Roles of Intracellular and Extracellular Calcium in the Kinetic Profile of Adrenocorticotropin Secretion by Perifused Rat Anterior Pituitary Cells. II. Arginine Vasopressin, Oxytocin, and Angiotensin-II Stimulation*

We examined the effects of removing extracellular Ca2+ (Ca2+e), depleting intracellular Ca2+ (Ca2+i), inhibiting cAMP-dependent calmodulin, and blocking voltage-sensitive Ca2+ channels on the secretion of ACTH by perifused dispersed rat anterior pituitary cells. The cells were stimulated with synthetic arginine vasopressin (AVP), oxytocin (OT), and angiotensin- II (All), all of which are thought to act via the Ca2+/inositol phosphate-dependent protein kinase-C pathway, with synthetic ovine CRF, which acts via the cAMP-dependent protein kinase- A pathway, and with dioctanoylglycerol, which directly activates protein kinase-C. All three secretagogues elicited an initial spike phase ACTH secretory response that peaked within 1 or 2 min and ended within 6 min. AVP and OT also elicited a sustained plateau phase response that lasted for as long as the cells were exposed to the secretagogue, but All did not. Removal of Ca2+e diminished the initial spike phase by 30-50%, but depletion of Ca2+i virtually abolished it. In contrast, the sustained phase of the response to AVP and OT was abolished by removal of Ca2+e. The effect of dioctanoylglycerol, which elicits a sustained progressive increase in ACTH release, but no initial spike phase, was also greatly inhibited by Ca2+e removal; no greater effect was observed when Ca2+i was depleted. Blockade of L-type voltage-sensitive Ca2+ channels with nimodipine, a dihydropyridine drug, had the same effect as Ca2+e removal on both the initial spike and sustained plateau phases of the response to AVP. Inhibiting cAMP-dependent calmodulin with penfluridol had no effect on the initial spike phase, but reduced the sustained plateau phase of the response to AVP. Removal of Ca2+e or depletion of Ca2+; did not abolish the synergistic ACTH secretory response to the combination of AVP and CRF. We conclude 1) that the initial spike phase of the response requires Ca2+i release, is independent of calmodulin, and presumably is mediated by activation of phospholipase-C with generation of inositol 1,4,5- trisphosphate, which mobilizes Ca2+; from the endoplasmic reticulum; Ca2+e influx via L-type Ca2+ channels also appears to play a role; 2) that the sustained response elicited by AVP and OT depends upon Ca2+e influx via L-type voltage-sensitive Ca2+ channels, perhaps as a result of protein kinase-C activation; 3) that either the inositol 1,4,5-trisphosphate and diacylglycerol arms of the phospholipase-C pathway can be dissociated or All acts via a different signal transduction pathway; and 4) that neither Ca2+i mobilization nor Ca2+e influx plays an essential role in the synergism between AVP and CRF, which presumably involves interactions between other elements of the protein kinase-C and protein kinase-A signal transduction pathways. (Endocrinology126: 858–868,1990)