This project is designed to assess the physiological importance of Ca2+-ions in the regulation of energy metabolism. Specifically, we have exposed isolated cardiac myocytes to a variety of conditions expected to alter cytosolic free Ca2+ concentration ([Ca2+]c), and have measured the amount of the active form of pyruvate dehydrogenase (PDH-A) which results, as well as estimating [Ca2+] by use of fluorescent chelating agents. Protocols used to increase [Ca2+]c include depolarization of the plasma membrane with KCl, abolition of the Na+c electrochemical gradient with ouabain and gramicidin, inhibition of sarcoplasmic reticulum Ca2+ uptake by caffeine and the use of metabolic inhibitors to deplete cellular ATP. We have demonstrated an increased value of PDH-A in response to each of these interventions, and an important role of Ca2+ transport into the mitochondria in mediating these increases. Further, we have taken advantage of the fluorescent chelating agent technology to characterize the mechanism of the increase in [Ca2+]c due to KCl-induced depolarization, and have established a sensitivity to beta -adrenergic agonists and to phorbol esters. We have also initiated a study of isolated hepatocytes, in order to investigate the importance of increased values of [Ca2+]c in mediating the increased activity of 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase which occurs in response to the hormones glucagon and vasopressin.