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Effects of Sodium Pump Inhibition on Contraction in Sheep Cardiac Purkinje Fibers
CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19
Effects of Sodium Pump Inhibitionon Contraction in Sheep Cardiac Purkinje Fibers D. A. EZSNER
Department of Physiology University College London London, United Kingdom
W. J . LEDERER Depanment of Physiology University of Maryland Baltimore, Maryland
R. D. VAUGHAN-JONES Department of Pharmacology Oxford, United Kingdom
I.
INTRODUCTION AND METHODS
I t i s w e l l e s t a b l i s h e d t h a t i n h i b i t i n g t h e sodium pump i n c r e a s e s t h e f o r c e of c o n t r a c t i o n o f c a r d i a c muscle. T h i s e f f e c t a p p e a r s t o be mediated v i a an i n crease o f i n t r a c e l l u l a r sodium a c t i v i t y , a i a . One t h e o r y f o r t h e mechanism of t h i s e f f e c t i n v o l v e s i a Na/Ca exchange ( R e u t e r and S e i t z , 1 9 6 8 ) . I n c r e a s i n g d N a would p r o d u c e an i n c r e a s e o f C a i n f l u x and d e c r e a s e C a e f f l u x t h r o u g h s u c h an e x c h a n g e , l e a d i n g t o i n c r e a s e d c o n t r a c tion. I n o r d e r t o i n v e s t i g a t e t h e mechanism of t h e pos i t i v e i n o t r o p i c e f f e c t s o f a r a i s e d d i a , one n e e d s t o measure a1 and t e n s i o n s i m u l t a n e o u s l y . As w e l l a s . i n Na v e s t i g a t i n g t h e s t e a d y - s t a t e r e l a t i o n s h i p between a i a and t e n s i o n , i t i s a l s o o f i n t e r e s t t o c h a r a c t e r i z e t h e e f f e c t s o f sydden c h a n g e s of a 1 The o n l y f e a s i b l e way t o measure a1 w i t h adequate N a t i m e r e s o l u t i o n i s w i t h a n ion-selec!?ve m i c r o e l e c t r o d e . W e used r e c e s s e d - t i p N a - s e n s i t i v e m i c r o e l e c t r o d e s (Thomas, 1 9 7 8 ) . The exp e r i m e n t s w e r e performed on v o l t a g e - c l a m p e d s h e e p c a r d i a c
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Copynght 0 1983 by Academic Press. Inc. All rights of reproductionin any form reserved. ISBN 0-12-153319-0
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F i g . 1 . S i m u l t a n e o u s measurement of c u r r e n t , aid, tension, and vol tage-clamped membrane p o t e n t i a l . A 500-msec d e p o l a r i z i n g p u l s e was a p p l i e d f r o m the h o l d i n g p o t e n t i a l ( - 6 8 mV) t o -35 mV a t 0.1 Hz t o e l i c i t a t w i t c h . F r o m Eisner e t a l . ( 1 9 8 0 ) .
P u r k i n j e f i b e r s . F u r t h e r d e t a i l s of t h e methods u s e d have a l r e a d y a p p e a r e d ( E i s n e r and L e d e r e r , 1979a; E i s n e r e t a l . , 1 9 8 1 ) . The use of t h e v o l t a g e clamp makes i t p o s s i b l e t o i n v e s t i g a t e t h e e f f e c t s of changes of aBa w i t h o u t s e c o n d a r y e f f e c t s due t o changes of membrane p o t e n t i a l . Sodium pump a c t i v i t y w a s c o n t r o l l e d by changing t h e e x t e r n a l rubidium c o n c e n t r a t i o n (K-free sol u t i o n s were used t h r o u g h o u t ) .
