Potentiation by cyclic GMP of beta-adrenergic effect on Ca2+ current in guinea-pig ventricular cells.

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1. Effects of cyclic GMP on L-type Ca2+ current (ICa) were investigated in myocytes isolated from guinea-pig ventricles using the patch clamp method in the whole-cell configuration combined with intracellular perfusion. 2. When ICa was increased by bath application of isoprenaline (0.001-0.1 microM) or forskolin (0.5-1 microM), or by intracellular dialysis with cyclic AMP (50-100 microM), dialysis with 10 microM-cyclic GMP resulted in an additional stimulation of ICa. Without these pre-treatments, cyclic GMP (1-100 microM) had no effect on the basal ICa. 5'-GMP was without effect. 3. The stimulatory effect of cyclic GMP was observed at concentrations higher than 0.1 microM with a maximum at around 10 microM in the pipette. The dose-response relation between isoprenaline and ICa was shifted to the left by (10 microM) cyclic GMP; the half-maximum isoprenaline concentration shifted from 16 to 4.6 nM. 4. The increase of ICa on dialysing 50 microM-cyclic AMP varied from cell to cell, probably due to a difference in phosphodiesterase activity. The cells responding weakly to cyclic AMP showed a greater response to cyclic GMP, and vice versa. In cells dialysed with hydrolysis-resistant derivatives (10-50 microM-8-(4-chlorophenylthio)-cyclic AMP or 50 microM-8-bromo-cyclic AMP), additional dialysis with cyclic GMP failed to modify ICa. Dialysis with cyclic GMP abolished the stimulatory effect of milrinone, a specific inhibitor of cyclic GMP-inhibited phosphodiesterase. These findings suggested that inhibition of cyclic GMP-sensitive phosphodiesterase was responsible for the stimulatory effect of cyclic GMP. 5. In the presence of isoprenaline, direct application of an active fragment of cyclic GMP-dependent protein kinase (PKG) failed to modify ICa in most cells. Activation of native PKG by intracellular dialysis with 8-bromo-cyclic GMP, or higher concentrations of cyclic GMP (100-1000 microM), depressed ICa in about 25% of the cells. Furthermore, dialysis of cyclic GMP reversed the increase of ICa by the non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX). These findings suggested the presence of antagonistic mechanisms of cyclic GMP, which are independent from the above synergistic action. PKG may be involved in this antagonistic effect.

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