ROLE OF INOSITOL TRIPHOSPHATE-SENSITIVE CALCIUM STORES IN THE REGULATION OF ADRENOCORTICOTROPIN SECRETION BY PERIFUSED RAT ANTERIOR-PITUITARY-CELLS

Intracellular Ca2+ (Ca-i(2+)) stores contribute significantly to Ca2+ signaling in many types of cells. We studied the role of inositol trisphosphate (InsP(3))-sensitive Ca2+ stores, a principal Ca-i(2+) store that presumably is within the endoplasmic reticulum (ER), in cell signaling by examining the effect of thapsigargin (Tg), an ER Ca2+ pump inhibitor that depletes the ER Ca2+ pool, on ACTH secretion. Preincubation for 6-24 h with 2-20 nM Tg had no effect on the resting cytosolic free Ca2+ concentrations ([Ca-i(2+)]) but inhibited the ionomycin-stimulated spike-type increase in [Ca-i(2+)], which is mediated by InsP(3)-independent Ca-i(2+) release from the ER, in a dose-dependent (IC50, 4 nM) and time-dependent manner. In ER Cai(2+)-depleted cells, the spike phase (initial 5 min) of the ACTH secretory response to arginine vasopressin (AVP), which is mediated by InsP(3)-induced Ca,(2+) release, was also attenuated (IC50, 7.3 nM). However, the spike phase of the ACTH secretory response to AVP was inhibited to a much greater degree than the spike-type response to ionomycin, suggesting that ER Ca-i(2+) stores might have functions other than simply providing Ca for InsP(3)-stimulated Ca-i(2+) release. Tg pretreatment (IC50, 12 nar) also markedly inhibited the sustained plateau (final 15-min) phase of the ACTH secretory response to AVP, which is mediated by diacylglycerol-induced activation of protein kinase C and subsequent influx of extracellular Ca2+ via L-type voltage-sensitive Ca2+ channels (VSCC), but had no effect on the sustained (full 20 min) response to dioctanoylglycerol that directly activates protein kinase C. Tg had no effect on specific cell binding of [I-125]AVp or On specific cell binding of [H-3]phorbol 12,13-dibutyrate (except at 20 nM Tg), an index of protein kinase C concentration, or on protein kinase C activity. AVP significantly stimulated inositol trisphosphate accumulation, but pretreatment with Tg completely abolished this effect of AVP, whereas [H-3]myoinositol incorporation into membrane-associated inositol lipids and inositol phosphates was unaffected. Thus, Tg-induced depletion of ER Ca-i(2+) stores inhibited both the spike and plateau phases of the ACTH secretory response to AVP, presumably by inhibiting phospholipase C activity and the resulting generation of InsP(3) and diacylglycerol. Preincubation with Tg inhibited, in a dose-dependent (IC50 13 nM) and time-dependent manner, the sustained ACTH secretory response to corticotropin-releasing hormone (CRH) that is mediated by cAMP-induced activation of protein kinase A and Ca(e)2+ influx via L-type VSCC, and the sustained response to forskolin, which directly activates adenylate cyclase. Tg preincubation had no effect on specific cell binding of [I-125]CRH or on the sustained response to 8-bromo-cAMP, which directly activates protein kinase A; the sustained response to KCl, which induces extracellular Ca2+ influx via all types of VSCC by depolarizing the cell membrane; or the kinase activity of protein kinase A. Tg pretreatment attenuated (IC50, 14 nM) CRH-stimulated cAMP accumulation, an effect that paralleled Tg's inhibition of the CRH-stimulated sustained ACTH secretory response. Thus, Tg-induced depletion of ER Ca-i(2+) stores suppressed CRH-stimulated ACTH secretion by inhibiting adenylate cyclase activation and consequent cAMP production. These results indicate that Ca2+ within the ER lumen, in addition to providing Ca2+ for InsP(3)-stimulated Ca-i(2+) release, is required for the sustained activity of both phospholipase C and adenylate cyclase.