SODIUM PUMP INHIBITION IN SHEEP CARDIAC PURKINJE FIBERS
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RESULTS AND D I S C U S S I O N
F i g u r e 1 shows s i m u l t a n e o u s measurements o f i n t r a c e l l u l a r N a a c t i v i t y , a h a , t e n s i o n and c u r r e n t i n a voltage-clamped s h e e p c a r d i a c P u r k i n j e f i b e r . The sodium pump w a s i n h i b i t e d by r e d u c i n g [ % l o from 1 0 t o 0 mM. T h i s produced a r i s e o f a 1 ( c f . D e i t m e r and E l l i s , 1978) and a l s o i n c r e a s e d Na t w i t c h and t o n i c t e n s i o n . Following t h i s e x p o s u r e t o Rb-free s o l u t i o n , [RbIo was i n c r e a s e d t o 4 mM r e s u l t i n g i n a decrease of a1 and both t w i t c h and t o n i c t e n s i o n . A t t h e same t i m e c u r r e n t t r a c e shows a peak of outward c u r r e n t which d e c a y s away. T h i s c u r r e n t t r a n s i e n t i s t h e e l e c t r o g e n i c Na-pump c u r r e n t t r a n s i e n t ( E i s n e r and L e d e r e r , 1979b; Gadsby and C r a n e f i e l d , 1 9 7 9 ) . A n a l y s i s of r e c o r d s s u c h a s F i g . 1 shows t h a t b o t h a i a and t h e c u r r e n t decay exp o n e n t i a l l y upon r e a c t i v a t i n g t h e N a pump w i t h rubidium. T h e h a l f - t i m e o f d e c a y , o f c u r r e n t i s always s i m i l a r t o t h a t o f t h e decay of a 1 ( E i s n e r et al., 1 9 8 1 ) . The r e l a t i o n s h i p @tween a N1 a and t e n s i o n i s s t u d i e d i n more d e t a i l i n F i g . 2 . Reducing [Rb], t o z e r o a g a i n produces a n i n c r e a s e o f p h a s i c t e n s i o n . F i g u r e 2 B shows p h a s i c t e n s i o n a s a f u n c t i o n of a 1 d u r i n g t h e e x p o s u r e It is clearN$hat t h e twitch tent o Rb-free s o l u t i o n . s i o n i s a n o n l i n e a r , f u n c t i o n of a i There i s v e r y l i t t l e t e n s i o n a t a i a v a l u e s o f 182s t h a n a b o u t 6 mM. I n c r e a s i n g a a above t h i s l e v e l i s a s s o c i a t e d w i t h a s t e e p rise o t e n s i o n . T h i s n o n l i n e a r r e l a t i o n s h i p between t e n s i o n and a 1 c o n t r a s t s w i t h t h e r e p o r t o f L e e et a i . ( 1 9 8 0 ) . The88 workers found t h a t t e n s i o n w a s a l i n e a r f u n c t i o n o f a k a d u r i n g N a pump i n h i b i t i o n by d i hydroouabain. W e found a n o n l i n e a r r e l a t i o n s h i p e v e n under comparable c o n d i t i o n s t o t h o s e u s e d by L e e e t a l . Although t h e r e a r e many p o s s i b l e e x p l a n a t i o n s f o r t h i s nonlinear r e l a t i o n s h i p , an a t t r a c t i v e p o s s i b i l i t y i s b a s e d upon t h e known p r o p e r t i e s of N a / C a exchange i n o t h e r t i s s u e s s u c h as t h e s q u i d axon. A r e c e n t model ( M u l l i n s , 1977) s u g g e s t s t h a t f o u r N a and one c a l c i u m i o n must complex w i t h t h e c a r r i e r f o r t r a n s p o r t t o OCc u r . I n t h i s . c a s e t h e c a l c i u m i n f l u x w i l l be a s t e e p T h i s might u p d e r l i e t h e s t e e p r e l a f u n c t i o n of a k a . I t i s , however, t i o n s h i p between t e n s i o n and a',. e q u a l l y p o s s i b l e t h a t i n c r e a s e i loading of i n t r a c e l l u l a r c a l c i u m s t o r e s o r a n i n c r e a s e of t h e calcium c u r r e n t c a n a l s o c o n t r i b u t e t o t h e s t r o n g dependence of t e n s i o n o n
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Na' W e have a l s o examined t h e r e l a t i o n s h i p between and t e n s i o n d u r i n g t h e r e a c t i v a t i o n o f t h e N a pump. The c o m p l i c a t e d r e s u l t o b t a i n e d can be s e e n i n F i g . 1. When t h e N a pump i s i n h i b i t e d , b o t h a k a and t e n s i o n rise
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i and twitch tension. The relationship between a ( A ) E f f e c t o f changing [Rb], from 10 t o o m~ on a i a and tension. Throughout the experiment a depolarizing pulse was applied a t 0 . 1 Hz from the holding p t e n t i h l (-70 mV) t o - 3 3 mV. The arrow denotes an a r t i f a c t due t o change o f solution l e v e l . ( B ) Twitch tension a s a function o f a i a during the exposure t o 0 [RbIo. Data from ( A ) . From Eisner e t al. ( 1 9 8 1 ) . F i g . 2.
