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 pre sent in chromaffin cells and we have shown that the triakontatetraneuropept ide 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 adre nal explants with graded concentrations of TTN induced a dose-dependent inc rease in cAMP formation, but did not affect polyphosphoinositide metabolism . Pretreatment of adrenal cells with the protein kinase A inhibitor H-89 ma rkedly reduced the stimulatory effect of TTN on corticosterone and aldoster one secretion by perifused cells, whereas the phospholipase C inhibitor U-7 3122 did not affect the TTN-evoked stimulation of corticosteroid output. In cubation of adrenal cells with cholera toxin abolished the stimulatory effe ct 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 in crease in the concentration of intracellular calcium ([Ca2+](i)). Repeated pulses of TTN resulted in a gradual attenuation of the responses, indicatin g the existence of a desensitization phenomenon. Incubation of the cells wi th the T-type calcium channel blocker mibefradil significantly reduced the TTN-evoked [Ca2+], increase, whereas the L-type calcium channel blocker nif edipine and the N-type calcium channel blocker omega-conotoxin GVIA had no effect. Incubation of adrenal cells with H-89 markedly reduced the stimulat ory effect of TTN on [Ca2+](i). The involvement of calcium in steroid secre tion induced by TTN has also been investigated. Administration of mibefradi l significantly reduced the TTN-evoked stimulation of steroid production, w hereas nifedipine was devoid of effect. Taken together, these data indicate that in frog adrenocortical cells, the endozepine TTN stimulates cAMP form ation and calcium entry through T-type calcium channels. The effects of TTN on the adenylyl cyclase/protein kinase A pathway and calcium influx both c ontribute to the stimulatory action of the peptide on corticosteroid secret ion.