane p r e p a r a t i o n s . [ H]0uabain b i n d i n g was c a r r i e d out i n a medium c o n t a i n i n g 1 mM Tris-EGTA, 4 mM M g C l 2 , 3 mM T r i s - p h o s p h a t e , and 50 mM T r i s - H C l b u f f e r (pH = 7.4). In t h i s experiment, 0.55 mg p r o t e i n of the 3 membrane p r e p a r a t i o n and 11.9-367.8 nM [ H]ouabain (1.75 Ci/mmol) was u t i l i z e d f o r the assay. The i n c u b a t i o n was c a r r i e d out f o r 1 h at room temperature (24°C) and was terminated by r a p i d f i l t r a t i o n through Whatman GF/B glass 3 m i c r o f i b e r f i l t e r s . S p e c i f i c [ H]ouabain b i n d i n g was de-3 f i n e d as the d i f f e r e n c e between the amount of [ H]ouabain -3 bound i n the absence and p r e s e n c e of 10 M u n l a b e l l e d ouabain. The values of the d i s s o c i a t i o n constants (K^; nM) and the maximum number of b i n d i n g s i t e s ( B m a x " pmol/mg protein) shown are mean ± S.E.M. of four observations. Note that neuraminidase treatment had no s i g n i f i c a n t e f f e c t on the c h a r a c t e r i s t i c s of ouabain binding. 123 ( • ) C O N T R O L (< ) N E U R A M I N I D A S E *D ("M) B m a x ( p m o l e / m g ) 9 5 . 7 * 2 . 1 10 .710 .6 9 8 . 8 * 2 . 3 1 0 . 6 * 0 . 5 • 1 0 l i . i 1 1 2 5 5 7 . 5 1 0 SPECIFIC BOUND (pmol/mg) 124 TABLE 6. E f f e c t of Neuraminidase Treatment on Basal (Na +-K +)ATPase and Mg 2 +-ATPase A c t i v i t i e s . Treatment ATPase A c t i v i t y (pmol P^ /mg protein/h) 2+ Mg -ATPase (Na+-K +)ATPase Control 0.94 ± 0.18 4.61 ± 0.58 Neuraminidase-treated (0.01 U/ml, lh and 3 7°C) 0.87 ± 0.06 4.79 ± 0.56 NOTE: (Na -K )ATPase and Mg -ATPase a c t i v i t i e s of control and neuraminidase-treated (Na +-K +)ATPase-containing membrane preparations of guinea pig l e f t v e n t r i c l e s (see Methods) were determined by measuring the amount of inorganic phos-phate (method of Martin and Doty, 1949) released from ATP following 30 min incubation at 37°C i n a medium containing 2.5 mM Tris-ATP, 3 mM MgCl 2, 1 mM Tris-EGTA, 50 mM Tris-HCl buffer (pH = 7.4), i n the presence and absence of 100 mM NaCl plus 10 mM KC1, respectively. Results are mean values ± S.E.M. of 5 observations. Note that neuraminidase treatment had no s i g n i f i c a n t e f f e c t (p>0.05) on the ATPase a c t i v i t i e s measured. 125 FIG. 16. E f f e c t of neuraminidase treatment on ouabain i n h i b i t i o n o f the enzyme (Na + -K + ) A T P a s e . ( N a + - K + ) A T P a s e a c t i v i -ty of c o n t r o l ( f i l l e d t r i a n g l e s ) and neuraminidase-treated (0.01 U/ml, 1 h and 37°C; open squares) (Na +-K +)ATPase-c o n t a i n i n g membrane p r e p a r a t i o n s o f guinea p i g l e f t ven-t r i c l e s was assayed by measuring the amount of i n o r g a n i c phosphate (method of Martin and Doty, 1949) relea s e d from ATP following 30 min incubation at 37°C i n a medium contain-i n g 2.5 mM T r i s - A T P , 3 mM MgCl 2, 1 mM Tris-EGTA, 50 mM T r i s - H C l b u f f e r (pH = 7.4), 100 mM NaCl, 10 mM KC1, and varying concentrations of ouabain. Data are mean values and bars r e p r e s e n t S.E.M. of 5 o b s e r v a t i o n s . The c a l c u l a t e d c o n c e n t r a t i o n of ouabain that produced 50% i n h i b i t i o n of t o t a l (Na +-K +)ATPase a c t i v i t y (IC,-Q) of neuraminidase-t r e a t e d p r e p a r a t i o n s was not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) from control. 126 I C S Q (UM) ( « ) CONTROL <n«5) 1 . 9 * 0 . 1 (•) NEURAMINIDASE <n«5) 1 . 7 * 0 . 2 OUABAIN (log M) 127 S E C T I O N I V D I S C U S S I O N 128 A. E f f e c t of Lanthanum on the Inotropic Response of  Isoproterenol: Role of the Superficially-Bound  Calcium: In 1910 Mines noted that lanthanum was a potent i n h i b i -t o r of force development i n the frog heart. Later, lanthanum i n h i b i t i o n of myocardial c o n t r a c t i l i t y was found to be ac-companied by the displacement of calcium from a s u p e r f i c i a l -l y located calcium pool (Sanborn and Langer, 1970; Ong and Bailey, 1972). The l a t t e r observations provided s u b s t a n t i a l evidence f o r an important r o l e of the s u p e r f i c i a l l y - b o u n d calcium i n the r e g u l a t i o n of myocardial c o n t r a c t i l i t y . In t h i s study I used lanthanum as a t o o l to e l u c i d a t e the role of the superficially-bound calcium i n the i n o t r o p i c response of i s o p r o t e r e n o l . I f the s u p e r f i c i a l l y - b o u n d c a l c i u m i s i n v o l v e d i n the i n o t r o p i c response of i s o p r o t e r e n o l , then one would expect that displacement of calcium from these s i t e s by lanthanum would diminish or i n h i b i t the response to i s o p r o t e r e n o l . As shown i n Figs. 2 and 3, lanthanum i n h i b i -ted the i n o t r o p i c response of isoproterenol i n a noncompeti-t i v e manner. In an e a r l i e r study, Bockman et a l . (1973) have al s o shown a complete block of the response to isoproterenol by lanthanum. The f a c t that lanthanum i n h i b i t e d the i n o t r o -p i c response of isoproterenol suggests that an e x t r a c e l l u l a r c