SODIUM PUMP INHIBITION IN SHEEP CARDIAC PURKINJE FIBERS
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a t comparable r a t e s . However, on r e a c t i v a t i n g t h e pump w i t h e x t e r n a l R b , t o n i c t e n s i o n f a l l s much f a s t e r t h a n does a k a . I n o t h e r words t h e r e i s a h y s t e r e s i s i n t h e r e l a t i o n s h l p between a’ and t o n i c t e n s i o n . A g i v e n l e v e l of a i a i s a s s o c i g t e d w i t h a h i g h e r l e v e l of t e n s i o n d u r i n g Na-pump i n h i b i t i o n ( R b - f r e e ) t h a n d u r i n g N a pump r e a c t i v a t i o n . Although n o t a p p a r e n t i n F i g . 1 t h e r e . i s sometimes a h y s t e r e s i s between t w i t c h t e n s i o n although t h e h y s t e r e s i s f o r t o n i c tension is more and ramatic. T h i s h y s t e r e s i s i s n o t e a s i l y e x p l a i n e d on a N a / C a exchange model. On s u c h a scheme t e n s i o n s h o u l d f a l l e i t h e r a t t h e same r a t e o r more s l o w l y t h a n a i a . There a r e s e v e r a l p o s s i b l e e x p l a n a t i o n s f o r t h e o b s e r v e d hysteresis. A t r i v i a l p o s s i b i l i t y i s t h a t t h e N a a c t i v i t y s e e n by t h e N a / C a exchange changes more q u i c k l y t h a n t h a t r e c o r d e d by t h e N a e l e c t r o d e . It is possible that N a i n a subsarcolemmal s p a c e exchanges much more r a p i d l y than t h e bulk, cytoplasmic afi This explanation is, however, made less c r e d i b l e by t8e e x p e r i m e n t o f F i g . 1 which d e m o n s t r a t e s t h a t t h e e l e c t r o g e n i c N a pump c u r r e n t h a s t h e same k i n e t i c s as aka measured by t h e e l e c t r o d e . T h i s s u g g e s t s t h a t t h e N a pump sees t h e same a h a a s t h e e l e c t r o d e and t h e r e f o r e t h a t Na n e a r t h e membrane changes i n p h a s e w i t h t h a t r e c o r d e d by t h e e l e c t r o d e . I t i s t h e r e f o r e u n l i k e l y t h a t t h e Na/Ca exchange i s cont r o l l e d by a d i f f e r e n t N a a c t i v i t y from t h a t r e c o r d e d . An a l t e r n a t i v e p o s s i b i l i t y i s b a s e d on t h e o b s e r v a t i o n t h a t i n c r e a s e s of a1 produced by N a pump i n h i b i t i o n l e a d t o a n i n t r a c e l y g l a r a c i d i f i c a t i o n ( D e i t m e r and E l l i s , 1 9 8 0 ) . T h i s a c i d i f i c a t i o n . w i . 1 1 r e d u c e t h e amount ( F a b i a t o and Fabiato, of t e n s i o n developed a t a g i v e n a1 1 9 7 8 ) . I t i s t h e r e f o r e p Q s s i b l e % a t t h e r e i s no hyst e r e s i s between and a i a . In t h i s case the hysteresis between a 1 and t e n s i o n would r e f l e c t changes of t h e s e n s i t i v i t y 8? t h e c o n t r a c t i l e p r o t e i n s t o calcium. Direct measurements of a C1 a u s i n g e i t h e r m i c r o e l e c t r o d e s o r a e g u o r i n w i l l be r e q u i r e d t o s e t t l e t h i s problem.
