The plateau phase of the action potential in heart cells is due, in part, to an influx of Ca2+ ions through voltage dependent channels. Electrophysiological studies reveal that these Ca2+ channels are modulated by inotropic hormones (eg. norepinephrine) and drugs (eg. verapamil, nifedipine). Although mechanisms of Ca2+ channel regulation mediated by beta-agonists and cAMP are well characterized, there is little information on other cellular signal pathways and their role in modifying channel function. This lack of information has stimulated the proposed project. Recent evidence indicates that protein kinase C stimulates beating frequency, decreases peak tension and increases Ca2+ current in cultured cardiocytes. The mechanism of action of protein kinase C will be examined by measuring the effects of enzyme activation on the phosphorylation state of proteins, including the Ca2+ channel, in intact cultured cardiac myocytes. These results will be compared with the effect of the enzyme on whole-cell Ca2+ current, intracellular Ca2+ concentration, and tension. These experiments will examine the hypothesis that protein kinase C is an important modulator of cardiac Ca2+ channels. Angiotensin II (AII), a cardioactive peptide hormone will be used as a probe to study the role of the phosphoinositide hydrolysis pathway in Ca2+ channel modulation. AII binds to intact cultured cardiac cell (kd=0.6 nM), elevates the production of inositol phosphates, increases beating frequency and Ca2+ current in these cells. Underlying biochemical mechanisms will be examined by measuring the effects of AII on the production of inositol trisphosphates, 1,2-diacylglycerol, and protein kinase C activity in intact cardiac myocytes. These results will be correlated with the effects of AII beating frequency and Ca2+ current. Finally, the role of the phosinositide signalling pathway in the AII-induced desensitization of cardiac cells will be examined. These experiments will test the hypothesis that the effects of AII on cardiac cells are mediated by messengers derived from the phosphoinoside pathway. The long range goal of this research program is to elucidate molecular mechanisms of hormonal and pharmacological regulation of Ca2+ channels in heart cells which should provide information on the excitation-contraction coupling process. The successful completion of this study will give insights into the mechanism of action of a number of cardioactive drugs and hormones
