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Effect of verapamil on Ca2+-loading properties of sarcoplasmic reticulum in rat cardiac muscle
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R~HE Ca 2+ PARADOX ANDTHE NEONATAL RAT HEART. J.S. Elz, W.G. Nayler. Department of Medicine, Hospital, Heidelberg, Victoria 3084.
University
of Melbourne,
Austin
Repletion of adult rat hearts with Ca 2+ following even short periods of C 2+ depletion results in irreversible damage. This phenomenon, known as the Ca 2+ paradox, results in a massive cellular gain of Ca 2+, loss of intracellular constituents and extensive ultrastructural damage. Recent studies have reported that this response to Ca 2+ repletion following a period of Ca 2+ depletion is age dependent in rat hearts. The present study using isolated perfused hearts from 3 day to adult rats investigated this age dependence in more detail. In particular we have looked at a) the Ca 2+ gain and myoglobin release upon Ca 2+ repletion and b) the ultrastructural changes. Prel~inary results, in agreement with others, show an increased loss of myoglobin upon repletion with increasing rat age. The Ca 2+ gain appears to reach adult levels at an earlier age than the myoglobin loss. The time-dependency of the loss of myoglob~n and gain in Ca 2+ will be diseussedwith respect to the time course of the changes of ultrastructure.
11
THE ROLE OF THE CYTOPLASMIC PHOSPHORYLATION POTENTIAL IN THE CONTROL OF CARDIAC EVENTS. C.L. Gibbs. Department of Physiology, Monash University, Clayton, 3168, Victoria, Australia. Recent work, particularly that of Wilson and Ereclnska, has shown that the major determinants of the respiratory rate of tissues are- i) the extramitochondrial [ATP]/ [ADP][PI] ratio - the so called cytoplasmic phosphorylation potential 2) intramitochondrial [NADm]/[NADH] which reflects the availabillty of reducing substrates; and 3) the oxygen tension. There has been a discrepancy between the oxygen tensions that biochemists believe should limit mltochondrlal respiration (pO 2 < 0.2 torr) and the cellular ;pO2 levels that physiologists believe affect cellular performance. The early failure of the hypoxic heart does not correlate well with changes in cellular ATP, and this has led to many theories but recently with improvements in our ability to accurately estimate free ADP levels attention has been paid. to how the cytosolic phosphoryl~tion potential varies (a) under different metabolic conditions and (b) during the cardiac cycle. Although it has been well known, that the free energy of ATP hydrolysis is given by AG = AGob _~ + RT in [ATP]/[ADP][Pi] it is only recently that the thermodynamic consequences o~ changes in the phosphorylation potential have become apparent. It appears that AG in cardiac tissue is normally close to 60 kJ/mol but can fall to values close to 50 kJ/mol during episodes of hypoxla. It has become apparent that the cytoplasmic phosphorylation potential is a key parameter controlling not only respiration but also mechanical activlty and ion transport.
12
EFFECT OF VERAPAMIL ON Ca2+-LOADING PROPERTIES OF SARCOPLASMIC RETICULUM IN RAT CARDIAC MUSCLE. L.M. Gibson, I.R. Wendt, D.G. Stephenson, Department o f Zoology, La Trobe U n i v e r s i t y , Bundoora, V i c t o r i a . Mechanically disrupted cardiac muscle preparations frQ~ the l e f t v e n t r i c l e o f the r a t were used to i n v e s t i g a t e the e f f e c t s o f verapamil, a Caz - a n t a g o n i s t , on Ca2+ movements associated with the sarcoplasmic reticulum (SR). Preparations were loaded in i d e n t i c a l calcium buffered solutions (pCa 6.0) containing lOmM EGTA, with and w i t h o u t verapamil (2xlO-6M). All solutions contained (mM); Mg2+ i , K+ 117, Na+ 36, HEPES 60, ATP 8, CP I0, CK 15Uml- I , EGTA = CaEGTA + HDTA 50, pH 7.10 at 22-23~ Loading was i n t e r r u p t e d a f t e r 6 mins by s h o r t l y (2-5sec) r e l a x i n g the preparations in a s o l u t i o n containing lOmM EGTA (pCa>9.0). Subsequently the preparations were allowed to e q u i l i b r a t e f o r 2mins in a s o l u t i o n with 40uM EGTA (pCa>8.0) and f i n a l l y calcium was f u l l y released from the SR in the presence o f 30mM c a f f e i n e , lOOuM EGTA (pCa>8.0). Peak height and the area under the force t r a n s i e n t were used as i n d i c a t o r s o f the amount o f Ca in the SR p r i o r to release. The force - pCa r e l a t i o n was not modified by verapamil. When the SR was loaded in the presence of the drug, peak height and the i n t e g r a l o f the force t r a n s i e n t were reduced by between 15-45% and 30-45% r e s p e c t i v e l y . Half time f o r force t r a n s i e n t decay decreased by 8-25% a f t e r treatment with verapamil, but time to peak force was not a f f e c t e d . These r e s u l t s i n d i c a t e t h a t verapamil in the micromolar range depresses the Ca loading capacity of the SR in cardiac muscle and should be taken i n t o consideration when i n t e r p r e t i n g observations obtained with this drug on i n t a c t cardiac muscle. (Supported by National Heart Foundation).