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REFERENCES Deitmer, J. W . , a n d E l l i s , D (1978). The i n t r a c e l l u l a r sodium act i v i t y o f c a r d i a c P u r k i n j e f i b r e s d u r i n g i n h i b i t i o n and rea c t i v a t i o n of t h e Na-K pump. J. P h y s i o l . (London) 284, 241259. Deitmer, J . W . , and E l l i s , D. (1980). I n t e r a c t i o n s between t h e r e g u l a t i o n of t h e i n t r a c e l l u l a r pH a n d sodium a c t i v i t y of sheep c a r d i a c Purkinje f i b r e s . J. P h y s i o l , (London) 204, 471-488.
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E i s n e r , D. A . , and L e d e r e r , W. J. (1979a). I n o t r o p i c and arrhythmogenic e f f e c t s o f potassium d e p l e t e d s o l u t i o n s on mammalian c a r d i a c muscle. J. P h y s i o l . (London) 284, 255277. E i s n e r , D. A . , and L e d e r e r , W. J. (1979b). The r o l e o f t h e sodium pump i n t h e e f f e c t s o f p o t a s s i u m d e p l e t e d s o l u t i o n s on J. P h y s i o l . (London) 294, 279mammalian c a r d i a c muscle. 301. E i s n e r , D. A . , L e d e r e r , W. J . , and Vaughan-Jones, R. D. ( 1 9 8 0 ) . E l e c t r o g e n i c sodium pumping i n c a r d i a c muscle: Shultaneo u s measurement of i n t r a c e l l u l a r sodium a c t i v i t y , membrane J. P h y s i o l . (London) 3 0 0 , 42P-43P. c u r r e n t and t e n s i o n . E i s n e r , D. A., L e d e r e r , W. J., and Vaughan-Jones, R. D. (1981). The dependence of sodium pumping and t e n s i o n on i n t r a c e l l u l a r sodium a c t i v i t y i n voltage-clamped s h e e p P u r k i n j e J. P h y s i o l . (London) 3 1 7 , 163-187. fibres. F a b i a t o , A., and F a b i a t o , F. (1978). E f f e c t s of p H o n t h e myof i l a m e n t s and t h e s a r c o p l a s m i c r e t i c u l u m o f s k i n n e d cells from c a r d i a c and s k e l e t a l muscles. J. P h y s i o l . (London) 276, 233-255. Gadsby, D. C ., and C r a n e f i e l d , P. F. ( 1 9 7 9 ) . D i r e c t measurement o f changes i n sodium pump c u r r e n t i n c a n i n e cardiac P u r k i n j e f i b r e s . Proc. N a t l . Acad. S c i . USA 76, 1783-1787. Lee, C. O . , Kang, D. H . , Sokol, J. H . , and L e e , K. S . ( 1 9 8 0 ) . Rel a t i o n between i n t r a c e l l u l a r N a i o n a c t i v i t y and t e n s i o n of s h e e p c a r d i a c P u r k i n j e f i b r e s exposed t o dihydro-ouabain. B i o p h y s . J. 29, 315-330. J . Gen. M u l l i n s , L . J. ( 1 9 7 7 ) . A mechanism f o r Na/Ca t r a n s p o r t . Physiol 70, 681-695. R e u t e r , H . , and S e i t z , H. ( 1 9 6 8 ) . The dependence of calcium e f flux from c a r d i a c muscle on t e m p e r a t u r e and i o n i c composiJ. P h y s i o l . (London) 1 9 5 , 451-470. tion. Thomas, R. C. (1978). " I o n - S e n s i t i v e 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 : How t o Make and U s e Them." Academic Press, New York.
