Prakash V. Sulakhe

Isolation of skeletal muscle membrane fragments containing active Na+K+ stimulated ATPase: Comparison of normal and dystrophic muscle sarcolemma☆

Abstract A method for the isolation of skeletal muscle membrane is described. These membrane fragments were found to contain a highly active Na + K + stimulated Mg ++ dependent ATPase which is sensitive to ouabain. The Na + K + stimulated ATPase activities of the muscle membranes isolated from normal and genetically dystrophic hamsters (B10 14.6) were about 18 and 46 μmoles ATP hydrolyzed/mg protein/hr respectively.

Excitation-contraction coupling in heart VI. demonstration of calcium activated ATPase in the dog heart sarcolemma☆

Abstract In addition to Mg ++ -activated ATPase and Na + K + activated ATPase, we have shown the presence of Ca ++ -activated ATPase in the dog heart sarcolemma. The Ca ++ -activated ATPase of cardiac sarcolemma does not require the presence of Mg ++ and is not influenced by ouabain, a well known inhibitor of Na + K + activated ATPase. Various agents such as EGTA, NaF, HgCl 2 and NEM were found to depress the ATP hydrolysis due to Ca ++ by the cardiac sarcolemma whereas azide, Na + , K + and Mg ++ showed no effect. It is suggested that Ca ++ -activated ATPase may be involved in the movements of calcium across the cell membrane (calcium efflux) and thus plays an important role in the regulation of intracellular calcium in the heart.

Adenylate cyclase of heart sarcotubular membranes.

Abstract Subcellular distribution of dog and rabbit heart homogenates revealed highest specific activity of adenylate cyclase (ATP:AMP phosphotransferase, EC 2.7.4.3) in the sarcotubular membranes. Further fractionation of the sarcotubular membranes by sucrose density gradient revealed that the fraction possessing the highest calcium accumulating ability also showed the highest adenylate cyclase activity. The enzyme located in these membranes was activated by NaF and catecholamines. Ca2- was found to inhibit adenylate cyclase activity of the sarcotubular membranes in the absence or presence of NaF and catecholamines. Neither norepinephrine nor NaF changed the affinity of the enzyme for ATP (Km = 0.13 mM), however, the V (150 pmoles/mg protein per min) was increased 2- and 8-fold by these agents, respectively. Saturation of the enzyme was observed at 9 mM Mg2+. Extraction of these membranes by deoxycholate as well as treatment with phospholipase C (EC 3.1.4.3) or trypsin (EC 3.4.4.4) markedly reduced the adenylate cyclase activity in the absence or presence of NaF and norepinephrine. Addition of sonicated lipids did not restore the reduced activity due to the phospholipase C treatment. Treatment with phospholipase A (EC 3.1.1.4) selectively increased the NaF stimulated activity without any significant effect on the basal activity. Pretreatment of sarcotubular membranes with NaF or norepinephrine and subsequent incubation with and without these activators indicated that F-activation was partially irreversible and norepinephrine activation reversible. It is speculated that the adenylate cyclase present in the sarcotubular system may be intimately involved in the regulation of heart metabolism during the contraction-relaxation cycle.

Alterations in the activity of cardiac Na+K+-stimulated ATPase in congestive heart failure

Abstract The activities of Na + K + -stimulated ATPase, a membrane bound enzyme, from the hearts of control hamsters and genetically myopathic hamsters with a moderate degree of heart failure (BIO 14.6) were determined. The specific activity of the Na + K + ATPase in the failing hearts was higher than the control preparations at different steps of isolation. The enzyme activity of the failing heart was also observed to be elevated under different assay conditions obtained by changing the concentrations of Na + , K + , H + , and Mg 2+ ATP. The value for the maximal velocity of reaction for the Na + K + ATPase from the failing heart was markedly higher than the control but the K m values were not different from each other. The enzyme preparations from the control and failing hearts exhibited similar allosteric nature of activation kinetics. The inhibitory responses of the Na + K + ATPase from the failing hearts to both ouabain and calcium were also similar to those of the control. The increase in Na + K + ATPase activity in the myopathic hamster heart may be due to some adaptive change in the cell membrane in this model of heart failure.