a l c i u m i n f l u x p l a y s an e s s e n t i a l r o l e i n the i n o t r o p i c response of i s o p r o t e r e n o l . These r e s u l t s a l s o suggest that s u p e r f i c i a l l y - b o u n d calcium i s required for the i n o t r o p i c response of i s o p r o t e r e n o l . However, the e f f e c t of lanthanum 129 could be mediated by a l t e r i n g other c e l l u l a r processes and may not be related to the superficially-bound calcium. To i n v e s t i g a t e the p o s s i b l e e f f e c t of lanthanum on isoproterenol binding to the beta-receptor and a c t i v a t i o n of the enzyme adenylate c y c l a s e , I determined the e f f e c t of lanthanum on the i s o p r o t e r e n o l - i n d u c e d c y c l i c AMP l e v e l . Note that 3 uM lanthanum reduced basal c o n t r a c t i l i t y and the maximum response to isoproterenol by 97 and 95% (Figs. 1-3), r e s p e c t i v e l y , but had no s i g n i f i c a n t e f f e c t on b a s a l or i s o p r o t e r e n o l - i n d u c e d c y c l i c AMP l e v e l s ( F i g . 4). These r e s u l t s i n d i c a t e that lanthanum i n h i b i t i o n of isoproterenol i n o t r o p y i s not due t o an i n h i b i t i o n of i s o p r o t e r e n o l b i n d i n g and s t i m u l a t i o n of the enzyme adenylate c y c l a s e . Meanwhile, i t i s v e r y u n l i k e l y t h a t lanthanum would i n t e r f e r e with subsequent bi o c h e m i c a l events mediated by an elevated c e l l u l a r c y c l i c AMP l e v e l , since lanthanum i s r e s t r i c t e d to the e x t r a c e l l u l a r space (Martinez-Palomo et a l . , 1973). Lanthanum i s a l s o known to i n h i b i t the slow inward calcium current i n the heart (Katzung et a l . , 1973; Shige-nobu and S p e r e l a k i s , 1972; Kass and T s i e n , 1975). Based on such o b s e r v a t i o n s i t i s a l s o b e l i e v e d t h a t lanthanum d i r e c t l y blocks the potential-dependent calcium channels. Evidence f o r a d i r e c t a c t i o n of lanthanum on the calcium channels was l a t e r obtained from lanthanum i n h i b i t i o n of 3 [ H N i t r e n d i p i n e b i n d i n g i n the h e a r t ( E h l e r t et a l . , 130 1982). N i t r e n d i p i n e i s a p o t e n t c a l c i u m a n t a g o n i s t ; i t i n h i b i t s the slow inward current, and i t binds with high a f f i n i t y to c e r t a i n s i t e s on the e x t e r n a l s u r f a c e of the sarcolemma b e l i e v e d to be a s s o c i a t e d with the p o t e n t i a l -dependent calcium channels (see Janis and S c r i a b i n e , 1983, f o r review). Since catecholamines increase the slow inward calcium current (Reuter, 1974), then the lanthanum i n h i b i -t i o n of i s o p r o t e r e n o l i n o t r o p y could simply be due t o a d i r e c t i n h i b i t i o n of the calcium channels by lanthanum. To determine i f the negative i n o t r o p i c e f f e c t of lanthanum i s r e l a t e d to a d i r e c t i n t e r a c t i o n with the calcium channels, I i n v e s t i g a t e d the r e l a t i o n s h i p between the e f f e c t of lanthanum on myocardial c o n t r a c t i l i t y and lanthanum i n h i b i -3 t i o n of C H ] n i t r e n d i p i n e b i n d i n g . R e s u l t s of t h i s study summarized i n F i g . 8, c l e a r l y demonstrate that concentration of lanthanum as low as 1 |J.M i n h i b i t e d myocardial c o n t r a c t i -l i t y by 90%, and the maximum response to i s o p r o t e r e n o l by 70% ( F i g . 2), but had no e f f e c t on [ H ] n i t r e n d i p i n e bind-ing. Also note that 3 p.M lanthanum reduced basal myocardial c o n t r a c t i l i t y and i s o p r o t e r e n o l i n o t r o p y by 97 and 95%, r e s p e c t i v e l y (Figs. 1 and 2, r e s p e c t i v e l y ) , but reduced the 3 t o t a l number of [ H ] n i t r e n d i p i n e b i n d i n g s i t e s by 31% 3 ( T a b l e 3 ) . Lanthanum i n h i b i t i o n o f [ H ] n i t r e n d i p i n e b i n d i n g ( F i g . 8) suggests that lanthanum binds to a s i t e c l o s e to or on the calcium channels and d i r e c t l y i n h i b i t s the channels. Meanwhile since low concentrations of lantha-num ( < 3 \iM) g r e a t l y diminish myocardial c o n t r a c t i l i t y and 131 the i n o t r o p i c response to i s o p r o t e r e n o l with l i t t l e or no 3 e f f e c t on [ H ] n i t r e n d i p i n e b i n d i n g a l s o suggest that the e f f e c t of such low c o n c e n t r a t i o n s of lanthanum are not 3 r e l a t e d t o an i n t e r a c t i o n w i t h the [ H ] n i t r e n d i p i n e binding s i t e s , and therefore may not be r e l a t e d to a d i r e c t i n h i b i t i o n of the calcium channels. I t should be pointed out that these r e s u l t s do not e l i m i n a t e other mechanisms through which lanthanum might block the calcium channels 3 without a f f e c t i n g [ H ] n i t r e n d i p i n e b i n d i n g . Nevertheless 3 u s i n g [ H ] n i t r e n d i p i n e b i n d i n g as a t o o l no r e l a t i o n s h i p could be found between the e f f e c t s of low concentrations of lanthanum on myocardial c o n t r a c t i l i t y and a d i r e c t i n h i b i -t i o n of the p o t e n t i a l - d e p e n d e n t c a l c i u m channels i n the guinea pig heart. Lanthanum has been shown to i n h i b i t the f u n c t i o n of a number of membrane-bound enzymes. Nayler and H a r r i s (1976) reported that 25 \xM lanthanum produced a 23.5% i n h i b i t i o n of the a c t i v i t y of the enzyme (N a + - K + ) A T P a s e i n a m i c r o -somal f r a c t i o n from the guinea p i g h e a r t . Takeo et a l . (1979) reported that 25 uM lanthanum i n h i b i t e d the enzymes ( N a + - K + ) A T P a s e , M g 2 + - A T P a s e , and C a 2 + - A T P a s e i n a r a t heart sarcolemmal preparation by 55.6, 15.8, and 15.0%, res p e c t i v e l y . Note that the concentrations of lanthanum used i n t h i s study are much less than the concentrations required t o i n h i b i t the a c t i v i t y of these enzymes. Meanwhile, i n i n t a c t preparations, Ravens (1975) showed that during long exposure to high concentrations of lanthanum (50, 100 and 500 p.M) the c o n t r a c t i o n amplitude of guinea p i g p a p i l l a r y muscles was increase d f o l l o w i n g an i n i t i a l decrease, the r e s t i n g t e n s i o n was i n c r e a s e d , and the r e s t i n g membrane p o t e n t i a l was reduced. Ravens (1975) concluded t h a t the acti o n of lanthanum on the cardiac muscle cannot be explain-ed by a simple displacement of s u p e r f i c i a l l y - b o u n d calcium. I t should be pointed out that Ravens' (1975) observations were made following long periods of treatment with high con-cen t r a t i o n s of lanthanum. C o l l e c t i v e l y these observations, i n c l u d i n g those of the present study regarding lanthanum 3 i n h i b i t i o n of [ H]nitrendipine binding, i n d i c a t e that high c o n c e n t r a t i o n s of lanthanum i n h i b i t the f u n c t i o n of many c r i t i c a l sarcolemmal enzymes and r e g u l a t o r y p r o t e i n s and therefore cannot be used as a pharmacological t o o l to study the role of sarcolemmal-bound calcium i n cardiac c o n t r a c t i -l i t y . However the concentrations of lanthanum used i n t h i s study are very low. The f a c t that the f u n c t i o n a l e f f e c t s of lanthanum are produced by very low concentrations suggests t h a t the e f f e c t s of such low co n c e n t r a t i o n s of lanthanum are mediated by b i n d i n g to s e l e c t i v e s i t e ( s ) with a high a f f i n i t y f or lanthanum. Unlike other e f f e c t s of lanthanum, displacement of the superficially-bound calcium was observed at very low lanthanum concentrations. Ong and Bailey (1972) reported that 5 uM lanthanum i n h i b i t e d the development of c o n t r a c t i l e f o r c e i n Langendorff p r e p a r a t i o n s of k i t t e n h e arts and blocked calcium uptake i n a r a p i d l y exchanging calcium p o o l . Sanborn and Langer (1970) showed a concen-133 t r a t i o n - d e p e n d e n t i n h i b i t i o n of c o n t r a c t i l e f o r c e and displacement of a s u p e r f i c i a l l y - b o u n d calcium by 5-20 |j.M lanthanum i n a r t e r i a l l y perfused r a b b i t i n t e r v e n t r i c u l a r s e p t a . Therefore the s u p e r f i c i a l l y - b o u n d c a l c i u m i s the most l i k e l y s i t e of a c t i o n of the low c o n c e n t r a t i o n s of lanthanum (<_ 3 \iM) used i n t h i s study. I n h i b i t i o n of i s o -proterenol inotropy by such low concentrations of lanthanum th e r e f o r e suggests that the s u p e r f i c i a l l y - b o u n d calcium i s the primary source of calcium mobilized by i s o p r o t e r e n o l . These r e s u l t s a l s o suggest t h a t the s u p e r f i c i a l l y - b o u n d calcium i s entering the c e l l through the potential-dependent calcium channels. Note that 0.25 p.M lanthanum reduced basal c o n t r a c t i l i t y by 63% ( F i g . 1) but had no e f f e c t on the r e s p o n s e t o isoproterenol (Fig. 2). This observation would suggest that e i t h e r a s p e c i f i c calcium binding s i t e i s i n v o l v e d i n the response to i s o p r o t e r e n o l , or that l e s s than h a l f of the calcium contained i n the s u p e r f i c i a l pool i s s u f f i c i e n t for i s o p r o t e r e n o l a c t i o n . This observation i s c o n s i s t e n t with observations made i n a number of laboratories showing that a decrease of the e x t r a c e l l u l a r calcium concentration to h a l f the normal b u f f e r content had no e f f e c t on the response to i s o p r o t e r e n o l d e s p i t e the decrease of basal c o n t r a c t i l i t y (see e.g. Yao et a l . , 1984). 