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Role of oxygen free radicals in the calcium paradox. Tissue protection b by allopurinol
j Mol Cell C a r d i o l 18 ( S u p p l e m e n t 2) (1986)
4MITOCHONDRIAJ~CYCLE OF
PHOSPHOCREATINE SHUTTLE. V . A .
Saks,
V.V.
Kuprlyanov,
Z.A.
Khuchua. A.V. K u z n e t s o v . C a r d i o l o g y R e s e a r c h C e n t e r , ~oscow, USSR. M i t o c h o n d r i a l c r e a t J n e k J n a s e (CKmit) c y c l e i s c o n s i d e r e d t o be of c e n t r a l i m p o r t a n c e f o r e n e r g y c h a n n e l l i n g in c a r d i o m y o c y t e s . In t h i s work we s t u d i e d t h e structural basis of f u n c t i o n a l c o u p l i n g of CKmit w i t h a d e n i n e q u c l e o t i d e translocase (T). All a f f i n i t y m o d i f i c a t i o n method by u s i n g o x i d i z e d [OH]A~P was used to d e t e r m i n e CKmit and T c o n t e n t s which were bot h found t o be i n r a n g e of 2 . 5 - 2 . 9 m o l e / m o l e c y t . a a 3. I n h i b i t o r y a n t i b o d i e s a g a i n s t CKmit i n h i b i t e d T b u t d i d n o t change r e s p i r a t o r y c h a i n a c t i v i t i e s . Noninhihitory antibodies against CKmit d i d n o t a f f e c t T. I t i s supposed t h a t b o t h CKmlt and T a r e located in cardiolipin domains i n m i t o c h o n d r i a l membrane and may i n t e r a c t i n way of frequent collision. Using 31p-N~iR s a t u r a t i o n transfer t e c h n i q u e we ha ve i n v e s t i g a t e d p h o s p h o c r e a t i n e s h u t t l e in p e r f u s e d r a t h e a r t . C r e a t i n e k i n a s e f l u x in t h e h e a r t grew up a s w o r k l o a d and ATP t u r n o v e r r o s e due t o a c t i v a t i o n of m i t o c h o n d r i a l CK c y c l e . D e p l e t i o n of c y t o p l a s m i c a d e n i n e n u c l e o t i d e pool 5 - I 0 t i m e s by 2 - d e o x y g l u c o s e t r e a t m e n t d e c r e a s e d CK f l u x t w o f o l d a t 30~ d e c l i n e in c o n t r a c t i l e a c t i v i t y and PCr l e v e l . These d a t a d e m o n s t r a t e ATP c o m p a r t m e n t a t l o n and t h e i m p o r t a n c e of CKmit c y c l e f o r c a r d i a c b i o e n e r g e t l c s i n l i v i n g h e a r t s .
5 E n d o g e n o u s Noradrenaline Release Does Not Contribute to the Effects of the Calcium/ Oxygen Paradox. A M Dart, R A Riemersma. Cardiovascular Research Unit, University of Edinburgh U.K. Reperfusion of ischaemic hearts is associated with noradrenaline (NA) overflow and cellular damage,similar to that seen after reintroduction of calcium/oxygen {calcium paradox). Anoxic substrate free perfusion of rat hearts leads to marked NA overflow even in the absence of calcium and after 30 minutes anoxic acidotic (pH 6.5) calcium free substrate free perfusion NA output has risen to 137 • 20.3pmol/g/min. This overflow is almost completely suppressed (to 2.69 + 0.85 pmol/g/min) by 100nM desipramine. Reintroduction of normal perfusate {without change in flow) is accompanied by a return of elevated NA outputs to pre-anoxic levels but produces a marked LDH overflow which is not different between control and desipramine treated hearts {cumulative outputs 84 • 16, 125 ~ 23 u/g respectively). Reintroduction of normal perfusate after 10 minutes anoxic calcium free perfusion produces marked LDH overflow (184 22 u/g) despite only a minimal change in NA output (< 1.0pmol/g/min). These results suggest that nerve terminal and myocyte respond differently to the reintroduction of calcium/oxygen and also demonstrate that the prevailing level of endogenous NA stimulation does not influence the magnitude of myocyte damage (LDH overflow) produced.
