title: 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)
authors: Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
authors: J G
authors: JG
authors: Won
authors: D N
authors: DN
authors: Orth
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D003907:Dexamethasone / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008297:Male; D009553:Nimodipine / Q000494:pharmacology; D010395:Penfluridol / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D002147:Calmodulin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D010395:Penfluridol; D009553:Nimodipine; D003907:Dexamethasone; D000324:Adrenocorticotropic Hormone; D003346:Corticotropin-Releasing Hormone; D002118:Calcium