Mechanisms of desensitization of the adrenocorticotropin response to arginine vasopressin in ovine anterior pituitary cells

Abstract

Arginine vasopressin (AVP) stimulates adrenocorticotropin (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. Among 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 protein kinase C (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 µM Ro 31-8220 did not reduce the ability of a 10 nM AVP pretreatment to induce desensitization to a subsequent stimulation with 100 nM AVP. Pharmacologic blockade of receptor internalization by treatment with 0·25 mg/ml concanavalin A significantly impaired the ability of a 15-min pretreatment 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 pretreatment. 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.

Analytical construct of reversible desensitization of pituitary-testicular signaling: illustrative application in aging

“…The median time delay of 23 min from T secretory-burst onset to hysteretic downregulation would be consistent with rapid (within 20 -30 min) pharmacological desensitization observed in several other endocrine systems in vivo and in vitro (2,3,16,21,35). Here, reversible cycles of desensitization were consistently inferable (in 25 of 26 subjects) under physiological conditions for the endogenous LH-T system.…”

Separation of μ-Opioid Receptor Desensitization and Internalization: Endogenous Receptors in Primary Neuronal Cultures

“…This sequence could result in a blockade of signaling without internalization, however, the binding of arrestin to the ligand/receptor complex is thought to proceed to endocytosis via clathrin-coated pits (Law and Loh, 1999;Ferguson, 2001;von Zastrow et al, 2003). The separation of internalization and desensitization has been reported for other G-protein-coupled receptors such as ␤ 2 -adrenergic and neurokinin-1 receptors (Pippig et al, 1995;Bennett et al, 2002), but it has also been show that the desensitization of somatostatin and V1b vasopressin receptors requires internalization (Beaumont et al, 1998;Hassan and Mason, 2005). Although this sequence has been established in many heterologous systems, the role of this process in MOR signaling is the subject of some debate.…”

Low Affinity Analogs of Thyrotropin-releasing Hormone Are Super-agonists

“…Therefore, it was possible that the increased agonist efficacy of some analogs may have been due to decreased desensitization. Because desensitization is usually mediated by phosphorylation by one or more protein kinases (15)(16)(17), we tested the possibility that the interaction of the activated TRH-R with a specific protein kinase is ligand-specific and contributes to generation of the 4 S. Neumann, in preparation. efficacy differences between the TRH analogs.…”

“…5A, the specific protein kinase C inhibitor Ro-31-8425 (18), as well as the broad range protein kinase inhibitor HA-1077 (19), had no effect on the ligand specificity of the TRH-R1 response. Upon prolonged stimulation, the balance between the rates of receptor desensitization and resensitization determines the steady state level of active receptors on the cell surface and thus the efficacy of response (15). To answer the question whether the internalization (and recycling) of the agonist-TRH-R complex, as part of the desensitization/resensitization pathway, is affected by the nature of agonist, we compared the ability of TRH and R-Desaza-TRH to trigger TRH-R1 loss from the cell surface.…”

“…5B, no differences in the rates or extents of TRH-R1 internalization were observed using these agonists. During the course of resensitization, a phosphorylation(s) introduced by protein kinase(s) is reversed by the action of distinct endosomal phosphatases (15,20), and the unphosphorylated receptor is returned to the cell surface. We have used the pharmacological agents preferentially affecting the activity of endosomal phosphatases to test the possibility that the efficiency of dephosphorylation is dependent on the nature of the agonist-receptor complex internalized and contributes to the efficacy differences between TRH analogs.…”