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Intracellular Sodium Enhancement of Ouabain Binding to Na,K-ATPase and the Development of Glycoside Actions
CURReNT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19
lntracellular Sodium Enhancementof Ouabain Binding to Na,K-ATPase and the Development of Glycoside TAI AKERA, KYOSUKE TEMMA,
AND SATOSHI YAMAMOTO’ Depament of Pharmacology and Toxicology Michigan State University East Lansing, Michigan
I.
INTRODUCTION
The b i n d i n g of c a r d i a c g l y c o s i d e s t o Na,K-ATPase, observed i n v i t r o i n t h e p r e s e n c e of Mg2+ and ATP, i s enhanced by N a + (Matsui.and Schwartz, 1 9 6 8 ) . S i n c e Na+ a c t i v a t e s t h e enzyme a t t h e c y t o p l a s m i c s i d e of t h e c e l l membrane, it seems t o be i n t r a c e l l u l a r N a + ( N a + i ) t h a t s t i m u l a t e s t h e g l y c o s i d e b i n d i n g . Bodemann and Hoffman ( 1 9 7 6 1 , however, r e p o r t e d t h a t Na+i f a i l s t o s t i m u l a t e ouabain b i n d i n g i n r e s e a l e d e r y t h r o c y t e g h o s t s under c e r t a i n ionic conditions. I n myocardium, t h e p o s i t i v e i n o t r o p i c and t o x i c a c t i o n s of t h e g l y c o s i d e s develop f a s t e r when t h e muscle i s e l e c t r i c a l l y s t i m u l a t e d a t higher frequencies. Since s t i m u l a t i o n , and r e s u l t i n g membrane d e p o l a r i z a t i o n , i n c r e a s e s t h e amount of Na+i a v a i l a b l e t o Na,K-ATPase o r t h e sodium pump, t h e above f i n d i n g f a v o r s t h e c o n c e p t t h a t Na+i s t i m u l a t e s g l y c o s i d e b i n d i n g . Clausen and Hansen ( 1 9 7 7 ) r e p o r t e d t h a t glycoside-induced sodium pump i n h i b i t i o n i n s k e l e t a l muscle o r a d i p o c y t e s i s aug‘ P r e s e n t a d d r e s s : Department of Pharmacology, Keio L h i v e r s i t y School of M e d i c i n e , T o k y o , J a p a n . 235
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mented under t h e c o n d i t i o n s which enhance enzyme t u r n over. Since N a + i i s a f a c t o r stimulating t h e turnover, t h e s e f i n d i n g s a l s o s u p p o r t t h e above c o n c e p t . I f N a + i regulates t h e glycoside binding i n i n t a c t c e l l s , p o t e n c i e s of t h e g l y c o s i d e s which i n h i b i t i s o lated Na,K-ATPase, observed i n t h e presence of a r b i t r a r y N a + and K+ c o n c e n t r a t i o n s , c a n n o t be compared w i t h t h o s e which produce i n o t r o p i c e f f e c t s i n i n t a c t c a r d i a c c e l l s i n an a t t e m p t t o examine t h e r e l a t i o n s h i p between t h e s e two e v e n t s . S i n c e r e l e v a n t (asymmetric) l i g a n d c o n d i t i o n s c a n n o t be reproduced i n i s o l a t e d enzyme s t u d i e s , i n h i b i t o r y and i n o t r o p i c a c t i o n s might o n l y be compared i n i n t a c t c e l l s . T h e r e f o r e it i s i m p o r t a n t t o u n d e r s t a n d t h e e f f e c t s of N a + i on sodium pump a c t i v i t y ( t u r n o v e r o f Na,K-ATPase i n i n t a c t c e l l s ) , g l y c o s i d e s e n s i t i v i t y of t h e sodium pump, and t h e g l y c o s i d e b i n d i n g t o Na,K-ATPase i n b e a t i n g m y o c a r d i a l c e l l s . The p u r p o s e of t h i s a r t i c l e i s t o examine t h e abovementioned e f f e c t s o f N a + i and t o r e e v a l u a t e t h e o u a b a i n s e n s i t i v e 86Rb+- o r 42K+-uptake as a means o f e s t i m a t i n g sodium pump a c t i v i t y . 11.
