Studies of the mechanisms of action of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the ovine anterior pituitary: evidence that CRF and AVP stimulate protein phosphorylation and dephosphorylation

Abstract

This study was undertaken to determine the roles of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of adrenocorticotropin (ACTH) secretion in perifused ovine anterior pituitary (AP) cells and their ability to cause protein phosphorylation and dephosphorylation in these cells. Freshly dispersed ovine AP cells were maintained in a miniperifusion chamber and ACTH secretion was monitored every 20 s. When cells were perifused with CRF (1 nM, 5 min) or AVP (1 nM, 5 min), ACTH release was increased 20-fold and 12-fold, respectively. When an ovine AP cell membrane fraction was incubated with either CRF or AVP, CRF stimulated the phosphorylation of at least 11 proteins and the dephosphorylation of at least 5 phosphoproteins, whereas AVP caused the phosphorylation of at least 15 proteins and the dephosphorylation of 5 proteins. A comparison of the proteins phosphorylated by CRF or AVP with those phosphorylated by cAMP or protein kinase C activators suggested that the hormone-stimulated phosphorylation may also involve unidentified protein kinases. Additionally, at least eight proteins appeared to be phosphorylated by both CRF and AVP. Furthermore, in the case of four particular proteins both CRF and AVP stimulated phosphorylation at low concentrations of Ca 2+ (0.1−1 μM), but at high concentrations of Ca 2+ (10–100 μM) CRF or AVP triggered dephosphorylation of these proteins. In conclusion: (1) CRF and AVP are both effective ACTH secretagogues with CRF more potent than AVP in the perifused ovine AP cells; (2) CRF and AVP stimulate phosphorylation and dephosphorylation of specific membrane proteins that may be involved in the hormone-mediated ACTH release; (3) the hormone-regulated phosphorylation of the substrate proteins is dually controlled by intracellular calcium concentrations; (4) In addition to cAMP-dependent protein kinase and protein kinase C, other protein kinase and phosphatase are also involved in the actions of CRF and AVP.