Roles of intracellular and extracellular calcium in the kinetic profile of adrenocorticotropin secretion by perifused rat anterior pituitary cells. I. Corticotropin-releasing factor stimulation

Abstract

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-bromo-cAMP (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-Br-cAMP-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 microM 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-Br-cAMP-stimulated ACTH release by 82% and 83%, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)