134 B. Effects of Calcium and Lanthanum on 3 [ H]Nitrendipine Binding i n the Guinea Pig  Left V e n t r i c l e : The use of r a d i o l a b e l l e d nitrendipine and other organic calcium antagonists has greatly enhanced the i n v e s t i g a t i o n of the molecular mechanism of action of these compounds and the regulation of the function of calcium channels. Results 3 o b t a i n e d from e l e c t r o p h y s i o l o g i c a l and [ H ] n i t r e n d i p i n e b i n d i n g s t u d i e s suggest that the major s i t e of a c t i o n of n i t r e n d i p i n e i n c a r d i a c c e l l s i s a sarcolemmal c a l c i u m channel ( J a n i s and S c r i a b i n e , 1983). R e g u l a t i o n of the calcium channels by calcium and i n o r g a n i c calcium channel blockers has been studied i n a number of l a b o r a t o r i e s using 3 [ H ] n i t r e n d i p i n e b i n d i n g ( E h l e r t et a l . , 1982; Glossmann e t a l . , 1982; Gould et a l . , 1982). Gould et a l . (1982) 3 reported that [ H N i t r e n d i p i n e binding to b r a i n membranes of the r a t has an absolute requirement f o r p h y s i o l o g i c a l concentrations of calcium. These investigators also reported that ions such as strontium and barium, which mimic calcium p h y s i o l o g i c a l l y , share the a c t i o n of calcium i n enhancing 3 [ H ] n i t r e n d i p i n e b i n d i n g . Ions such as lanthanum and c o b a l t , which block the e f f e c t s of calcium, can i n h i b i t 3 [ H]nitrendipine binding and block the s t i m u l a t i n g actions of c a l c i u m . Hence, Gould et a l . (1982) suggested t h a t 3 [ H ] n i t r e n d i p i n e binding could be used as a t o o l to study the r e g u l a t i o n of the calcium channels by calcium and the i n o r g a n i c calcium antagonists. Glossman et a l . (1982) also 135 rep o r t e d t h a t the channels i n guinea p i g b r a i n membranes have a need f o r a d i v a l e n t c a t i o n i n order to b i n d the r a d i o l a b e l l e d c a l c i u m a n t a g o n i s t . However, Gould et a l . 3 (1982) d i d not detect calcium dependence of [ H ] n i t r e n d i -pine b i n d i n g i n r a t h e a r t membranes. This was s u r p r i s i n g s i n c e e x t r a c e l l u l a r c a l c i u m has been known t o p l a y an important role i n the regulation of cardiac c o n t r a c t i l i t y . In t h i s study i t was noted that calcium i s required for 3 [ H]nitrendipine binding i n the guinea p i g l e f t v e n t r i c l e . 3 C a l c i u m s l i g h t l y i n c r e a s e d the f o r [ H ] n i t r e n d i p i n e b i n d i n g , but i t s main e f f e c t was on the B (Table 2). 3 max 3 I n h i b i t i o n of [ H ] n i t r e n d i p i n e b i n d i n g by EDTA and the r e v e r s a l of t h i s e f f e c t by calcium (Fig. 6) provide further 3 evidence f o r the requirement of calcium f o r [ H ] n i t r e n d i -pine binding. However extensive washing of the membrane with 3 EDTA d i d not e l i m i n a t e b a s a l [ H ] n i t r e n d i p i n e b i n d i n g . These r e s u l t s suggest t h a t a t i g h t l y bound c a l c i u m i s 3 r e q u i r e d f o r [ H N i t r e n d i p i n e b i n d i n g . Since Gould et a l . 3 (1982) did not observe a calcium dependence of [ H] n i t r e n -d i p i n e b i n d i n g i n the r a t heart. They a l s o suggested that calcium i n the rat heart was probably t i g h t l y bound so that treatment with c h e l a t i n g agents could not remove a l l the calcium. Interestingly, the main e f f e c t of lanthanum was also on 3 the B m a x of L H ] n i t r e n d i p i n e b i n d i n g (Table 2). S i n c e the decrease of the B m a x produced by lanthanum was par-1 3 6 t i a l l y reversed by calcium and the lanthanum concentration-3 dependent i n h i b i t i o n of [ H ] n i t r e n d i p i n e b i n d i n g was s h i f t e d to the r i g h t by c a l c i u m ( F i g . 7), these r e s u l t s suggest that calcium and lanthanum are probably a c t i n g on the same s i t e i n an agonist-antagonist fashion. The s e n s i t i -3 v i t y of [ H N i t r e n d i p i n e binding to low concentrations of lanthanum (uM) and the fac t that 1 0 mM calcium had a p a r t i a l e f f e c t on lanthanum e f f e c t s suggest that lanthanum has a much higher a f f i n i t y than calcium for these s i t e s . Based on c a l c i u m c u r r e n t measurements i n i s o l a t e d guinea p i g heart c e l l s , Lee and Tsien ( 1 9 8 3 ) suggested that nitrendipine may bind to activated calcium channels. In l i n e with t h i s proposal, our r e s u l t s would suggest that calcium binding to c e r t a i n s i t e s i s required to maintain the calcium channels i n an a c t i v e s t a t e and that removal of calcium by ch e l a t i o n with EDTA or displacement of calcium with lantha-num would transfer the channels to an i n a c t i v e state.Calcium 3 enhancement of [ H N i t r e n d i p i n e binding a l s o suggests that 3 c a l c i u m and [ H ] n i t r e n d i p i n e are not competing f o r the same b i n d i n g s i t e . I t should a l s o be noted t h a t i n the guinea pig heart an increase of e x t r a c e l l u l a r calcium would incre a s e myocardial c o n t r a c t i l i t y (EC,-Q = 2 . 6 mM calcium; maximum e f f e c t at 15 mM calcium; data shown F i g . 