The overall objective of this project is to understand the mechanisms of the positive inotropic and toxic actions of the cardiac glycosides. It is now clear that Na pump inhibition is a cause of the positive inotropic and arrhythmogenic effects of the cardiac glycosides; however, the steps, which link Na pump inhibition to the inotropic effect, are not clear. In addition, whether Na,K-ATPase inhibition is the sole mechanism for the positive inotropic effect is unknown. Proposed studies are to evaluate if an enhancement of a putative """"""""phasic"""""""" increase in [Na+]i which may occur during membrane depolarization at the inner surface of the sarcolemma is responsible for the enhancement of intracellular Ca2+ transients. Alternatively, a slight """"""""tonic"""""""" increase in [Na+]i which is observed during the diastolic phase of the cardiac cycle may enhance the Ca2+ transient which occurs during membrane depolarization. Because the proposed mechanism which links Na pump inhibition to an enhancement of intracellular Ca2+ transients is the Na/Ca exchange reaction, the direction and magnitude of this reaction will be examined in isolated myocytes obtained from guinea pig and rat heart under conditions in which normal Na+, Ca2+ and electrical gradients are maintained. 13-Propylberberine, an agent which is claimed to inhibit adenylate cyclase in other types of tissues, eliminates the positive inotropic effect of ouabain. Thus, we will examine the mechanism by which this effect is brought about. This will define a critical step required for the expression of the inotropic effect. The possible presence of a second """"""""digitalis receptor"""""""" which is unrelated to Na,K-ATPase and might have been overlooked because of the lack of appropriate ligands which are required for glycoside binding in cell fragment preparations will be examined using myocytes. Additionally, we will test the hypothesis that Ca2+ pools responsible for the positive inotropic effect of the cardiac glycosides are separate from those involved in Ca2+ overload and digitalis toxicity. Finally, the possible presence of an endogenous ligand for glycoside binding sites on Na,K-ATPase will be examined. These studies are designed to fill the missing links in the present scheme representing our understanding of the positive inotropic and toxic effects of the cardiac glycosides. Because the glycoside produces its positive inotropic effect by increasing the apparent efficiency of excitation-contraction coupling, understanding the mechanism of action of the glycoside will shed light on the coupling mechanism in the cardiac muscle.
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