RESULTS AND DISCUSSION
A r e d u c t i o n o r a u m e n t a t i o n o f t h e ouabains e n s i t i v e ( s p e c i f i c ) 8iRb+- o r 42K+-uptake h a s been f r e q u e n t l y i n t e r p r e t e d t o i n d i c a t e i n h i b i t i o n and s t i m u l a t i o n , r e s p e c t i v e l y , of t h e sodium pump. A d d i t i o n a l l y , c o n c e n t r a t i o n s of o u a b a i n which c a u s e a 5 0 % i n h i b i t i o n of t h e u p t a k e are used t o r e p r e s e n t t h e g l y c o s i d e s e n s i t i v i t y of t h e sodium pump. The f o l l o w i n g c o n s i d e r a t i o n s however, s u g g e s t t h a t t h e s e might n o t b e t h e case. Under normal c o n d i t i o n s , t h e m y o c a r d i a l sodium pump seems t o have r e s e r v e c a p a c i t y because n e i t h e r a modera t e enhancement o f t h e sodium i n f l u x , caused by a n i n crease i n h e a r t r a t e o r by a g e n t s which i n c r e a s e sodium i n f l u x ( e . g . , monensin, v e r a t r i d i n e , o r g r a y a n o t o x i n ) , nor a p a r t i a l i n h i b i t i o n o f t h e sodium pump by i o n o t r o p i c c o n c e n t r a t i o n s of c a r d i a c g l y c o s i d e s markedly e l e v a t e s m y o c a r d i a l sodium. T h e r e f o r e , when t h e s p e c i f i c Rb+ or K+ u p t a k e i s e s t i m a t e d i n q u i e s c e n t p r e p a r a t i o n s w i t h o u t a Na+ p r e l o a d i n g , t h e o b s e r v e d u p t a k e r e p r e s e n t s ongoing sodium pump a c t i v i t y , which i s determined by, and e q u i v a l e n t t o , t h e r a t e o f sodium i n f l u x ( F i g . 1 ) . Thus, t h e Rb+ o r K+ u p t a k e may be a l t e r e d by changes i n t h e N a + i n f l u x . Moreover, i f t h e sodium pump h a s res e r v e c a p a c i t y , pump i n h i b i t i o n o f a d e g r e e less t h a n t h e r e s e r v e c a p a c i t y may n o t r e s u l t i n a r e d u c t i o n o f
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F i g . 1 . R e s e r v c c a p a c i t y of the s o d i u m pump and ouabain s e n s i t i v i t y of 86Rb+ u p t a k e .
