Desensitization of the adrenocorticotropin response to arginine vasopressin
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The hypothalamic peptide arginine vasopressin (AVP) is an important regulator of adrenocorticotropin (ACTH) release from the anterior pituitary. AVP stimulates ACTH secretion from corticotroph cells by activating the V1b AVP receptor, a member of the G protein-coupled receptor (GPCR) family which activates the phosphoinositide signalling pathway. In vivo, persistent or repeated stress can result in reduced ACTH responsiveness and it appears that regulation of the V1b receptor plays an important role in this process. Similarly, repeated or prolonged stimulation of anterior pituitary cells with AVP in vitro results in a reduction in ACTH responsiveness, or desensitization. The aims of this study were to characterize this desensitization process and to investigate the mechanisms underlying it. Desensitization of the ACTH response to AVP was investigated in perifused dispersed ovine anterior pituitary cells. Desensitization was found to be rapid, readily reversible and occurred at relatively low AVP concentrations. Treatment with 5 nM AVP for 10 min reduced the response to a subsequent stimulation with 100 nM AVP by 39.4 ± 8.6%, but treatment for longer durations at this concentration did not increase the magnitude of desensitization. Recovery of responsiveness to a subsequent stimulation with AVP was complete within 40 min following pre-treatment with 10 nM AVP for 15 min. Desensitization was found to occur at concentrations and durations of AVP pre-treatment which are within the range of endogenous AVP pulses, suggesting that it plays an important physiological role in the regulation of ACTH secretion. The very rapid kinetics of desensitization and resensitization of the ACTH response to AVP suggest that these processes are more important in regulating the acute response to AVP than the alterations of corticotroph responsiveness which have been observed during chronic stress. The characteristics of the desensitization process suggested that it might be caused by uncoupling of the pituitary AVP receptor from its signalling pathway, a common mechanism of desensitization amongst GPCRs which is often mediated by receptor phosphorylation. Investigations showed that the protein kinases protein kinase C (PKC) and casein kinase lα, were not involved in desensitization. Activation of PKC by treatment with the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol did not result in desensitization and inhibition of PKC activity by treatment with Ro 31-8220 did not prevent AVP-induced desensitization. Similarly, treatment with the casein kinase 1 inhibitor CK1-7 did not prevent AVP-induced desensitization. Receptor internalization and dephosphorylation by protein phosphatases are important in the resensitization of many GPCRs. Interestingly, pharmacological inhibition of receptor internalization reduced the extent of desensitization which could be induced by AVP pre-treatment, suggesting that sequestration of receptors to an intracellular compartment is important in the desensitization process. Inhibition of protein phosphatase 2B by treatment with FK506 slowed the rate of recovery following desensitization, suggesting that this enzyme plays an important role in the resensitization process. No evidence could be found for the involvement of protein phosphatase 2A in resensitization of the ACTH response to AVP.