Acetylcholine and adenosine regulate cardiac function by direct receptor mediated effects and by receptor mediated modulation of catecholamine effects on the heart. Both agents inhibit catecholamine-stimulated adenylate cyclase, but it is postulated that they inhibit other effects of cyclic AMP by a receptor mediated effect at an intracellular site or sites beyond adenylate cyclase. This hypothesis is supported by prior studies where acetylcholine and adenosine inhibit cyclic AMP mediated events without altering cyclic AMP levels. To directly test this hypothesis, the effects of acetylcholine and adenosine on isolated guinea pig ventricular myocytes where the concentrations of cyclic AMP or cyclic AMP-dependent protein kinase can be elevated by direct intracellular injection will be studied. For these studies, calcium tolerant isolated myocytes are prepared by enzymatic dissolution. Conventional microelectrode techniques are used to record intracellular action potentials from isolated myocytes. Action potentials increase in duration and the plateau elevates during superfusion of isoproterenol. This effect is antagonized by acetylcholine and adenosine. Cyclic AMP or the catalytic subunit of cyclic AMP-dependent protein kinase will be pressure injected into the myocytes and are expected to produce effects on action potential characteristics similar to isoproterenol. Effects of acetylcholine and adenosine on myocytes injected with these substances will be determined. Inhibition by acetylcholine and adenosine of transient depolarizations induced by isoproterenol, injected cyclic AMP or injected catalytic subunit will be studied. Lastly, two microelectrode voltage clamp studies in isolated myocytes of effects of antagonists on catecholamine-enhanced slow inward and transient inward currents will be performed. These studies will document if acetylcholine and adenosine antagonize cyclic AMP mediated events at an intracellular site or sites distal to adenylate cyclase and define the ionic basis for suppression of catecholamine-enhanced transient inward currents.