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Effects of Sodium Pump Inhibitionon Contraction in Sheep Cardiac Purkinje Fibers D. A. EZSNER
Department of Physiology University College London London, United Kingdom
W. J . LEDERER Depanment of Physiology University of Maryland Baltimore, Maryland
R. D. VAUGHAN-JONES Department of Pharmacology Oxford, United Kingdom
I.
INTRODUCTION AND METHODS
I t i s w e l l e s t a b l i s h e d t h a t i n h i b i t i n g t h e sodium pump i n c r e a s e s t h e f o r c e of c o n t r a c t i o n o f c a r d i a c muscle. T h i s e f f e c t a p p e a r s t o be mediated v i a an i n crease o f i n t r a c e l l u l a r sodium a c t i v i t y , a i a . One t h e o r y f o r t h e mechanism of t h i s e f f e c t i n v o l v e s i a Na/Ca exchange ( R e u t e r and S e i t z , 1 9 6 8 ) . I n c r e a s i n g d N a would p r o d u c e an i n c r e a s e o f C a i n f l u x and d e c r e a s e C a e f f l u x t h r o u g h s u c h an e x c h a n g e , l e a d i n g t o i n c r e a s e d c o n t r a c tion. I n o r d e r t o i n v e s t i g a t e t h e mechanism of t h e pos i t i v e i n o t r o p i c e f f e c t s o f a r a i s e d d i a , one n e e d s t o measure a1 and t e n s i o n s i m u l t a n e o u s l y . As w e l l a s . i n Na v e s t i g a t i n g t h e s t e a d y - s t a t e r e l a t i o n s h i p between a i a and t e n s i o n , i t i s a l s o o f i n t e r e s t t o c h a r a c t e r i z e t h e e f f e c t s o f sydden c h a n g e s of a 1 The o n l y f e a s i b l e way t o measure a1 w i t h adequate N a t i m e r e s o l u t i o n i s w i t h a n ion-selec!?ve m i c r o e l e c t r o d e . W e used r e c e s s e d - t i p N a - s e n s i t i v e m i c r o e l e c t r o d e s (Thomas, 1 9 7 8 ) . The exp e r i m e n t s w e r e performed on v o l t a g e - c l a m p e d s h e e p c a r d i a c
.
885
Copynght 0 1983 by Academic Press. Inc. All rights of reproductionin any form reserved. ISBN 0-12-153319-0
D. A. EISNER eta/.
886
10
0
'
6min
1
4
'
F i g . 1 . S i m u l t a n e o u s measurement of c u r r e n t , aid, tension, and vol tage-clamped membrane p o t e n t i a l . A 500-msec d e p o l a r i z i n g p u l s e was a p p l i e d f r o m the h o l d i n g p o t e n t i a l ( - 6 8 mV) t o -35 mV a t 0.1 Hz t o e l i c i t a t w i t c h . F r o m Eisner e t a l . ( 1 9 8 0 ) .
P u r k i n j e f i b e r s . F u r t h e r d e t a i l s of t h e methods u s e d have a l r e a d y a p p e a r e d ( E i s n e r and L e d e r e r , 1979a; E i s n e r e t a l . , 1 9 8 1 ) . The use of t h e v o l t a g e clamp makes i t p o s s i b l e t o i n v e s t i g a t e t h e e f f e c t s of changes of aBa w i t h o u t s e c o n d a r y e f f e c t s due t o changes of membrane p o t e n t i a l . Sodium pump a c t i v i t y w a s c o n t r o l l e d by changing t h e e x t e r n a l rubidium c o n c e n t r a t i o n (K-free sol u t i o n s were used t h r o u g h o u t ) .
