Mechanisms of desensitization of the adrenocorticotrophin response to arginine vasopressin in ovine anterior pituitary cells
Ali Hassan and Drusilla R. Mason
School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.
Arginine vasopressin (AVP) stimulates adrenocorticotrophin (ACTH) secretion from corticotroph cells of the anterior pituitary via activation of the V1b vasopressin receptor, a member of the G protein-coupled receptor (GPCR) family. Recently we have shown that treatment of ovine anterior pituitary cells with AVP for short periods results in reduced responsiveness to subsequent stimulation with AVP. The aim of this study was to investigate mechanisms involved in this desensitization process. Amongst the GPCR family rapid desensitization is commonly mediated by receptor phosphorylation, with resensitization being mediated by internalization and subsequent dephosphorylation of the receptors by protein phosphatases. Since desensitization of V1a vasopressin receptors is mediated by protein kinase C-mediated receptor phosphorylation, we investigated the involvement of this enzyme in desensitization of the ACTH response to AVP. Treatment of perifused ovine anterior pituitary cells with the specific PKC activator 1,2-dioctanoyl-sn-glycerol (300 µM) did not induce any reduction in response to a subsequent 5 min stimulation with 100 nM AVP, despite potently stimulating ACTH secretion. Likewise, the results obtained using the PKC inhibitor Ro 31-8220 were not consistent with involvement of PKC in AVP desensitization: 2 mM Ro 31-8220 did not reduce the ability of a 10 nM AVP pre-treatment to induce desensitization to a subsequent stimulation with 100 nM AVP. Pharmacological blockade of receptor internalization by treatment with 0.25 mg/ml ConA significantly impaired the ability of a 15 min pre-treatment with 10 nM AVP to induce desensitization, rather than affecting resensitization. Treatment with 10 nM okadaic acid, an inhibitor of protein phosphatase 1 and 2A, had no effect on either resensitization or desensitization. In contrast, inhibition of protein phosphatase 2B (PP2B) with 1 µM FK506 decreased the rate of resensitization: complete recovery from desensitization took 40 min, whereas in controls recovery was complete 20 min after termination of the pre-treatment. These results indicate that desensitization of the ACTH response to AVP is not mediated by PKC-catalyzed phosphorylation, suggesting subtype-specific differences in the regulation of V1a and V1b vasopressin receptors. The data demonstrate that desensitization was dependent, at least in part, upon receptor internalization and that resensitization was dependent upon PP2B- mediated receptor dephosphorylation.