Ecto-ATPase activity in the rat cardiac muscle: biochemical characteristics and histocytochemical localization

We used a combined biochemical and histocytochemical approach to study ecto -ATPase in the rat cardiac muscle. The reaction medium employed for histocy tochemical detection was optimized in biochemical assays to achieve the hig hest enzyme activity and lowest inhibition by the capture agent used for vi sualization of the reaction product. Approximately 70% of the enzyme activi ty was retained in samples after the fixation procedure. Divalent cations s timulated ecto-ATPase. High activity was detectable within a wide pH range. Histocytochemical reaction was observed at sites at which extracellular AT P can potentially exert its actions on the cardiac muscle: nerve endings, p lasma membranes of cardiac myocytes and capillary endothelial cells, and T- tubules, Product of the reaction was found exclusively at the outer surface of the cells. In controls, enzyme activity was abolished by diethyl pyroca rbonate and slightly stimulated by digitonin and concanavalin A, whereas so dium orthovanadate, N-ethylmaleimide, and sodium azide yielded no effect.. Our results support the view that cardiac ecto-ATPase is involved in import ant physiological functions and suggest that its activity may be regulated by the release of ATP from nerve endings.