SODIUM PUMP INHIBITION IN SHEEP CARDIAC PURKINJE FIBERS
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RESULTS AND D I S C U S S I O N
F i g u r e 1 shows s i m u l t a n e o u s measurements o f i n t r a c e l l u l a r N a a c t i v i t y , a h a , t e n s i o n and c u r r e n t i n a voltage-clamped s h e e p c a r d i a c P u r k i n j e f i b e r . The sodium pump w a s i n h i b i t e d by r e d u c i n g [ % l o from 1 0 t o 0 mM. T h i s produced a r i s e o f a 1 ( c f . D e i t m e r and E l l i s , 1978) and a l s o i n c r e a s e d Na t w i t c h and t o n i c t e n s i o n . Following t h i s e x p o s u r e t o Rb-free s o l u t i o n , [RbIo was i n c r e a s e d t o 4 mM r e s u l t i n g i n a decrease of a1 and both t w i t c h and t o n i c t e n s i o n . A t t h e same t i m e c u r r e n t t r a c e shows a peak of outward c u r r e n t which d e c a y s away. T h i s c u r r e n t t r a n s i e n t i s t h e e l e c t r o g e n i c Na-pump c u r r e n t t r a n s i e n t ( E i s n e r and L e d e r e r , 1979b; Gadsby and C r a n e f i e l d , 1 9 7 9 ) . A n a l y s i s of r e c o r d s s u c h a s F i g . 1 shows t h a t b o t h a i a and t h e c u r r e n t decay exp o n e n t i a l l y upon r e a c t i v a t i n g t h e N a pump w i t h rubidium. T h e h a l f - t i m e o f d e c a y , o f c u r r e n t i s always s i m i l a r t o t h a t o f t h e decay of a 1 ( E i s n e r et al., 1 9 8 1 ) . The r e l a t i o n s h i p @tween a N1 a and t e n s i o n i s s t u d i e d i n more d e t a i l i n F i g . 2 . Reducing [Rb], t o z e r o a g a i n produces a n i n c r e a s e o f p h a s i c t e n s i o n . F i g u r e 2 B shows p h a s i c t e n s i o n a s a f u n c t i o n of a 1 d u r i n g t h e e x p o s u r e It is clearN$hat t h e twitch tent o Rb-free s o l u t i o n . s i o n i s a n o n l i n e a r , f u n c t i o n of a i There i s v e r y l i t t l e t e n s i o n a t a i a v a l u e s o f 182s t h a n a b o u t 6 mM. I n c r e a s i n g a a above t h i s l e v e l i s a s s o c i a t e d w i t h a s t e e p rise o t e n s i o n . T h i s n o n l i n e a r r e l a t i o n s h i p between t e n s i o n and a 1 c o n t r a s t s w i t h t h e r e p o r t o f L e e et a i . ( 1 9 8 0 ) . The88 workers found t h a t t e n s i o n w a s a l i n e a r f u n c t i o n o f a k a d u r i n g N a pump i n h i b i t i o n by d i hydroouabain. W e found a n o n l i n e a r r e l a t i o n s h i p e v e n under comparable c o n d i t i o n s t o t h o s e u s e d by L e e e t a l . Although t h e r e a r e many p o s s i b l e e x p l a n a t i o n s f o r t h i s nonlinear r e l a t i o n s h i p , an a t t r a c t i v e p o s s i b i l i t y i s b a s e d upon t h e known p r o p e r t i e s of N a / C a exchange i n o t h e r t i s s u e s s u c h as t h e s q u i d axon. A r e c e n t model ( M u l l i n s , 1977) s u g g e s t s t h a t f o u r N a and one c a l c i u m i o n must complex w i t h t h e c a r r i e r f o r t r a n s p o r t t o OCc u r . I n t h i s . c a s e t h e c a l c i u m i n f l u x w i l l be a s t e e p T h i s might u p d e r l i e t h e s t e e p r e l a f u n c t i o n of a k a . I t i s , however, t i o n s h i p between t e n s i o n and a',. e q u a l l y p o s s i b l e t h a t i n c r e a s e i loading of i n t r a c e l l u l a r c a l c i u m s t o r e s o r a n i n c r e a s e of t h e calcium c u r r e n t c a n a l s o c o n t r i b u t e t o t h e s t r o n g dependence of t e n s i o n o n
tIa
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a1
aia
Na' W e have a l s o examined t h e r e l a t i o n s h i p between and t e n s i o n d u r i n g t h e r e a c t i v a t i o n o f t h e N a pump. The c o m p l i c a t e d r e s u l t o b t a i n e d can be s e e n i n F i g . 1. When t h e N a pump i s i n h i b i t e d , b o t h a k a and t e n s i o n rise