10
R~HE Ca 2+ PARADOX ANDTHE NEONATAL RAT HEART. J.S. Elz, W.G. Nayler. Department of Medicine, Hospital, Heidelberg, Victoria 3084.
University
of Melbourne,
Austin
Repletion of adult rat hearts with Ca 2+ following even short periods of C 2+ depletion results in irreversible damage. This phenomenon, known as the Ca 2+ paradox, results in a massive cellular gain of Ca 2+, loss of intracellular constituents and extensive ultrastructural damage. Recent studies have reported that this response to Ca 2+ repletion following a period of Ca 2+ depletion is age dependent in rat hearts. The present study using isolated perfused hearts from 3 day to adult rats investigated this age dependence in more detail. In particular we have looked at a) the Ca 2+ gain and myoglobin release upon Ca 2+ repletion and b) the ultrastructural changes. Prel~inary results, in agreement with others, show an increased loss of myoglobin upon repletion with increasing rat age. The Ca 2+ gain appears to reach adult levels at an earlier age than the myoglobin loss. The time-dependency of the loss of myoglob~n and gain in Ca 2+ will be diseussedwith respect to the time course of the changes of ultrastructure.
11
THE ROLE OF THE CYTOPLASMIC PHOSPHORYLATION POTENTIAL IN THE CONTROL OF CARDIAC EVENTS. C.L. Gibbs. Department of Physiology, Monash University, Clayton, 3168, Victoria, Australia. Recent work, particularly that of Wilson and Ereclnska, has shown that the major determinants of the respiratory rate of tissues are- i) the extramitochondrial [ATP]/ [ADP][PI] ratio - the so called cytoplasmic phosphorylation potential 2) intramitochondrial [NADm]/[NADH] which reflects the availabillty of reducing substrates; and 3) the oxygen tension. There has been a discrepancy between the oxygen tensions that biochemists believe should limit mltochondrlal respiration (pO 2 < 0.2 torr) and the cellular ;pO2 levels that physiologists believe affect cellular performance. The early failure of the hypoxic heart does not correlate well with changes in cellular ATP, and this has led to many theories but recently with improvements in our ability to accurately estimate free ADP levels attention has been paid. to how the cytosolic phosphoryl~tion potential varies (a) under different metabolic conditions and (b) during the cardiac cycle. Although it has been well known, that the free energy of ATP hydrolysis is given by AG = AGob _~ + RT in [ATP]/[ADP][Pi] it is only recently that the thermodynamic consequences o~ changes in the phosphorylation potential have become apparent. It appears that AG in cardiac tissue is normally close to 60 kJ/mol but can fall to values close to 50 kJ/mol during episodes of hypoxla. It has become apparent that the cytoplasmic phosphorylation potential is a key parameter controlling not only respiration but also mechanical activlty and ion transport.
12
EFFECT OF VERAPAMIL ON Ca2+-LOADING PROPERTIES OF SARCOPLASMIC RETICULUM IN RAT CARDIAC MUSCLE. L.M. Gibson, I.R. Wendt, D.G. Stephenson, Department o f Zoology, La Trobe U n i v e r s i t y , Bundoora, V i c t o r i a . Mechanically disrupted cardiac muscle preparations frQ~ the l e f t v e n t r i c l e o f the r a t were used to i n v e s t i g a t e the e f f e c t s o f verapamil, a Caz - a n t a g o n i s t , on Ca2+ movements associated with the sarcoplasmic reticulum (SR). Preparations were loaded in i d e n t i c a l calcium buffered solutions (pCa 6.0) containing lOmM EGTA, with and w i t h o u t verapamil (2xlO-6M). All solutions contained (mM); Mg2+ i , K+ 117, Na+ 36, HEPES 60, ATP 8, CP I0, CK 15Uml- I , EGTA = CaEGTA + HDTA 50, pH 7.10 at 22-23~ Loading was i n t e r r u p t e d a f t e r 6 mins by s h o r t l y (2-5sec) r e l a x i n g the preparations in a s o l u t i o n containing lOmM EGTA (pCa>9.0). Subsequently the preparations were allowed to e q u i l i b r a t e f o r 2mins in a s o l u t i o n with 40uM EGTA (pCa>8.0) and f i n a l l y calcium was f u l l y released from the SR in the presence o f 30mM c a f f e i n e , lOOuM EGTA (pCa>8.0). Peak height and the area under the force t r a n s i e n t were used as i n d i c a t o r s o f the amount o f Ca in the SR p r i o r to release. The force - pCa r e l a t i o n was not modified by verapamil. When the SR was loaded in the presence of the drug, peak height and the i n t e g r a l o f the force t r a n s i e n t were reduced by between 15-45% and 30-45% r e s p e c t i v e l y . Half time f o r force t r a n s i e n t decay decreased by 8-25% a f t e r treatment with verapamil, but time to peak force was not a f f e c t e d . These r e s u l t s i n d i c a t e t h a t verapamil in the micromolar range depresses the Ca loading capacity of the SR in cardiac muscle and should be taken i n t o consideration when i n t e r p r e t i n g observations obtained with this drug on i n t a c t cardiac muscle. (Supported by National Heart Foundation).