ACTIVATION OF 5-HYDROXYTRYPTAMINE(4) RECEPTORS CAUSES CALCIUM INFLUX IN ADRENOCORTICAL-CELLS - INVOLVEMENT OF CALCIUM IN 5-HYDROXYTRYPTAMINE-INDUCED STEROID-SECRETION

5-Hydroxytryptamine (5-HT) stimulates corticosteroid secretion from ad renal cells through activation of 5-HT4 receptors positively coupled t o adenylyl-cyclase. In the present study, we investigated in frog adre nocortical cells the effect of 5-HT4 receptor agonists on cytosolic ca lcium concentration ([Ca2+](i)) and determined the sequence of events associated with 5-HT4 receptor activation. The application of 5-HT or the 5-HT4 receptor agonist zacopride (10(-8) to 10(-5) M each) in the vicinity of cultured adrenocortical cells caused a dose-dependent incr ease in [Ca2+](i). Preincubation of the cells with the selective 5-HT4 receptor antagonist peridinyl]methyl-1--methyl-1H-indole-3-carboxylat e maleate totally blocked the 5-HT-induced stimulation of [Ca2+](i). C helation of extracellular calcium with ethylene glycol bis(beta-aminoe thyl ether)-N,N,N',N'-tetraacetic acid (10 mM) suppressed the stimulat ory effect of 5-HT on [Ca2+](i). Conversely, thapsigargin, an inhibito r of calcium ATPase activity, had no effect on the [Ca2+](i) rise. The calcium influx induced by 5-HT4 receptor agonists was not affected by nifedipine and omega-conotoxin GVIA but was totally blocked by pimozi de, a T-type calcium channel antagonist. The [Ca2+](i) response to zac opride was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1 -methylxanthine and markedly reduced by the protein kinase A inhibitor adenosine-3',5'-cyclic monophosphorothioate. We studied in perifused frog adrenal slices the involvement of [Ca2+](i) rise and cAMP formati on in the mechanism of action of 5-HT4 receptor agonists. Zacopride-in duced steroidogenesis was significantly reduced in the presence of ade nosine-3',5'-cyclic monophosphorothioate or after suppression of calci um in the perifusion medium. The stimulatory effect of zacopride on co rticosteroid secretion was not affected by nifedipine and omega-conoto xin GVIA but was significantly inhibited by pimozide. Taken together, these data indicate that activation of 5-HT4 receptors in adrenocortic al cells causes stimulation of adenylyl cyclase and subsequently incre ases calcium influx through a T-type calcium channel. Both the increas ed in cAMP formation and the calcium rise are involved in the stimulat ory effect of 5-HT on corticosteroid secretion.