888
D. A. EISNER eta/.
5 min
0
4
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12
i and twitch tension. The relationship between a ( A ) E f f e c t o f changing [Rb], from 10 t o o m~ on a i a and tension. Throughout the experiment a depolarizing pulse was applied a t 0 . 1 Hz from the holding p t e n t i h l (-70 mV) t o - 3 3 mV. The arrow denotes an a r t i f a c t due t o change o f solution l e v e l . ( B ) Twitch tension a s a function o f a i a during the exposure t o 0 [RbIo. Data from ( A ) . From Eisner e t al. ( 1 9 8 1 ) . F i g . 2.
SODIUM PUMP INHIBITION IN SHEEP CARDIAC PURKINJE FIBERS
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a t comparable r a t e s . However, on r e a c t i v a t i n g t h e pump w i t h e x t e r n a l R b , t o n i c t e n s i o n f a l l s much f a s t e r t h a n does a k a . I n o t h e r words t h e r e i s a h y s t e r e s i s i n t h e r e l a t i o n s h l p between a’ and t o n i c t e n s i o n . A g i v e n l e v e l of a i a i s a s s o c i g t e d w i t h a h i g h e r l e v e l of t e n s i o n d u r i n g Na-pump i n h i b i t i o n ( R b - f r e e ) t h a n d u r i n g N a pump r e a c t i v a t i o n . Although n o t a p p a r e n t i n F i g . 1 t h e r e . i s sometimes a h y s t e r e s i s between t w i t c h t e n s i o n although t h e h y s t e r e s i s f o r t o n i c tension is more and ramatic. T h i s h y s t e r e s i s i s n o t e a s i l y e x p l a i n e d on a N a / C a exchange model. On s u c h a scheme t e n s i o n s h o u l d f a l l e i t h e r a t t h e same r a t e o r more s l o w l y t h a n a i a . There a r e s e v e r a l p o s s i b l e e x p l a n a t i o n s f o r t h e o b s e r v e d hysteresis. A t r i v i a l p o s s i b i l i t y i s t h a t t h e N a a c t i v i t y s e e n by t h e N a / C a exchange changes more q u i c k l y t h a n t h a t r e c o r d e d by t h e N a e l e c t r o d e . It is possible that N a i n a subsarcolemmal s p a c e exchanges much more r a p i d l y than t h e bulk, cytoplasmic afi This explanation is, however, made less c r e d i b l e by t8e e x p e r i m e n t o f F i g . 1 which d e m o n s t r a t e s t h a t t h e e l e c t r o g e n i c N a pump c u r r e n t h a s t h e same k i n e t i c s as aka measured by t h e e l e c t r o d e . T h i s s u g g e s t s t h a t t h e N a pump sees t h e same a h a a s t h e e l e c t r o d e and t h e r e f o r e t h a t Na n e a r t h e membrane changes i n p h a s e w i t h t h a t r e c o r d e d by t h e e l e c t r o d e . I t i s t h e r e f o r e u n l i k e l y t h a t t h e Na/Ca exchange i s cont r o l l e d by a d i f f e r e n t N a a c t i v i t y from t h a t r e c o r d e d . An a l t e r n a t i v e p o s s i b i l i t y i s b a s e d on t h e o b s e r v a t i o n t h a t i n c r e a s e s of a1 produced by N a pump i n h i b i t i o n l e a d t o a n i n t r a c e l y g l a r a c i d i f i c a t i o n ( D e i t m e r and E l l i s , 1 9 8 0 ) . T h i s a c i d i f i c a t i o n . w i . 1 1 r e d u c e t h e amount ( F a b i a t o and Fabiato, of t e n s i o n developed a t a g i v e n a1 1 9 7 8 ) . I t i s t h e r e f o r e p Q s s i b l e % a t t h e r e i s no hyst e r e s i s between and a i a . In t h i s case the hysteresis between a 1 and t e n s i o n would r e f l e c t changes of t h e s e n s i t i v i t y 8? t h e c o n t r a c t i l e p r o t e i n s t o calcium. Direct measurements of a C1 a u s i n g e i t h e r m i c r o e l e c t r o d e s o r a e g u o r i n w i l l be r e q u i r e d t o s e t t l e t h i s problem.