1 3 ) and the amplitude of calcium current (Kohlhardt, 1 9 8 3 ) . In contrast, 3 the e f f e c t of calcium on [ H]n i t r e n d i p i n e binding observed i n t h i s study reached a maximum l e v e l at 1 mM calcium (Fig. 6 ) and a further increase of calcium did not cause a further 137 3 increase but i n f a c t decreased the b i n d i n g of [ ^ n i t r e n -d i p i n e . Therefore the f u n c t i o n a l e f f e c t observed following a l t e r a t i o n s of e x t r a c e l l u l a r calcium (0.1-15 mM) could not be due to a p a r a l l e l a l t e r a t i o n of the number of f u n c t i o n a l calcium channels. These r e s u l t s , however, suggest that only a t i g h t l y bound calcium i s required for maintenance of the calcium channels i n an a c t i v e state. Therefore t h i s calcium 3 b i n d i n g s i t e a s s o c i a t e d w i t h [ H ] n i t r e n d i p i n e b i n d i n g could not be the metal cation co-ordination s i t e involved i n the genesis of c a l c i u m c u r r e n t proposed by Hagiwara and Takahashi (1967). C. S t a b i l i t y and Effectiveness of Neuraminidase: Due to the c o n f l i c t i n g r e s u l t s regarding the e f f e c t of neuraminidase treatment on the i n o t r o p i c response of cardiac glycosides, I began t h i s study with the following questions: Is the a c t i v i t y of the enzyme as s t a t e d by the s u p p l i e r ? Is i t stable i n s o l u t i o n , and does i t maintain i t s a c t i v i t y d u r i n g the course of a p p l i c a t i o n ? Does neuraminidase treatment of an i n t a c t heart preparation from adult animals actually remove the s i a l i c acids ? Results shown i n F i g . 9 c l e a r l y demonstrate that the neuraminidase used i n t h i s study ( p u r i f i e d from Clostridium  perfringens, Sigma type X) i s not stable i n s o l u t i o n . These observations are c o n s i s t e n t with those of Cassidy et a l . (1965). They reported that the p u r i f i e d enzyme prepared from 138 C l o s t r i d i u m p e r f r i n g e n s r a p i d l y l o s t i t s a c t i v i t y , even i n the assay procedure, unless protected with serum albumin. The f a c t that the presence of serum albumin maintained the a c t i v i t y of the enzyme i n s o l u t i o n (Fig. 9) raised the ques-t i o n that the neuraminidase product may contain proteases. However, using Remazobrilliant Blue-hide as a substrate, no protease a c t i v i t y was detected. The s u p p l i e r ' s report also indicated that using casein as a substrate the neuraminidase product d i d not contain s i g n i f i c a n t protease a c t i v i t y . It s h o u l d be p o i n t e d out t h a t most i n v e s t i g a t o r s used the enzyme n e u r a m i n i d a s e p r e p a r e d from V i b r i o c h o l e rae obtained from d i f f e r e n t suppliers, and thus t h i s observation cannot n e c e s s a r i l y be extrapolated to t h e i r i n v e s t i g a t i o n s . However, Drzenieck (1972) reported that commercially a v a i l -a b l e V i b r i o c h o l e r a e n e u r a m i n i d a s e (Behringwerke AG, Marburg) l o s t 1/3 of i t s a c t i v i t y at 37°C at pH 7 w i t h i n 24 h and Ada et a l . (1961) have shown that the s o l u t i o n of p u r i f i e d V i b r i o c h o l e r a e neuraminidase at pH 5.6 or 6.7 l o s t about 20% of i t s a c t i v i t y over a period of 2 h at 37°C. In any event, u n c e r t a i n t y r e g a r d i n g the e f f e c t i v e n e s s of neuraminidase treatment makes i t d i f f i c u l t to i n t e r p r e t some of the reported observations and perhaps could e x p l a i n , at l e a s t i n part, the f a i l u r e of some i n v e s t i g a t o r s to observe any e f f e c t of neuraminidase treatment. The f a c t that neuraminidase perfusion of i n t a c t hearts from a d u l t guinea p i g s reduced t o t a l t i s s u e c o n t e n t of s i a l i c a c i ds by about 65-71% (Table 4) c l e a r l y i n d i c a t e s 139 that neuraminidase gained access to the s i a l i c acid residues of the g l y c o c a l y x i n t h i s i n v e s t i g a t i o n . Using e l e c t r o n microscopy and lanthanum to s t a i n the s i a l i c a c i d residues of the glycocalyx, i t was also noted that lanthanum staining of the g l y c o c a l y x of c a r d i a c muscle c e l l s was diminished f o l l o w i n g a s i m i l a r n e uraminidase t r e a t m e n t (data not shown). D. E f f e c t of Neuraminidase Treatment on Ouabain Inotropy: In c o n t r a s t to Grupp et a l . (1980) and Harding and H a l l i d a y ' s (1980) observations, I found that neuraminidase treatment of Langendorff p r e p a r a t i o n s of the guinea p i g heart reduced the magnitude of the p o s i t i v e i n o t r o p i c e f f e c t of s u b t o x i c c o n c e n t r a t i o n s of ouabain ( F i g . 