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.

Following repeated or prolonged exposure to either corticotrophin-releasing hormone (CRH) or arginine vasopressin (AVP), pituitary adrenocorticotrophin (ACTH) responsiveness is reduced. This study compared the characteristics of desensitization to CRH and AVP in perifused ovine anterior pituitary cells. Desensitization to AVP occurred at relatively low AVP concentrations and was both rapid and readily reversible. Treatment for 25 min with AVP at concentrations greater than 2 nM caused significant reductions in the response to a subsequent 5 min 100 nM AVP pulse (IC(50)=6.54 nM). Significant desensitization was observed following pretreatment with 5 nM AVP for as briefly as 5 min. Desensitization was greater following a 10 min pretreatment, but longer exposures caused no further increase. Resensitization was complete within 40 min following 15 min treatment with 10 nM AVP. Continuous perifusion with 0.01 nM CRH had no effect on AVP-induced desensitization. Treatment with 0.1 nM CRH for either 25 or 50 min caused no reduction in the response to a subsequent 5 min stimulation with 10 nM CRH. When the pretreatment concentration was increased to 1 nM significant desensitization was observed, with a greater reduction in response occurring after 50 min treatment. Recovery of responsiveness was progressive following 50 min treatment with 1 nM CRH and was complete after 100 min. Our data show that in the sheep AVP desensitization can occur at concentrations and durations of AVP exposure within the endogenous ranges. This suggests that desensitization may play a key role in regulating ACTH secretion in vivo. If, as has been suggested, CRH acts to set corticotroph gain while AVP is the main dynamic regulator, any change in responsiveness to CRH may significantly influence the overall control of ACTH secretion.

The human ovarian surface epithelium (hOSE) is a mesothelial layer that surrounds the ovary and undergoes injury and repair cycles after ovulation-associated inflammation. We previously showed that IL4 is a key regulator of progesterone bioavailability during post-ovulatory hOSE repair as it differentially up-regulated 3 β -HSD1 and 3 β -HSD2 mRNA transcripts and total 3β-hydroxysteroid dehydrogenase activity whereas it inhibited androgen receptor (AR) expression. We now show that the pro-inflammatory effect of IL1α on 3 β -HSD1 expression is mediated by nuclear factor-κB (NF-κB), whereas its anti-inflammatory action on 3 β -HSD2 expression is exerted via p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and NF-κB signalling pathways. The anti-inflammatory IL4 effects on 3 β -HSD1 and 3 β -HSD2 mRNA expression are mediated through STAT6 and PI3K signalling networks. IL4 effects on AR and 3 β -HSD2 expression involve the p38 MAPK pathway. We also document that IL4 up-regulates lysyl oxidase (LOX) mRNA transcripts, a key gene for extracellular matrix (ECM) deposition and inhibits IL1α-induced expression of cyclooxygenase-2 (COX-2) mRNA, a gene involved in breakdown of ECM, showing a further role in post-ovulatory wound healing. We conclude that IL1α and IL4 actions in the post-ovulatory wound healing of hOSE cells are mediated by different signalling transduction pathways. The p38 MAPK signalling pathway may have possible therapeutic benefit in inflammation-associated disorders of the ovary, including cancer.