Role of the GTP-binding protein Gs in the beta-adrenergic modulation of cardiac Ca channels.

In the heart, the guanosine 5'-triphosphate (GTP)-binding protein Gs is activated by hormone binding to beta-adrenergic receptors and stimulates the intracellular cyclic adenosine 3',5'-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A. Additionally, Gs can modulate cardiac Ca channels directly in cell-free systems. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current (ICa) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained. First, typically, the ICa density increased from 12 to 40 microA/cm2 following application of 1 microM isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl2. However, 1 microM ISP enhanced ICa only from 9 to 17 microA/cm2 after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPs (the Rp-isomer of adenosine 3',5'-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on ICa. Thus, Rp-cAMPS unmasks a GTP-dependent component of the beta-adrenergic stimulation of ICa, which probably reflects the direct stimulation of Ca channels by Gs under block of cAMP-dependent phosphorylation. Second, in cells under dialysis with 100 or 200 microM cAMP, bath application of 20-40 microM 3-isobutyl-1-methylxanthine (IBMX) enhanced the ICa density to about 41 microA/cm2 indicating saturation of the cAMP pathway. Under this condition, 1 microM ISP was without significant effect on ICa. This result may suggest that direct Gs stimulation is rather ineffective on Ca channels after maximal cAMP-dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the Gs pathway in intact myocytes. Third, simultaneous application of 1 microM ISP and 40 microM IBMX enhanced ICa up to densities of around 75 microA/cm2 during cell dialysis with 100 microM cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that Gs is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of Gs may act to prime L-type Ca channels for cAMP-dependent phosphorylation during beta-adrenergic stimulation of cardiac myocytes.