6 R O L E OF OXYGEN FREE R A D I C A L S IN THE CALCIUM PARADOX. TIS S U E P R O T E C TION BY ALLOPURINOL. A . N . ~ ) k s e n d a l , P. J y n g e . D e p t . of P h a r m a c o l o g y a n d To~dc o l o g y , F a c u l t y of M e d i c i n e , U n i v e r s i t y of Trondheim, N o r w a y . T h e aim of t h e p r e s e n t s t u d y w a s to e v a l u a t e t h e p o t e n t i a l i n v o l v e m e n t of o x y g e n f r e e r a d i c a l s in t h e m y o c a r d i a l t i s s u e i n j u r y in t h e c a l c i u m p a r a d o x ( C a P a ) . Two i n j u r y l e v e l s (minimal a n d t o t a l C a P a ) c a u s e d b y d i f f e r e n t v o l u m e s (5 a n d 45 ml) of c a l c i u m - f r e e p e r f u s i o n (5 s i n ) p r i o r to c a l c i u m r e p l e t i o n (15 s i n ) w e r e e x a m i n e d + a l l o p u r i n o l (20 r a g / l ) in t h e n o r m o t h e r m i c i s o l a t e d r a t h e a r t m o d e l . A l l o p u r i n o l s u p p l e m e n t a t i o n (5 s i n p r i o r t o , d u r i n g a n d 5 s i n f o l l o w i n g C a Z + - f r e e p e r f u s i o n ) h a d no e f f e c t u p o n t i s s u e i n j u r y in t h e t o t a l C a P a , b u t a f f o r d e d c o n s i d e r a b l e p r o t e c t i o n a s a s s e s s e d b y e n z y m a t i c , p h y s i o l o g i c a l a n d m e t a b o l i c p a r a m e t e r s in t h e minimal C a P a . When a l l o p u r i n o l w a s o m i t t e d p r i o r t o c a l c i u m r e p l e t i o n , t h e r e w a s no p r o t e c t i v e e f f e c t . T h e p r e s e n c e of v e r a p a m i l ( 1 . 0 r a g / l ) in a d d i t i o n t o a l l o p u r i n o l (5 min p r i o r t o , d u r i n g a n d 5 s i n f o l l o w i n g c a l c i u m d e p l e t i o n ) a f f o r d e d no f u r t h e r p r o t e c t i o n in t h e minimal C a P a . I t is c o n c l u d e d from t h e p r e s e n t s t u d y t h a t t i s s u e p r o t e c t i o n b y a l l o p u r i n o l in t h e CaPa is l i m i t e d to minimal o r " l e s s s e v e r e " CaPa m o d e l s a n d t h a t x a n t h i n e o x i d a s e m a y be a s o u r c e of f r e e r a d i c a l d a m a g e d u r i n g c a l c i u m r e p l e t i o n in t h e C a P a .
L
4MITOCHONDRIAJ~CYCLE OF
PHOSPHOCREATINE SHUTTLE. V . A .
Saks,
V.V.
Kuprlyanov,
Z.A.
Khuchua. A.V. K u z n e t s o v . C a r d i o l o g y R e s e a r c h C e n t e r , ~oscow, USSR. M i t o c h o n d r i a l c r e a t J n e k J n a s e (CKmit) c y c l e i s c o n s i d e r e d t o be of c e n t r a l i m p o r t a n c e f o r e n e r g y c h a n n e l l i n g in c a r d i o m y o c y t e s . In t h i s work we s t u d i e d t h e structural basis of f u n c t i o n a l c o u p l i n g of CKmit w i t h a d e n i n e q u c l e o t i d e translocase (T). All a f f i n i t y m o d i f i c a t i o n method by u s i n g o x i d i z e d [OH]A~P was used to d e t e r m i n e CKmit and T c o n t e n t s which were bot h found t o be i n r a n g e of 2 . 5 - 2 . 9 m o l e / m o l e c y t . a a 3. I n h i b i t o r y a n t i b o d i e s a g a i n s t CKmit i n h i b i t e d T b u t d i d n o t change r e s p i r a t o r y c h a i n a c t i v i t i e s . Noninhihitory antibodies against CKmit d i d n o t a f f e c t T. I t i s supposed t h a t b o t h CKmlt and T a r e located in cardiolipin domains i n m i t o c h o n d r i a l membrane and may i n t e r a c t i n way of frequent collision. Using 31p-N~iR s a t u r a t i o n transfer t e c h n i q u e we ha ve i n v e s t i g a t e d p h o s p h o c r e a t i n e s h u t t l e in p e r f u s e d r a t h e a r t . C r e a t i n e k i n a s e f l u x in t h e h e a r t grew up a s w o r k l o a d and ATP t u r n o v e r r o s e due t o a c t i v a t i o n of m i t o c h o n d r i a l CK c y c l e . D e p l e t i o n of c y t o p l a s m i c a d e n i n e n u c l e o t i d e pool 5 - I 0 t i m e s by 2 - d e o x y g l u c o s e t r e a t m e n t d e c r e a s e d CK f l u x t w o f o l d a t 30~ d e c l i n e in c o n t r a c t i l e a c t i v i t y and PCr l e v e l . These d a t a d e m o n s t r a t e ATP c o m p a r t m e n t a t l o n and t h e i m p o r t a n c e of CKmit c y c l e f o r c a r d i a c b i o e n e r g e t l c s i n l i v i n g h e a r t s .
