The plateau phase of the action potential in heart cells is due to an influx of Ca2+ ions through voltage dependent channels. These channels have been extensively studied electrophysiologically and in addition to their being time- and voltage-dependent, they are modulated by inotropic hormones (eg. norepinephrine, histamine, angiotensin II) and drugs (eg. verapamil, nifedipine). There is little biochemical information on the channel or its regulation and this lack of data has stimulated the proposed work. Angiotensin II (AII), a positive inotropic hormone and Ca2+ channel agonist, will be used as a probe to study hormonal mechanisms of Ca2+ channel modulation. High affinity AII receptors (Kd = 0.59 nM) have been identified in cultured rat myocytes, in suspension, and now these studies will be extended to the characterization of 125I-AII binding to spontaneously beating cultured myocytes. The functional responses of these cells to AII will be identified by measuring changes in action potentials in these cells using single-electrode recording methods. The biochemical responses of myocytes to AII will be explored by examining changes in transsarcolemmal Ca2+ fluxes, cAMP levels, and protein phosphorylation. These experiments will be designed so that direct correlations can be examined between AII receptor occupancy, functional responses, and biochemical responses of the intact cultured myocytes under identical conditions. The Ca2+ antagonists will be used as probes to study pharmacological regulation of Ca2+ channel activity. Charged, impermeant derivatives of verapamil, diltiazem, nifedipine, and bepridil will be synthesized to explore the possibility that these drugs have intracellular sites of action. The relative potencies of these compounds when applied intra- versus extracellularly will be compared. Molecular components of the Ca2+ antagonist receptor in membranes and intact myocytes will be identified by synthesizing a photoaffinity label from carboxy nifedipine, a derivative recently prepared by the applicant. In further studies, carboxy nifedipine will be coupled to Sepharose beads. This support will be used in affinity chromatographic procedures to identify proteins of the high affinity nifedipine receptor. The long range goal is to characterize molecular mechanisms of hormonal and pharmacological regulation of slow 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.