11). Only Harding and H a l l i d a y (1980) i n v e s t i g a t e d the e f f e c t i v e n e s s of neuraminidase treatment i n t h e i r study. They reported t h a t removal of up to 79% of t o t a l t i s s u e s i a l i c a c i d c o n t e n t of guinea p i g l e f t a t r i a had no e f f e c t on the _ 7 i n o t r o p i c response to ouabain (2x10 M). The d i f f e r e n c e observed i n our study could be due to the difference between the guinea p i g l e f t atrium and the l e f t v e n t r i c l e regarding the response t o c a r d i a c g l y c o s i d e s . N e v e r t h e l e s s , the d i f f e r e n c e s i n the r e s u l t s of Harding and H a l l i d a y and the present study are d i f f i c u l t to reconcile. Contrary to the statement of Grupp et a l . (1980) the p r e p a r a t i o n used i n t h i s study was found to be s t a b l e for 140 p e r i o d s g r e a t e r than 6 h. It should a l s o be noted t h a t , i n our hands, a ouabain response of approximately 194% of c o n t r o l was obtained i n c o n t r o l preparations i n contrast to the 122% of control obtained by Grupp et a l . (1980). To determine the s i t e of action of neuraminidase on the p o s i t i v e i n o t r o p i c e f f e c t of ouabain I i n v e s t i g a t e d the e f f e c t of neuraminidase treatment on ouabain b i n d i n g and i n h i b i t i o n of the enzyme (Na +-K +)ATPase and the f u n c t i o n + 2 + o f the Na -Ca exchanger. R e s u l t s o b t a i n e d from the 3 [ H]ouabain b i n d i n g study are summarized i n F i g . 15 and c l e a r l y demonstrate that neuraminidase treatment had no ef-f e c t on [ 3 H ] o u a b a i n b i n d i n g to ( N a + - K + ) A T P a s e - c o n t a i n -ing membrane preparations of the guinea p i g l e f t v e n t r i c l e . Furthermore, neuraminidase treatment of (Na +-K +)ATPase-c o n t a i n i n g membrane p r e p a r a t i o n s of the guinea p i g l e f t v e n t r i c l e had no e f f e c t on b a s a l ( N a + - K + ) A T P a s e and 2+ Mg -ATPase a c t i v i t i e s (Table 6), nor d i d i t in f l u e n c e the s e n s i t i v i t y of the enzyme (Na +-K +)ATPase to i n h i b i t i o n by ouabain ( F i g . 16). In order to t e s t the func t i o n of the + 2 + Na -Ca exchanger i n an i n t a c t p r e p a r a t i o n I s t u d i e d the i n o t r o p i c e f f e c t of reducing the e x t r a c e l l u l a r sodium concentration i n Langendorff preparations of the guinea pig h e a r t . Reducing the e x t r a c e l l u l a r sodium concentration i s known to increase the i n t r a c e l l u l a r calcium concentration and i s accompanied by an i n o t r o p i c e f f e c t mediated by the + 2 + Na -Ca exchanger (Langer, 1982).Results of t h i s study, shown i n F i g . 14, c l e a r l y demonstrate that neuraminidase 141 treatment had no s i g n i f i c a n t e f f e c t on the i n o t r o p i c respon-se induced by decreasing the e x t r a c e l l u l a r sodium concentra-t i o n . C o l l e c t i v e l y , these re s u l t s suggest that the e f f e c t of neuraminidase treatment on ouabain inotropy i s not r e l a t e d to an a l t e r a t i o n i n ouabain b i n d i n g and i n h i b i t i o n of the enzyme (Na +-K +)ATPase or s t i m u l a t i o n of the sarcolemmal Na +-Ca 2 + exchanger. I t should be noted that neuraminidase treatment did not completely block the i n o t r o p i c response to ouabain. This could be due either to incomplete removal of s i a l i c acids or to the presence of neuraminidase-insensitive mechanism(s) which contribute to ouabain inotropy. The f i r s t p o s s i b i l i t y can not t o t a l l y e x p l a i n the i n a b i l i t y of neuraminidase t r e a t m e n t t o c o m p l e t e l y b l o c k the i n o t r o p i c e f f e c t of ouabain since neuraminidase treatment removed about 71% of the t o t a l t i s s u e content of s i a l i c a c i ds but reduced the - 7 response t o 2.5x10 M ouabain by only 46%. About 60-80% of the t o t a l t i s s u e content of s i a l i c a c i ds i n d i f f e r e n t t i s s u e s has been found to be a s s o c i a t e d with c e l l surface s t r u c t u r e s (Warren, 1976), and neuraminidase i s unable to cross the c e l l membrane (Kemp, 1970). Thus, the removal of 65-71% of t o t a l t i s s u e content of s i a l i c acids i n t h i s study suggests that the majority of s i a l i c acids residues of the gycocalyx were indeed removed. These observations accompa-n i e d by the f a c t t h a t neuraminidase t r e a t m e n t d i d not prevent ouabain t o x i c i t y ( c a r d i a c a r r e s t i n c o n t r a c t u r e ) suggest that the p o s i t i v e i n o t r o p i c e f f e c t of ouabain i s 142 mediated by at le a s t two major mechanisms: (1) a neuramini-dase-sensitive mechanism which accounts for about 1/2 of the p o s i t i v e i n o t r o p i c e f f e c t of s u b t o x i c c o n c e n t r a t i o n s of ouabain i n the adult guinea p i g l e f t v e n t r i c l e and i s n e i -+ 2 + t h e r mediated by the Na -Ca exchanger, nor r e l a t e d to ouabain t o x i c i t y ; (2) neuraminidase-insensitive mechanism(s) which i s p r o b a b l y r e l a t e d t o i n h i b i t i o n of the enzyme (Na +-K +)ATPase and ouabain t o x i c i t y . The neuraminidase-sensitive mechanism i s very l i k e l y r e l a t e d to a calcium i n f l u x mechanism across the sarcolemma associated with the s i a l i c acids of the glycocalyx and stim-u l a t e d by ouabain. In any event i t should be pointed out that none of the above findings provide any evidence for an ass o c i a t i o n or a d i s s o c i a t i o n of the function of t h i s mecha-nism from ouabain b i n d i n g to the enzyme (Na +-K +)ATPase, and i n h i b i t i o n of t h i s enzyme. Although t h i s study was not designed to investigate the e f f e c t of neuraminidase treatment on ouabain t o x i c i t y , sub-t o x i c and toxic concentrations