t h e o b s e r v e d Rb+ o r K+ u p t a k e : t h e u p t a k e may be i n h i b i t e d o n l y when t h e reserve c a p a c i t y i s e x h a u s t e d . Thus, t h e s e n s i t i v i t y of t h e sodium pump t o an i n h i b i t o r may be u n d e r e s t i m a t e d ( F i g . 1 ) . The f o l l o w i n g o b s e r v a t i o n s s u p p o r t t h e above concept: (1) N a + l o a d i n g o f l e f t a t r i a l muscle p r e p a r a t i o n s o f g u i n e a - p i g h e a r t ( e l e v a t e d N a + i due t o a 30-min i n c u b a t i o n a t O°C i n a K+- and Ca2+-free medium) enhanced t h e s p e c i f i c Rb+ u p t a k e o b s e r v e d w i t h a 7-min i n c u b a t i o n a t 37'C. A f t e r a 35-min i n c u b a t i o n , however, t h e u p t a k e was n o t s i g n i f i c a n t l y a f f e c t e d by t h e N a + loading, indicating t h a t t h e e x t r a N a + i is a v a i l a b l e t o t h e pump o n l y d u r i n g t h e f i r s t s e v e r a l m i n u t e s . ( 2 ) a - D i h y d r o g r a y a n o t o x i n (0.5-5 L I M ) , monensin (1-10 L I M ) , o r 1.5-3 Hz e l e c t r i c a l s t i m u l a t i o n enhanced t h e s p e c i f i c Rb+ u p t a k e i n p r e p a r a t i o n s which were n o t N a + l o a d e d . ( 3 ) The s t i m u l a t e d p o r t i o n w a s i n h i b i t e d by h i g h concent r a t i o n s ( 1 0 - 1 0 0 U M ) o f v e r a p a m i l , which i s known t o i n h i b i t Na+ influx. ( 4 ) R e l a t i v e l y h i g h s p e c i f i c Rb+ upt a k e of e l e c t r i c a l l y s t i m u l a t e d p r e p a r a t i o n s was n o t a f -
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f e c t e d by monensin a t 2 . 5 U M and w a s r a t h e r i n h i b i t e d a t 1 0 VM. ( 5 ) Monensin o r g r a y a n o t o x i n d i d n o t a f f e c t Na,K-ATPase a c t i v i t y i n v i t r o , i n d i c a t i n g t h a t t h e obs e r v e d e f f e c t s o f t h e s e a g e n t s are n o t due t o a d i r e c t s t i m u l a t i o n of t h e sodium pump. (6) Concentrations of ouabain r e q u i r e d t o cause a 50% i n h i b i t i o n of s p e c i f i c Rb+ u p t a k e were markedly lowered by 2 . 5 V M monensin o r 1.5-3 Hz e l e c t r i c a l s t i m u l a t i o n . E l e c t r i c a l s t i m u l a t i o n a l s o i n c r e a s e d o u a b a i n F s e n s i t i v i t y of t h e s p e c i f i c 42K+ uptake. Exposure o f q u i e s c e n t l e f t a t r i a l muscle p r e p a r a t i o n s o f g u i n e a p i g h e a r t t o 0 . 5 V M o u a b a i n f o r 60 min a t 3 O o C r e s u l t e d i n a 1 3 . 4 % occupancy o f t h e g l y c o s i d e Monensin ( 1 0 v M ) enhanced b i n d i n g s i t e s on Na,K-ATPase. t h e o u a b a i n b i n d i n g , r e s u l t i n g i n a 34.3% occupancy. E l e c t r i c a l s t i m u l a t i o n a t 2 Hz caused a f a s t e r and g r e a t e r o u a b a i n b i n d i n g t o Na,K-ATPase t h a n t h a t obs e r v e d w i t h 0.5-Hz s t i m u l a t i o n . Concomitantly, t h e pos i t i v e i n o t r o p i c e f f e c t o f o u a b a i n developed more r a p i d l y when p r e p a r a t i o n s w e r e s t i m u l a t e d a t , h i g h e r f r e q u e n c i e s , o r i n t h e p r e s e n c e of e i t h e r g r a y a n o t o x i n o r monensin. Monensin ( 1 0 U M ) enhanced t h e development o f c o n t r a c t u r e due t o 5 P M o u a b a i n i n q u i e s c e n t muscle, i n d i c a t i n g t h a t t h e development o f t h e t o x i c e f f e c t of o u a b a i n i s a l s o enhanced by Na'i.
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SUMMARY
These r e s u l t s i n d i c a t e t h a t Na'i available t o the sodium pump i s t h e d e t e r m i n a n t of t h e s p e c i f i c 86Rb+o r 42K+-uptake i n c a r d i a c muscle p r e p a r a t i o n s which a r e not preloaded with Na+. The s p e c i f i c u p t a k e , t h e r e f o r e , may r e p r e s e n t t h e r a t e of N a + i n f l u x which i s e q u i v a l e n t t o N a + e f f l u x i n s t e a d y - s t a t e p r e p a r a t i o n s . The condit i o n s which i n c r e a s e N a + i a v a i l a b l e t o t h e sodium pump a f f e c t t h e c o n c e n t r a t i o n of o u a b a i n r e q u i r e d t o i n h i b i t t h e s p e c i f i c Rb+- or K+-uptake by enhancing t h e g l y c o s i d e b i v d i n g and a l s o by r e d u c i n g t h e r e s e r v e c a p a c i t y . In Na,-loaded p r e p a r a t i o n s , ouabain s e n s i t i v i t y i s probably overestimated. I n t r a c e l l u l a r N a + enhances Na,K-ATPase i n h i b i t i o n by o u a b a i n and i t s i n o t r o p i c and t o x i c actions.
