ROLES OF INTRACELLULAR AND EXTRACELLULAR CALCIUM IN THE KINETIC PROFILE OF ADRENOCORTICOTROPIN SECRETION BY PERIFUSED RAT ANTERIOR-PITUITARY-CELLS .1. CORTICOTROPIN-RELEASING FACTOR STIMULATION

We examined the effects of removing extracellular Ca2+ (Ca2+e), depleting intracellular Ca2+ (Ca2+i), inhibiting Ca2+-dependent calmodulin, blocking voltage-sensitive Ca2+ channels, and combining Ca2+i depletion with exposure to glucocorticoid on the secretion of ACTH by perifused dispersed rat anterior pituitary cells stimulated with ovine CRF and 8-bromocAMP (8-Br-cAMP). A time-course analysis of the effect of perifusing the cells for 60 min with Ca2+-free medium on 10 nM CRF-stimulated ACTH release revealed that inhibition required about 3 min to begin and about 40 min to reach maximal effect. Within 2 min of restoring Ca2+ to the medium, ACTH secretion rebounded for about 5 min before falling to the pre-Ca2+e removal rate. A similar pattern and time course were observed when Ca2+e was more completely removed by perifusing the cells with Ca2+-free medium containing 2 mM EGTA, except that greater suppression was observed. Removing Ca2+e reduced CRF- and 5 mM 8-Br-cAMP-induced ACTH release by 54% and 49%, respectively, and delayed by 1 min the response to 8-Br-cAMP, but not that to CRF. Perifusing 0.2 mM nimodipine, a dihydropyridine Ca2+ channel blocker, before and after restoration of Ca2+ to the Ca2+-free medium inhibited ACTH release by 40-48%, and the blockade persisted for at least 70 min after nimodipine was removed from the medium. When intracellular Ca2+ was depleted by perifusing the cells with Ca2+-free/EGTA medium containing the Ca2+ ionophore A23187 to facilitate the efflux of Ca2+I(CRF- and 8-BrcAMP- stimulated ACTH release were reduced by 70% and 71%, respectively, and the responses to both agents were delayed by 1 min. Preperifusion of the cells with 5 pM penfluridol, a calmodulin inhibitor, reduced CRF- and 8-Br-cAMP-induced ACTH release by 54% and 41%, respectively. The combination of Ca2+i depletion and perifusion with 100 nM dexamethasone, a maximally inhibitory concentration, inhibited CRF- and 8-BrcAMP- stimulated ACTH release by 82% and 83%, respectively. These results indicate that 1) CRF and 8-Br-cAMP both require Ca2+e influx to elicit maximal ACTH release, consistent with the concept that CRF acts mainly via activation of the adenylate cyclase/cAMP-dependent protein kinase-A pathway, causing influx of Ca2+e; 2) CRF- and 8-Br-cAMP-stimulated entry of Ca2+e proceeds via both L- and non-L-type voltageregulated Ca2+ channels; 3) at least one site of Ca2+’s action in effecting ACTH release lies distal to cAMP generation; 4) Ca2+; may be involved in the initial incremental phase of the response to CRF that is analogous to the transient spike phase of the response to agents, such as arginine vasopressin, which act via the Ca2+/inositol phosphate-dependent protein kinase-C pathway; 5) both phases of the response to CRF appear to involve Ca2+-dependent calmodulin; 6) at least one site at which glucocorticoids exert their inhibitory effect lies distal to cAMP generation; 7) glucocorticoids appear to inhibit secretion of the Ca2+-regulated ACTH pool; and, finally, 8) there appears to be a fraction of CRF-stimulated ACTH secretion that is both Ca2+ independent and glucocorticoid nonsuppressible. © 1990 by The Endocrine Society.