of ouabain were used i n order to compare our r e s u l t s with those of Harding and H a l l i d a y (1980) and Grupp et a l . (1980). Therefore signs of ouabain t o x i c i t y were observed. These signs include e l e c t r i c a l t o x i -c i t y ( e x t r a s y s t o l e s and other arrhythmias) and mechanical t o x i c i t y ( d e c r e a s e d developed p r e s s u r e and +dP/dt , J r r i max i n c r e a s e d end d i a s t o l i c p r e s s s u r e and c a r d i a c a r r e s t i n c o n t r a c t u r e ) . I t i s obvious from t h i s study that neurami-143 nidase treatment neither i n h i b i t e d mechanical t o x i c i t y nor prevented the development of arrhythmias produced at high c o n c e n t r a t i o n s of ouabain (5x10 and 10 M). However, i t was s t r i k i n g to note that: 1) arrhythmias developed i n _ 7 two h e a r t s durxng the i n i t i a l treatment w i t h 2.5x10 M ouabain were not observed following neuraminidase treatment; and, 2) two of the BSA-treated h e a r t s developed severe -7 arrhythmias during treatment with 5x10 M ouabain and were a r r e s t e d i n c o n t r a c t u r e p r i o r t o the end o f the 30 min period of treatment, while none of the neuraminidase-treated h e a r t s was a r r e s t e d i n c o n t r a c t u r e during treatment with -7 5x10 M ouabain d e s p i t e development of a r r h y t h m i a s . I t should be p o i n t e d out t h a t the number of ob s e r v a t i o n s i n t h i s study (n = 5) i s small and these observations may not have any s t a t i s t i c a l relevance. Nevertheless, these r e s u l t s may s u g g e s t t h a t n e u r a m i n i d a s e t r e a t m e n t d e l a y e d the development and decreased the i n t e n s i t y of arrhythmias produced by subtoxic and t o x i c concentrations of ouabain, r e s p e c t i v e l y . I t i s also i n t e r e s t i n g to note that Nathan et a l . (1980) observed that neuraminidase treatment of cultured 7-day chick embryo heart c e l l s resulted i n hyperpolarization o f the maximum d i a s t o l i c p o t e n t i a l and r e d u c t i o n i n the s l o p e of d i a s t o l i c d e p o l a r i z a t i o n . These e f f e c t s are the opposite of that produced by t o x i c concentrations of cardiac g l y c o s i d e s (Rosen et a l . , 1975). In any event, f u r t h e r studies are required to evaluate the e f f e c t of neuraminidase treatment on the arrhythmogenic e f f e c t of ouabain and the 144 c e l l u l a r mechanisms involved i n that process. E. E f f e c t of Neuraminidase Treatment on the  Response to Isoproterenol and Calcium: The absence of a s i g n i f i c a n t e f f e c t of neuraminidase — 8 treatment on the i n o t r o p i c response t o as h i g h as 10 M i s o p r o t e r e n o l s u g g e s t s t h a t the s i a l i c a c i d s are not involved i n the calcium i n f l u x mechanism a c t i v a t e d by lower concentrations of isoproterenol. The f a c t that neuraminidase treatment has no e f f e c t on the calcium response up to 5 mM calcium also suggests that the s i a l i c acids residues of the g l y c o c a l y x do not play any 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 coupling at p h y s i o l o g i c a l concentrations of calcium. These r e s u l t s suggest that the s i a l i c acids are not i n v o l v e d i n the genesis of the slow inward calcium current at physiolo-g i c a l calcium concentrations. Isenberg and Klockner (1980) have shown t h a t the c h a r a c t e r i s t i c s of the slow inward cal c i u m c u r r e n t i n i s o l a t e d a d u l t r a t h e a r t myocytes, i n which the g l y c o c a l y x was removed by the c e l l i s o l a t i o n procedure, were s i m i l a r to those displayed by i n t a c t t i s s u e . They concluded that the g l y c o c a l y x i s u n l i k e l y to be the structure o r i g i n a t i n g or c o n t r o l l i n g the slow inward calcium c u r r e n t . This observation i s c o n s i s t e n t with our observa-tions regarding the e f f e c t of neuraminidase treatment on the magnitude of c o n t r a c t i l i t y developed by the h e a r t at low calcium and isoproterenol concentrations since Isenberg and Klockner (1980) measured the slow inward calcium current at 145 p h y s i o l o g i c a l calcium concentrations. The diminished mag-nitude of myocardial c o n t r a c t i l i t y at calcium concentrations higher than 5 mM and at the maximum concentration of isopro-—8 t e r e n o l (5x10 M), however, suggests that the s i a l i c acids may play a p a r t i a l but s i g n i f i c a n t role i n the regulation of myocardial c o n t r a c t i l i t y under these c o n d i t i o n s . Further studies are required to eluc i d a t e the s i t e ( s ) and mechanism (s) influenced by neuraminidase treatment. The r o l e of the sarcolemmal c a l c i u m b i n d i n g i n the r e g u l a t i o n of myocardial c o n t r a c t i l i t y i s w e l l documented (see Langer, 1980 and 1984 for review). In p a r t i c u l a r the magnitude of the force developed by the heart was shown to be r e l a t e d to the amount of calcium bound to the sarcolemma ( P h i l i p s o n and Langer, 