Multiple second messenger routes enhance two high-voltage-activated calcium currents in molluscan neuroendocrine cells.

Two types of high-voltage-activated calcium currents were identified in whole-cell voltage-clamp recordings of the neuroendocrine caudodorsal cells, which control egg-laying in the freshwater snail Lymnaea stagnalis. The currents were: (i) a rapidly inactivating high-voltage-activated current, with an activation threshold of -40 mV and maximal amplitude at +10 mV; and (ii) a slowly inactivating high-voltage-activated current, with a threshold of -10 mV and a peak at +30 mV. Both currents were reduced by nifedipine and verapamil, but not by omega-conotoxin GVIA, suggesting that they belong to the L-type family of calcium currents. The voltage-dependence of inactivation of the rapidly inactivating high-voltage-activated current was bell-shaped. Time-constants of inactivation ranged from 10 to 25 ms. Steady-state inactivation was characterized by a potential of half maximal inactivation of -21.7 +/- 3.4 mV and a slope factor of 8.1 +/- 1.7 mV. The voltage-dependence of inactivation of the slowly inactivating high-voltage-activated current was S-shaped. Time-constants of inactivation increased with depolarization up to a maximum of 300 ms. The steady-state inactivation parameters were a potential of half maximal inactivation of +6.8 +/- 2.2 mV and a slope factor of 6.0 +/- 1.1 mV. The membrane-permeable analog of cAMP, 8-chlorophenylthio-cyclic AMP, predominantly increased the slowly inactivating high-voltage-activated current, and shifted its voltage-dependence of activation and inactivation 10 mV to the left. The rapidly inactivating high-voltage-activated current was slightly increased by 8-chlorophenylthio-cyclic AMP. 8-Bromo-cyclic GMP and the phorbol ester, 12-O-tetradecanoyl-13-phorbol acetate, had qualitatively similar effects. Both agents enhanced the rapidly inactivating current and, to a lesser degree, the slowly inactivating current, without affecting their voltage-dependence. The cyclic AMP-dependent protein kinase inhibitor, Walsh inhibitor peptide, antagonized the stimulating effect of 8-chlorophenylthio-cyclic AMP. The broad-spectrum protein kinase inhibitor 1-(5-isoquino-linylsulfonyl)-2-methyl-piperazine (H-7) strongly attenuated the effects of 8-chlorophenylthio-cyclic AMP, 8-bromo-cyclic GMP and 12-O-tetradecanoyl-13-phorbol acetate, suggesting that all treatments increase both types of high-voltage-activated calcium currents through phosphorylation of the channel-complex.