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ACKNOWLEDGMENTS T h i s work w a s s u p p o r t e d by a USPHS g r a n t , HL 16052 from t h e N a t i o n a l H e a r t , Lung, and Blood I n s t i t u t e .
REFERENCES BOdemann, H. H . , and Hoffman, J. F. ( 1 9 7 6 ) . Comparison of t h e side-dependent e f f e c t s o f N a and K on o r t h o p h o s p h a t e - , UTP-, a n d ATP-promoted o u a b a i n b i n d i n g t o r e c o n s t i t u t e d human r e d blood c e l l g h o s t s . J. Gen. P h y s i o l . 6 7 , 527-545. Clausen, T . , and Hansen, 0. (1977). A c t i v e Na-K t r a n s p o r t and t h e r a t e o f o u a b a i n b i n d i n g . The e f f e c t o f i n s u l i n and o t h e r s t i m u l i on s k e l e t a l muscle and a d i p o c y t e s . J. P h y s i o l . (London) 2 7 0 , 415-430. Matsui, H., and Schwartz, A. ( 1 9 6 8 ) . Mechanism of c a r d i a c g l y c o s i d e i n h i b i t i o n of t h e (Na+-K+)-dependent A T P a s e from c a r d i a c t i s s u e . B i o c h i m . B i o p h y s . A c t a ( A m s t e r d a m ) 1 5 1 , 655-663.
lntracellular Sodium Enhancementof Ouabain Binding to Na,K-ATPase and the Development of Glycoside TAI AKERA, KYOSUKE TEMMA,
AND SATOSHI YAMAMOTO’ Depament of Pharmacology and Toxicology Michigan State University East Lansing, Michigan
I.
INTRODUCTION
The b i n d i n g of c a r d i a c g l y c o s i d e s t o Na,K-ATPase, observed i n v i t r o i n t h e p r e s e n c e of Mg2+ and ATP, i s enhanced by N a + (Matsui.and Schwartz, 1 9 6 8 ) . S i n c e Na+ a c t i v a t e s t h e enzyme a t t h e c y t o p l a s m i c s i d e of t h e c e l l membrane, it seems t o be i n t r a c e l l u l a r N a + ( N a + i ) t h a t s t i m u l a t e s t h e g l y c o s i d e b i n d i n g . Bodemann and Hoffman ( 1 9 7 6 1 , however, r e p o r t e d t h a t Na+i f a i l s t o s t i m u l a t e ouabain b i n d i n g i n r e s e a l e d e r y t h r o c y t e g h o s t s under c e r t a i n ionic conditions. I n myocardium, t h e p o s i t i v e i n o t r o p i c and t o x i c a c t i o n s of t h e g l y c o s i d e s develop f a s t e r when t h e muscle i s e l e c t r i c a l l y s t i m u l a t e d a t higher frequencies. Since s t i m u l a t i o n , and r e s u l t i n g membrane d e p o l a r i z a t i o n , i n c r e a s e s t h e amount of Na+i a v a i l a b l e t o Na,K-ATPase o r t h e sodium pump, t h e above f i n d i n g f a v o r s t h e c o n c e p t t h a t Na+i s t i m u l a t e s g l y c o s i d e b i n d i n g . Clausen and Hansen ( 1 9 7 7 ) r e p o r t e d t h a t glycoside-induced sodium pump i n h i b i t i o n i n s k e l e t a l muscle o r a d i p o c y t e s i s aug‘ P r e s e n t a d d r e s s : Department of Pharmacology, Keio L h i v e r s i t y School of M e d i c i n e , T o k y o , J a p a n . 235
