Interactive effects of isoprenaline, forskolin and acetylcholine on Ca2+ current in frog ventricular myocytes.

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Abstract
1. Calcium currents (ICa) were measured in single cells isolated from frog ventricle using the whole-cell patch-clamp technique and a perfused pipette. The dose-dependent stimulatory effects of isoprenaline (Iso, 0.1-100 .mu.M) and forskolin (Fo, 0.1-50 .mu.M) on ICa were determined in the presence and absence of acetylcholine (ACh, 10 .mu.M) and/or threshold concentrations of Fo (0.2 .mu.M) and Iso (0.05 .mu.M), respectively. EC50 (i.e. concentration of Iso or Fo at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of ICa expressed as percentage increase in ICa with respect to control) were measured under each condition. 2. ACh increased EC50 for the stimulatory action of Iso on ICa from 0.84 to 3.72 .mu.M while it reduced Emax from 658 to 185%. Thus, ACh mainly reduced the efficacy of Iso to stimulate ICa. 3. ACh increased EC50 for the stimulatory action of Fo on ICa from 2.06 to 10.26 .mu.M but only slightly reduced Emax from 893 to 778%. Thus, ACh mainly reduced the potency of Fo to stimulate ICa. 4. Intracellular perfusion with 100 .mu.M of hydrolysis-resistant GTP analogues, GTP-.gamma.-S [guanosine-5''-O-(3-thiotriphosphate)] and Gpp(NH)p (5''-guanylylimidodiphosphate), had no effect on basal ICa but reduced by > 50% the stimulatory effect of 2 .mu.M-Iso on ICa. 5. In the presence of Gpp(NH)p or GTP-.gamma.-S, Fo (3 .mu.M) reversibly increased ICa by 490%, as compared to a 717% increase in control (GTP) intracellular solution. Although ACh could still inhibit Fo-stimulated ICa, the degree of inhibition was significantly smaller than in the presence of GTP. 6. Extracellular perfusion with low concentration of a combination of Iso (33 nM) and Fo (330 nM) enhanced ICa to a much greater extent than did either agent alone at 3 times higher concentrations. Thus, low concentrations of Iso and Fo appear to increase ICa in a synergistic fashion. 7. ICa stimulated by a combination of Iso and Fo appeared to be more resistant to inhibition by ACh than when stimulated by either alone. It was the efficacy, rather than the potency, of ACh to inhibit, ICa that was reduced upon dual stimulation of ICa. 8. In the presence of 0.2 .mu.M-Fo, EC50 and Emax for the effects of Iso on ICa were 0.27 .mu.M and 619%, respectively. By comparison with the effects of Iso alone. Fo reduced EC50 .apprxeq. 3 times with no significant change in maximal stimulation. ACh shifted the curve downwards (EC50 = 0.15 .mu.M and Emax = 218%). 9. In the presence of 0.05 .mu.M-Iso, EC50 and Emax for the effect of Fo on ICa were 0.91 .mu.M and 614%, respectively. By comparison with the effects of Fo alone, Iso reduced EC50 by .apprxeq. 50% with no significant change in Emax. In the presence of ACh, the curve had an EC50 of 3.51 .mu.M and an Emax of 528%. 10. The results will be discussed in terms of (i) an interaction between binding sites on adenylate cyclase for Fo and the stimulatory and inhibitory G proteins and (ii) an ACh inhibition of adenylate cyclase and some additional action of ACh at a subsequent level in the cascade leading to phosphorylation of the Ca2+ channel.