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 G(s) 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 [28]. Additionally, G(s) can modulate cardiac Ca channels directly in cell-free systems [57]. 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 (I(Ca)) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained. First, typically, the I(Ca) density increased from 12 to 40-mu-A/cm2 following application of 1-mu-M isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl2. However, 1-mu-M ISP enhanced I(Ca) only from 9 to 17-mu-A/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 I(Ca). Thus, Rp-cAMPS unmasks a GTP-dependent component of the beta-adrenergic stimulation of I(Ca), which probably reflects the direct stimulation of Ca channels by G(s) under block of cAMP-dependent phosphorylation. Second, in cells under dialysis with 100 or 200-mu-M cAMP, bath application of 20-40-mu-M 3-isobutyl-1-methylxanthine (IBMX) enhanced the I(Ca) density to about 41-mu-A/cm2 indicating saturation of the cAMP pathway. Under this condition, 1-mu-M ISP was without significant effect on I(Ca). This result may suggests that direct G(s) 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 G(s) pathway in intact myocytes. Third, simultaneous application of 1-mu-M ISP and 40-mu-M IBMX enhanced I(Ca) up to densities of around 75-mu-A/cm2 during cell dialysis with 100-mu-M cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that G(s) is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of G(s) may act to prime L-type Ca channels for cAMP-dependent phosphorylation during beta-adrenergic stimulation of cardiac myocytes.