The effect of the endozepine triakontatetraneuropeptide on corticosteroid secretion by the frog adrenal gland is mediated by activation of adenylyl cyclase and calcium influx through T-type calcium channels.

We have recently found that, in the frog adrenal gland, endozepines are present in chromaffin cells and we have shown that the triakontatetraneuropeptide TTN is a potent stimulator of corticosteroid secretion in vitro. In the present study, we have investigated the transduction mechanisms mediating the corticotropic effect of TTN on adrenocortical cells. Incubation of adrenal explants with graded concentrations of TTN induced a dose-dependent increase in cAMP formation, but did not affect polyphosphoinositide metabolism. Pretreatment of adrenal cells with the protein kinase A inhibitor H-89 markedly reduced the stimulatory effect of TTN on corticosterone and aldosterone secretion by perifused cells, whereas the phospholipase C inhibitor U-73122 did not affect the TTN-evoked stimulation of corticosteroid output. Incubation of adrenal cells with cholera toxin abolished the stimulatory effect of TTN on steroid secretion. Administration of a brief pulse of TTN (10(-6) M) in the vicinity of cultured adrenocortical cells induced a robust increase in the concentration of intracellular calcium ([Ca2+]i). Repeated pulses of TTN resulted in a gradual attenuation of the responses, indicating the existence of a desensitization phenomenon. Incubation of the cells with the T-type calcium channel blocker mibefradil significantly reduced the TTN-evoked [Ca2+]i increase, whereas the L-type calcium channel blocker nifedipine and the N-type calcium channel blocker omega-conotoxin GVIA had no effect. Incubation of adrenal cells with H-89 markedly reduced the stimulatory effect of TTN on [Ca2+]i. The involvement of calcium in steroid secretion induced by TTN has also been investigated. Administration of mibefradil significantly reduced the TTN-evoked stimulation of steroid production, whereas nifedipine was devoid of effect. Taken together, these data indicate that in frog adrenocortical cells, the endozepine TTN stimulates cAMP formation and calcium entry through T-type calcium channels. The effects of TTN on the adenylyl cyclase/protein kinase A pathway and calcium influx both contribute to the stimulatory action of the peptide on corticosteroid secretion.