MULTIPLE 2ND MESSENGER ROUTES ENHANCE 2 HIGH-VOLTAGE-ACTIVATED CALCIUM CURRENTS IN MOLLUSCAN NEUROENDOCRINE CELLS

Two types of high-voltage-activated calcium currents were identified i n whole-cell voltage-clamp recordings of the neuroendocrine caudodorsa l cells, which control egg-laying in the freshwater snail Lymnaea stag nalis. The currents were: (i) a rapidly inactivating high-voltage-acti vated current, with an activation threshold of -40 mV and maximal ampl itude 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 curren ts were reduced by nifedipine and verapamil, but not by omega-conotoxi n GVIA, suggesting that they belong to the L-type family of calcium cu rrents. The voltage-dependence of inactivation of the rapidly inactiva ting high-voltage-activated current was bell-shaped. Time-constants of inactivation ranged from 10 to 25 ms. Steady-state inactivation was c haracterized 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 curren t was S-shaped. Time-constants of inactivation increased with depolari zation up to a maximum of 300 ms. The steady-state inactivation parame ters 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 slow ly inactivating high-voltage-activated current, and shifted its voltag e-dependence of activation and inactivation 10 mV to the left. The rap idly inactivating high-voltage-activated current was slightly increase d by 8-chlorophenylthio-cyclic AMP. 8-Bromo-cyclic GMP and the phorbol ester, 12-O-tetradecanoyl-13-phorbol acetate, had qualitatively simil ar effects. Both agents enhanced the rapidly inactivating current and, to a lesser degree, the slowly inactivating current, without affectin g their voltage-dependence. The cyclic AMP-dependent protein kinase in hibitor, Walsh inhibitor peptide, antagonized the stimulating effect o f 8-chlorophenylthio-cyclic AMP. The broad-spectrum protein kinase inh ibitor 1-(5-isoquino-linylsulfonyl)-2-methyl-piperazine (H-7) strongly attenuated the effects of 8-chlorophenylthio-cyclic AMP, 8-bromo-cycl ic GMP and 12-O-tetradecanoyl-13-phorbol acetate, suggesting that all treatments increase both types of high-voltage-activated calcium curre nts through phosphorylation of the channel-complex.