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mented under t h e c o n d i t i o n s which enhance enzyme t u r n over. Since N a + i i s a f a c t o r stimulating t h e turnover, t h e s e f i n d i n g s a l s o s u p p o r t t h e above c o n c e p t . I f N a + i regulates t h e glycoside binding i n i n t a c t c e l l s , p o t e n c i e s of t h e g l y c o s i d e s which i n h i b i t i s o lated Na,K-ATPase, observed i n t h e presence of a r b i t r a r y N a + and K+ c o n c e n t r a t i o n s , c a n n o t be compared w i t h t h o s e which produce i n o t r o p i c e f f e c t s i n i n t a c t c a r d i a c c e l l s i n an a t t e m p t t o examine t h e r e l a t i o n s h i p between t h e s e two e v e n t s . S i n c e r e l e v a n t (asymmetric) l i g a n d c o n d i t i o n s c a n n o t be reproduced i n i s o l a t e d enzyme s t u d i e s , i n h i b i t o r y and i n o t r o p i c a c t i o n s might o n l y be compared i n i n t a c t c e l l s . T h e r e f o r e it i s i m p o r t a n t t o u n d e r s t a n d t h e e f f e c t s of N a + i on sodium pump a c t i v i t y ( t u r n o v e r o f Na,K-ATPase i n i n t a c t c e l l s ) , g l y c o s i d e s e n s i t i v i t y of t h e sodium pump, and t h e g l y c o s i d e b i n d i n g t o Na,K-ATPase i n b e a t i n g m y o c a r d i a l c e l l s . The p u r p o s e of t h i s a r t i c l e i s t o examine t h e abovementioned e f f e c t s o f N a + i and t o r e e v a l u a t e t h e o u a b a i n s e n s i t i v e 86Rb+- o r 42K+-uptake as a means o f e s t i m a t i n g sodium pump a c t i v i t y . 11.
RESULTS AND DISCUSSION
A r e d u c t i o n o r a u m e n t a t i o n o f t h e ouabains e n s i t i v e ( s p e c i f i c ) 8iRb+- o r 42K+-uptake h a s been f r e q u e n t l y i n t e r p r e t e d t o i n d i c a t e i n h i b i t i o n and s t i m u l a t i o n , r e s p e c t i v e l y , of t h e sodium pump. A d d i t i o n a l l y , c o n c e n t r a t i o n s of o u a b a i n which c a u s e a 5 0 % i n h i b i t i o n of t h e u p t a k e are used t o r e p r e s e n t t h e g l y c o s i d e s e n s i t i v i t y of t h e sodium pump. The f o l l o w i n g c o n s i d e r a t i o n s however, s u g g e s t t h a t t h e s e might n o t b e t h e case. Under normal c o n d i t i o n s , t h e m y o c a r d i a l sodium pump seems t o have r e s e r v e c a p a c i t y because n e i t h e r a modera t e enhancement o f t h e sodium i n f l u x , caused by a n i n crease i n h e a r t r a t e o r by a g e n t s which i n c r e a s e sodium i n f l u x ( e . g . , monensin, v e r a t r i d i n e , o r g r a y a n o t o x i n ) , nor a p a r t i a l i n h i b i t i o n o f t h e sodium pump by i o n o t r o p i c c o n c e n t r a t i o n s of c a r d i a c g l y c o s i d e s markedly e l e v a t e s m y o c a r d i a l sodium. T h e r e f o r e , when t h e s p e c i f i c Rb+ or K+ u p t a k e i s e s t i m a t e d i n q u i e s c e n t p r e p a r a t i o n s w i t h o u t a Na+ p r e l o a d i n g , t h e o b s e r v e d u p t a k e r e p r e s e n t s ongoing sodium pump a c t i v i t y , which i s determined by, and e q u i v a l e n t t o , t h e r a t e o f sodium i n f l u x ( F i g . 1 ) . Thus, t h e Rb+ o r K+ u p t a k e may be a l t e r e d by changes i n t h e N a + i n f l u x . Moreover, i f t h e sodium pump h a s res e r v e c a p a c i t y , pump i n h i b i t i o n o f a d e g r e e less t h a n t h e r e s e r v e c a p a c i t y may n o t r e s u l t i n a r e d u c t i o n o f
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F i g . 1 . R e s e r v c c a p a c i t y of the s o d i u m pump and ouabain s e n s i t i v i t y of 86Rb+ u p t a k e .
