Effect of protein kinase-C depletion on inositol trisphosphate-mediated and cyclic adenosine 3',5'-monophosphate-dependent protein kinase- mediated adrenocorticotropin secretion

Abstract

We studied the effect of Ca2+/phospholipid-dependent protein kinase-C (protein kinase-C) down-regulation by chronic exposure to phorbol 12-myristate 13-acetate (PMA) on ACTH secretion by dispersed male rat anterior pituitary cells in a microperifusion system. Preincubation for 24 h and preperifusion for 3 h with 0.1 and 1 microM PMA significantly inhibited (by 85% and 91%, respectively) the specific cell binding of [3H]phorbol 12,13-dibutyrate, an index of protein kinase-C concentration, and significantly reduced (by 101% and 20%, respectively) the sustained plateau (final 15-min) phase of the ACTH response to arginine vasopressin (AVP) and (by 56% and 54%, respectively) the sustained (full 20-min) response to dioctanoylglycerol (DOG), both of which are mediated by protein kinase-C activation. In contrast, the spike (initial 5-min) phase of the response to AVP, which is mediated by intracellular Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive stores, was significantly increased (by 112% and 99%, respectively), but the spike-type response to ionomycin, which releases intracellular Ca2+ by an InsP3-independent mechanism, was unaffected. AVP significantly stimulated inositol bisphosphate and InsP3, but not inositol monophosphate, accumulation, and PMA pretreatment significantly enhanced their AVP-stimulated accumulation (by 86%, 34%, and 78%, respectively), an effect that was abolished by simultaneous preperifusion with PMA and cycloheximide to inhibit new protein synthesis. Enhancement of the spike phase response to AVP and AVP-stimulated InsP3 accumulation were lost within 1 h of PMA removal, but [3H]phorbol 12,13-dibutyrate binding and the sustained responses to AVP and DOG remained suppressed after 3 h. Pretreatment with 0.1 and 1 microM PMA slightly reduced the sustained responses to CRF (by 29% and 16%, respectively) and 8-bromo-cAMP (by 8% and 12%, respectively), which are mediated by protein kinase-A activation and extracellular Ca2+ influx via L-type voltage-sensitive Ca2+ channels, but not the response to KCl, which is mediated by extracellular Ca2+ influx via all types of voltage-sensitive Ca2+ channels. The sustained response to CRF was still suppressed 1 h after PMA removal, but returned to the control level by 3 h. When new protein synthesis was inhibited by preperifusion with cycloheximide alone for 3 h after 24-h PMA pretreatment, recovery from the effects of PMA was abolished. Three-hour exposure to cycloheximide without PMA pretreatment inhibited the sustained responses to CRF, AVP, and DOG, but not the spite response to AVP.