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ata
REFERENCES Deitmer, J. W . , a n d E l l i s , D (1978). The i n t r a c e l l u l a r sodium act i v i t y o f c a r d i a c P u r k i n j e f i b r e s d u r i n g i n h i b i t i o n and rea c t i v a t i o n of t h e Na-K pump. J. P h y s i o l . (London) 284, 241259. Deitmer, J . W . , and E l l i s , D. (1980). I n t e r a c t i o n s between t h e r e g u l a t i o n of t h e i n t r a c e l l u l a r pH a n d sodium a c t i v i t y of sheep c a r d i a c Purkinje f i b r e s . J. P h y s i o l , (London) 204, 471-488.
D. A. EISNER eta/.
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E i s n e r , D. A . , and L e d e r e r , W. J. (1979a). I n o t r o p i c and arrhythmogenic e f f e c t s o f potassium d e p l e t e d s o l u t i o n s on mammalian c a r d i a c muscle. J. P h y s i o l . (London) 284, 255277. E i s n e r , D. A . , and L e d e r e r , W. J. (1979b). The r o l e o f t h e sodium pump i n t h e e f f e c t s o f p o t a s s i u m d e p l e t e d s o l u t i o n s on J. P h y s i o l . (London) 294, 279mammalian c a r d i a c muscle. 301. E i s n e r , D. A . , L e d e r e r , W. J . , and Vaughan-Jones, R. D. ( 1 9 8 0 ) . E l e c t r o g e n i c sodium pumping i n c a r d i a c muscle: Shultaneo u s measurement of i n t r a c e l l u l a r sodium a c t i v i t y , membrane J. P h y s i o l . (London) 3 0 0 , 42P-43P. c u r r e n t and t e n s i o n . E i s n e r , D. A., L e d e r e r , W. J., and Vaughan-Jones, R. D. (1981). The dependence of sodium pumping and t e n s i o n on i n t r a c e l l u l a r sodium a c t i v i t y i n voltage-clamped s h e e p P u r k i n j e J. P h y s i o l . (London) 3 1 7 , 163-187. fibres. F a b i a t o , A., and F a b i a t o , F. (1978). E f f e c t s of p H o n t h e myof i l a m e n t s and t h e s a r c o p l a s m i c r e t i c u l u m o f s k i n n e d cells from c a r d i a c and s k e l e t a l muscles. J. P h y s i o l . (London) 276, 233-255. Gadsby, D. C ., and C r a n e f i e l d , P. F. ( 1 9 7 9 ) . D i r e c t measurement o f changes i n sodium pump c u r r e n t i n c a n i n e cardiac P u r k i n j e f i b r e s . Proc. N a t l . Acad. S c i . USA 76, 1783-1787. Lee, C. O . , Kang, D. H . , Sokol, J. H . , and L e e , K. S . ( 1 9 8 0 ) . Rel a t i o n between i n t r a c e l l u l a r N a i o n a c t i v i t y and t e n s i o n of s h e e p c a r d i a c P u r k i n j e f i b r e s exposed t o dihydro-ouabain. B i o p h y s . J. 29, 315-330. J . Gen. M u l l i n s , L . J. ( 1 9 7 7 ) . A mechanism f o r Na/Ca t r a n s p o r t . Physiol 70, 681-695. R e u t e r , H . , and S e i t z , H. ( 1 9 6 8 ) . The dependence of calcium e f flux from c a r d i a c muscle on t e m p e r a t u r e and i o n i c composiJ. P h y s i o l . (London) 1 9 5 , 451-470. tion. Thomas, R. C. (1978). " I o n - S e n s i t i v e 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 : How t o Make and U s e Them." Academic Press, New York.
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