t h e o b s e r v e d Rb+ o r K+ u p t a k e : t h e u p t a k e may be i n h i b i t e d o n l y when t h e reserve c a p a c i t y i s e x h a u s t e d . Thus, t h e s e n s i t i v i t y of t h e sodium pump t o an i n h i b i t o r may be u n d e r e s t i m a t e d ( F i g . 1 ) . The f o l l o w i n g o b s e r v a t i o n s s u p p o r t t h e above concept: (1) N a + l o a d i n g o f l e f t a t r i a l muscle p r e p a r a t i o n s o f g u i n e a - p i g h e a r t ( e l e v a t e d N a + i due t o a 30-min i n c u b a t i o n a t O°C i n a K+- and Ca2+-free medium) enhanced t h e s p e c i f i c Rb+ u p t a k e o b s e r v e d w i t h a 7-min i n c u b a t i o n a t 37'C. A f t e r a 35-min i n c u b a t i o n , however, t h e u p t a k e was n o t s i g n i f i c a n t l y a f f e c t e d by t h e N a + loading, indicating t h a t t h e e x t r a N a + i is a v a i l a b l e t o t h e pump o n l y d u r i n g t h e f i r s t s e v e r a l m i n u t e s . ( 2 ) a - D i h y d r o g r a y a n o t o x i n (0.5-5 L I M ) , monensin (1-10 L I M ) , o r 1.5-3 Hz e l e c t r i c a l s t i m u l a t i o n enhanced t h e s p e c i f i c Rb+ u p t a k e i n p r e p a r a t i o n s which were n o t N a + l o a d e d . ( 3 ) The s t i m u l a t e d p o r t i o n w a s i n h i b i t e d by h i g h concent r a t i o n s ( 1 0 - 1 0 0 U M ) o f v e r a p a m i l , which i s known t o i n h i b i t Na+ influx. ( 4 ) R e l a t i v e l y h i g h s p e c i f i c Rb+ upt a k e of e l e c t r i c a l l y s t i m u l a t e d p r e p a r a t i o n s was n o t a f -
TAI AKERA eta/.
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f e c t e d by monensin a t 2 . 5 U M and w a s r a t h e r i n h i b i t e d a t 1 0 VM. ( 5 ) Monensin o r g r a y a n o t o x i n d i d n o t a f f e c t Na,K-ATPase a c t i v i t y i n v i t r o , i n d i c a t i n g t h a t t h e obs e r v e d e f f e c t s o f t h e s e a g e n t s are n o t due t o a d i r e c t s t i m u l a t i o n of t h e sodium pump. (6) Concentrations of ouabain r e q u i r e d t o cause a 50% i n h i b i t i o n of s p e c i f i c Rb+ u p t a k e were markedly lowered by 2 . 5 V M monensin o r 1.5-3 Hz e l e c t r i c a l s t i m u l a t i o n . E l e c t r i c a l s t i m u l a t i o n a l s o i n c r e a s e d o u a b a i n F s e n s i t i v i t y of t h e s p e c i f i c 42K+ uptake. Exposure o f q u i e s c e n t l e f t a t r i a l muscle p r e p a r a t i o n s o f g u i n e a p i g h e a r t t o 0 . 5 V M o u a b a i n f o r 60 min a t 3 O o C r e s u l t e d i n a 1 3 . 