Adenyl cyclase activity in failing hearts of genetically myopathic hamsters

Abstract The activities of adenyl cyclase, a membrane-bound enzyme important for the regulation of myocardial metabolism, were determined in the hearts of genetically myopathic hamsters (BIO 14.6 strain) with moderate and severe degrees of heart failure. The enzyme activities in both heart homogenates and washed cell particulate fraction were measured in the absence or presence of various concentrations of norepinephrine and NaF. No appreciable change in enzyme activities in homogenates and washed cell particles was observed in hearts with a moderate degree of failure. However, at the advanced stages of heart failure, the responses of this enzyme to fluoride were markedly reduced and those to norepinephrine were not apparent. In another strain of myopathic hamsters (BIO 82.62) with moderate degree of heart failure, the adenyl cyclase activities of the heart homogenate and washed cell particles in the presence of norepinephrine or NaF were not different from the control. The basal adenyl cyclase activities and phosphodiesterase activities in the failing hearts were also not different from the control. These results indicate that alterations in adenyl cyclase activities in the presence of NaF and norepinephrine are secondary to heart failure in myopathic hamsters but strengthen our belief concerning a defect in cardiac membranes in heart failure.

Excitation-Contraction Coupling in Heart

On perfusing the isolated rat heart with a medium containing Ca45, mitochondrial fraction was found to contain more radioactivity than sarcoplasmic reticulum. Not only the calcium contents

Studies on the relationship between adenylate cyclase activity and calcium transport by cardiac sarcotubular membranes

Abstract The relationship between changes in adenylate cyclase activity and calcium transporting ability of the dog heart sarcotubular membrane was examined under different experimental conditions. Treatment of the sarcotubular membranes with phospholipace C or trypsin decreased adenylate cyclase activity more than calcium uptake. In low concentrations both nonrepinephrine (10−7−10−4 M) and NaF (1–5 mM) increased adenylate cyclase activity without any appreciable changes in calcium uptake; whereas in high concentrations, these agents decreased calcium uptake by the sarcotubular membranes. Different concentrations of glucagon and prostaglandins E1 and F2α increased adenylate cyclase activity without changing calcium uptake. The calcium binding ability of the sarcotubular membranes did not alter when examined in the presence of norepinephrine, glucagon and prostaglandins E1 and F2α. Increasing the concentration of calcium in the incubation medium increased calcium binding by the sarcotubular membranes without appreciably affecting the adenylate cyclase activity. Adenosine 3′,5′-monophosphate (cyclic AMP) neither influenced the calcium binding constant nor the number of calcium binding sites. Although we have failed to show a direct relation between changes in adenylate cyclase and calcium transport under the experimental conditions employed in this study, the results suggest that adenylate cyclase-cyclic AMP and calcium transport systems of the heart sarcotubular membranes are independently controlled.

Influence of fluothane on calcium accumulation by the heavy microsomal fraction of human skeletal muscle: Comparison with a patient with malignant hyperpyrexia☆

Abstract The effect of fluothane on calcium accumulation by the heavy microsomal fraction of skeletal muscle was studied in specimens obtained from normal subjects and from a patient who had an episode of malignant hyperpyrexia. The extent of energy-linked calcium accumulation by normal human muscle microsomes was markedly decreased by fluothane both in the presence and absence of oxalate. Although the rate and extent of calcium accumulation by the heavy microsomal fraction of the patients muscle were similar to those in normal muscle, the inhibition of calcium uptake caused by fluothane was considerably less than normal. In a further series of experiments it has been shown that calcium binding, uptake and calcium-stimulated ATPase activities of the heavy microsomal fraction of guinea pig skeletal muscle were inhibited by fluothane. These results are consistent with the view that the effects of fluothane on skeletal muscle are mediated in part through its action on the calcium transport system.

Excitation contraction coupling in heart II. Studies on the role of adenyl cyclase in the calcium transport by dog heart sarcoplasmic reticulum

Abstract The relation between the activation of adenyl cyclase and calcium uptake by dog heart sarcoplasmic reticulum was investigated in this study. Incubation of the reticulum with various concentrations of sodium fluoride or epinephrine was found to increase adenyl cyclase activity without increasing the ability of reticulum to accumulate calcium. High concentration of sodium fluoride (5 mM) considerably decreased calcium uptake. Different concentrations of calcium were found to have an inhibitory action on the adenyl cyclase activity of sarcoplasmic reticulum. These results do not suggest any role of adenyl cyclase in the transport of calcium in the heart sarcoplasmic reticulum.

Calcium transport by the subcellular particles of the skeletal muscle of genetically dystrophic hamster

Abstract In about 220-day-old genetically dystrophic hamsters (BIO strain 14.6) the abilities of hind leg muscle mitochondrial and sarcoplasmic reticular fractions to hydrolyze ATP as well as to bind and accumulate calcium were not different from those of the control. The calcium uptake by the muscle sarcoplasmic reticulum as well as its abilities to hydrolyze ATP and retain calcium were depressed in about 260-day-old dystrophic hamsters. The alterations in “calcium pump” mechanisms appear to be secondary to the pathogenesis of muscular dystrophy in BIO 14.6 strain of Syrian hamsters.