1979; P h i l i p s o n et a l . , 1980b; Bers et a l . , 1981). If the sarcolemmal calcium binding s i t e s play a 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 , at l e a s t as calcium stores, then one would expect that removal of such s i t e s should reduce the force of c o n t r a c t i o n developed by the h e a r t . R e s u l t s obtained i n t h i s study show th a t the s i a l i c acids do not serve as calcium stores at p h y s i o l o g i c a l c o n c e n t r a t i o n s of c a l c i u m . However, the absence of a s i g n i f i c a n t r o l e for the s i a l i c acids i n the r e g u l a t i o n of myocardial c o n t r a c t i l i t y at p h y s i o l o g i c a l calcium concen-t r a t i o n s does not negate the r o l e of sarcolemmal calcium binding i n the regulation of myocardial c o n t r a c t i l i t y . Note that about 80% of t o t a l sarcolemmal-bound calcium was found to be bound to n e g a t i v e l y charged phospholipids i n r a b b i t 146 cardiac sarcolemmal preparations ( P h i l i p s o n et a l . , 1980a). Therefore calcium binding to the negatively charged phospho-l i p i d s i s the most l i k e l y candidate f o r the r e g u l a t i o n of myocardial c o n t r a c t i l i t y at p h y s i o l o g i c a l calcium concentra-tions and may be the s i t e involved i n the p o s i t i v e inotropic response of isoproterenol. I t has been p r e v i o u s l y demonstrated (Langer et a l . , 1976; Frank et a l . , 1977) that removal of s i a l i c acids from the myoblast c e l l s u r f a c e of neonatal r a t c u l t u r e d heart c e l l s markedly i n c r e a s e d c e l l u l a r c a l c i u m p e r m e a b i l i t y w ithout a f f e c t i n g potassium p e r m e a b i l i t y . In an i n t a c t p r e p a r a t i o n an i n c r e a s e i n calcium p e r m e a b i l i t y could be detected as an increase i n the force of contraction develop-ed, an increase i n end d i a s t o l i c tension, and/or the deve-lopment of contracture. As judged from basal c o n t r a c t i l i t y developed f o l l o w i n g neuraminidase treatment i n t h i s study (Table 5), there was a s l i g h t i n c r e a s e i n +dP/dt and ^ ' max end d i a s t o l i c pressure f o l l o w i n g neuraminidase treatment. These e f f e c t s were only s i g n i f i c a n t when compared to pre-treated controls using a paired t - t e s t , and the magnitude of the e f f e c t was about 12% of c o n t r o l . These observations are consistent with the hypothesis that neuraminidase treatment i n c r e a s e s c a l c i u m exchange. However, the f a c t t h a t the extent of t h i s e f f e c t i s very l i t t l e , suggests t h a t the glycocalyx s i a l i c acids i n the guinea p i g l e f t v e n t r i c l e may play a very minor role i n the r e g u l a t i o n of calcium permea-b i l i t y . As proposed by Langer and co-workers (1981) such a 147 d i f f e r e n c e among these s t u d i e s suggests that the r o l e of s i a l i c acids may vary with preparation, species and age. 148 SUMMARY AND CONCLUSIONS Mammalian myocardial c o n t r a c t i l i t y i s b e l i e v e d to be re g u l a t e d by the amount of calcium contained i n a h i g h l y l a b i l e s u p e r f i c i a l calcium pool. The purpose of t h i s study was to determine the role and the biochemical nature of such s i t e s i n the i n o t r o p i c response of c a r d i o t o n i c agents. In Langendorff preparations of the guinea p i g heart, low con-c e n t r a t i o n s of lanthanum (_<_ 3 u-M) , an i o n r e s t r i c t e d to the e x t r a c e l l u l a r space and which displaces the s u p e r f i c i a l -ly-bound calcium, reduced basal myocardial c o n t r a c t i l i t y i n a concentration-dependent fashion and blocked the i n o t r o p i c response of isoproterenol i n a non-competitive manner. These e f f e c t s of lanthanum were neither r e l a t e d to an i n h i b i t i o n of isoproterenol-induced increases i n c y c l i c AMP l e v e l s , nor 3 the r e d u c t i o n of [ H ] n i t r e n d i p i n e b i n d i n g . Hence these r e s u l t s suggest that superficially-bound calcium i s required for the inotropic action of isoproterenol. Neuraminidase treatment brought about removal of s i a l i c a c i d residues, which are candidates for s u p e r f i c i a l calcium b i n d i n g s i t e s , and reduced the i n o t r o p i c response to sub-t o x i c c o n c e n t r a t i o n s of ouabain by about 50% but d i d not prevent ouabain t o x i c i t y . Neuraminidase treatment had no e f f e c t on: 1) b a s a l myocardial c o n t r a c t i l i t y at p h y s i o l o -g i c a l calcium concentrations, 2) the i n o t r o p i c response of i s o p r o t e r e n o l , 3) the i n o t r o p i c e f f e c t of r e d u c i n g the e x t r a c e l l u l a r sodium c o n c e n t r a t i o n , 4) b a s a l ( N a + - K + ) -149 2 + ATPase and Mg -ATPase a c t i v i t i e s , 5) the s e n s i t i v i t y of ( N a + - K + ) A T P a s e t o i n h i b i t i o n by o u a b a i n , and 6) the 3 c h a r a c t e r i s t i c s of [ H ] o u a b a i n b i n d i n g . 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