5 E n d o g e n o u s Noradrenaline Release Does Not Contribute to the Effects of the Calcium/ Oxygen Paradox. A M Dart, R A Riemersma. Cardiovascular Research Unit, University of Edinburgh U.K. Reperfusion of ischaemic hearts is associated with noradrenaline (NA) overflow and cellular damage,similar to that seen after reintroduction of calcium/oxygen {calcium paradox). Anoxic substrate free perfusion of rat hearts leads to marked NA overflow even in the absence of calcium and after 30 minutes anoxic acidotic (pH 6.5) calcium free substrate free perfusion NA output has risen to 137 • 20.3pmol/g/min. This overflow is almost completely suppressed (to 2.69 + 0.85 pmol/g/min) by 100nM desipramine. Reintroduction of normal perfusate {without change in flow) is accompanied by a return of elevated NA outputs to pre-anoxic levels but produces a marked LDH overflow which is not different between control and desipramine treated hearts {cumulative outputs 84 • 16, 125 ~ 23 u/g respectively). Reintroduction of normal perfusate after 10 minutes anoxic calcium free perfusion produces marked LDH overflow (184 22 u/g) despite only a minimal change in NA output (< 1.0pmol/g/min). These results suggest that nerve terminal and myocyte respond differently to the reintroduction of calcium/oxygen and also demonstrate that the prevailing level of endogenous NA stimulation does not influence the magnitude of myocyte damage (LDH overflow) produced.
6 R O L E OF OXYGEN FREE R A D I C A L S IN THE CALCIUM PARADOX. TIS S U E P R O T E C TION BY ALLOPURINOL. A . N . ~ ) k s e n d a l , P. J y n g e . D e p t . of P h a r m a c o l o g y a n d To~dc o l o g y , F a c u l t y of M e d i c i n e , U n i v e r s i t y of Trondheim, N o r w a y . T h e aim of t h e p r e s e n t s t u d y w a s to e v a l u a t e t h e p o t e n t i a l i n v o l v e m e n t of o x y g e n f r e e r a d i c a l s in t h e m y o c a r d i a l t i s s u e i n j u r y in t h e c a l c i u m p a r a d o x ( C a P a ) . Two i n j u r y l e v e l s (minimal a n d t o t a l C a P a ) c a u s e d b y d i f f e r e n t v o l u m e s (5 a n d 45 ml) of c a l c i u m - f r e e p e r f u s i o n (5 s i n ) p r i o r to c a l c i u m r e p l e t i o n (15 s i n ) w e r e e x a m i n e d + a l l o p u r i n o l (20 r a g / l ) in t h e n o r m o t h e r m i c i s o l a t e d r a t h e a r t m o d e l . A l l o p u r i n o l s u p p l e m e n t a t i o n (5 s i n p r i o r t o , d u r i n g a n d 5 s i n f o l l o w i n g C a Z + - f r e e p e r f u s i o n ) h a d no e f f e c t u p o n t i s s u e i n j u r y in t h e t o t a l C a P a , b u t a f f o r d e d c o n s i d e r a b l e p r o t e c t i o n a s a s s e s s e d b y e n z y m a t i c , p h y s i o l o g i c a l a n d m e t a b o l i c p a r a m e t e r s in t h e minimal C a P a . When a l l o p u r i n o l w a s o m i t t e d p r i o r t o c a l c i u m r e p l e t i o n , t h e r e w a s no p r o t e c t i v e e f f e c t . T h e p r e s e n c e of v e r a p a m i l ( 1 . 0 r a g / l ) in a d d i t i o n t o a l l o p u r i n o l (5 min p r i o r t o , d u r i n g a n d 5 s i n f o l l o w i n g c a l c i u m d e p l e t i o n ) a f f o r d e d no f u r t h e r p r o t e c t i o n in t h e minimal C a P a . I t is c o n c l u d e d from t h e p r e s e n t s t u d y t h a t t i s s u e p r o t e c t i o n b y a l l o p u r i n o l in t h e CaPa is l i m i t e d to minimal o r " l e s s s e v e r e " CaPa m o d e l s a n d t h a t x a n t h i n e o x i d a s e m a y be a s o u r c e of f r e e r a d i c a l d a m a g e d u r i n g c a l c i u m r e p l e t i o n in t h e C a P a .
L