4 % occupancy o f t h e g l y c o s i d e Monensin ( 1 0 v M ) enhanced b i n d i n g s i t e s on Na,K-ATPase. t h e o u a b a i n b i n d i n g , r e s u l t i n g i n a 34.3% occupancy. E l e c t r i c a l s t i m u l a t i o n a t 2 Hz caused a f a s t e r and g r e a t e r o u a b a i n b i n d i n g t o Na,K-ATPase t h a n t h a t obs e r v e d w i t h 0.5-Hz s t i m u l a t i o n . Concomitantly, t h e pos i t i v e i n o t r o p i c e f f e c t o f o u a b a i n developed more r a p i d l y when p r e p a r a t i o n s w e r e s t i m u l a t e d a t , h i g h e r f r e q u e n c i e s , o r i n t h e p r e s e n c e of e i t h e r g r a y a n o t o x i n o r monensin. Monensin ( 1 0 U M ) enhanced t h e development o f c o n t r a c t u r e due t o 5 P M o u a b a i n i n q u i e s c e n t muscle, i n d i c a t i n g t h a t t h e development o f t h e t o x i c e f f e c t of o u a b a i n i s a l s o enhanced by Na'i.
111.
SUMMARY
These r e s u l t s i n d i c a t e t h a t Na'i available t o the sodium pump i s t h e d e t e r m i n a n t of t h e s p e c i f i c 86Rb+o r 42K+-uptake i n c a r d i a c muscle p r e p a r a t i o n s which a r e not preloaded with Na+. The s p e c i f i c u p t a k e , t h e r e f o r e , may r e p r e s e n t t h e r a t e of N a + i n f l u x which i s e q u i v a l e n t t o N a + e f f l u x i n s t e a d y - s t a t e p r e p a r a t i o n s . The condit i o n s which i n c r e a s e N a + i a v a i l a b l e t o t h e sodium pump a f f e c t t h e c o n c e n t r a t i o n of o u a b a i n r e q u i r e d t o i n h i b i t t h e s p e c i f i c Rb+- or K+-uptake by enhancing t h e g l y c o s i d e b i v d i n g and a l s o by r e d u c i n g t h e r e s e r v e c a p a c i t y . In Na,-loaded p r e p a r a t i o n s , ouabain s e n s i t i v i t y i s probably overestimated. I n t r a c e l l u l a r N a + enhances Na,K-ATPase i n h i b i t i o n by o u a b a i n and i t s i n o t r o p i c and t o x i c actions.
INTRACELLULAR Na, OUABAIN BINDING,AND Na PUMP
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ACKNOWLEDGMENTS T h i s work w a s s u p p o r t e d by a USPHS g r a n t , HL 16052 from t h e N a t i o n a l H e a r t , Lung, and Blood I n s t i t u t e .
REFERENCES BOdemann, H. H . , and Hoffman, J. F. ( 1 9 7 6 ) . Comparison of t h e side-dependent e f f e c t s o f N a and K on o r t h o p h o s p h a t e - , UTP-, a n d ATP-promoted o u a b a i n b i n d i n g t o r e c o n s t i t u t e d human r e d blood c e l l g h o s t s . J. Gen. P h y s i o l . 6 7 , 527-545. Clausen, T . , and Hansen, 0. (1977). A c t i v e Na-K t r a n s p o r t and t h e r a t e o f o u a b a i n b i n d i n g . The e f f e c t o f i n s u l i n and o t h e r s t i m u l i on s k e l e t a l muscle and a d i p o c y t e s . J. P h y s i o l . (London) 2 7 0 , 415-430. Matsui, H., and Schwartz, A. ( 1 9 6 8 ) . Mechanism of c a r d i a c g l y c o s i d e i n h i b i t i o n of t h e (Na+-K+)-dependent A T P a s e from c a r d i a c t i s s u e . B i o c h i m . B i o p h y s . A c t a ( A m s t e r d a m ) 1 5 1 , 655-663.