Studies On Fast Glucocorticoids Feedback And Its Mechnism In Pituitary Corticotroph

Hypothalamic-pituitary-adrenal axis (HPA) is an important part of the livingbody endocrine system, involves in controlling stress response, and has an importantphysiological role in regulating digestion, immune system, mood and emotions,sexuality, and energy storage and consumption. A major target of glucocorticoidfeedback for HPA axis activity is inhibition of adrenocorticotropic hormone (ACTH)secretion and transcription of the ACTH precursor, proopiomelanocortin (POMC)hnRNA in the pituitary corticotroph. The mechanism is complicated and involves inglucocorticoids, related receptors, protein kinases and other cytokines and it wasreported that rapid inhibition of ACTH secretion during ultradian variations mayinvolve non-genomic effects.In order to clarify the non-genomic mechanism of rapid inhibition of ACTHsecretion, the study was conducted in rat pituitary cells co-cultured with micro-carrierbeads. To mimic the dynamic physiological condition, cells were loaded in cellperfusion system for studying the effect of CRH and corticosterone on ACTHsecretion and the regulatory mechanism. The relative important role of the aminoacids on membrane association, trafficking and ligand-binding of the glucocorticoidreceptor (GR) was studied in4B cells, expressing endogenous GR and Cos-7cellstransfected EGFP-GR constructs.Cell perfusion data reveals concentration-dependent effects of CRH on ACTHsecretion, with sustained elevations with expected basal concentrations (30pM). Thesensitivity of glucocorticoids feedback depended on pulse concentrations, andprevious degree of glucocorticoid exposure. Prolonged preincubation in serum freemedium resulted in lower glucocorticoids, basal corticosterone levels (10nM) immediately inhibited CRH-stimulated ACTH secretion, returning to stimulated levelssoon after corticosterone withdrawal. Repeated corticosterone pulses resulted inpulsatile ACTH release. These data suggests the rapid glucocorticoid feedback at thepituitary corticotroph is an important part of the mechanism of ultradian pulsegeneration, and this could have important implications for HPA axis regulation duringchronic stress or disorders associated with elevated basal circulating levels ofglucocorticoids.At the same time, the data showed the inhibitory effect of corticosterone onACTH secretion was non-genomic action, unaffected by transcription or proteinsynthesis inhibitor. Corticosterone had an early inhibition on CRH–stimulated Srcphosphorylation and late potentiation. Src inhibitors had no effect on the rapidinhibition of CRH stimulated ACTH secretion by corticosterone, but the inhibitoralone caused marked inhibition of CRH-stimulated ACTH secretion suggesting thatsuppression of early CRH induced Src phosphorylation by corticosterone maycontribute to rapid glucocorticoid feedback.Western blot showed ligand-dependent rapid association of GR to membranefractions, with low but not stress corticosterone levels inducing GR membranelocalization and membrane clearance, and the GR receptor antagonist RU486preventsthe rapid inhibitory effect of corticosterone on CRH-stimulated ACTH secretion,suggesting that these non-genomic actions of glucocorticoids were mediated by theclassical GR.Unlike the membrane association mechanism of steroid hormonereceptor family members, mutation C683A (corresponding to the ER palmitoylationsite) did not affect corticosterone-induced membrane or nuclear GR trafficking. GRimmunoprecipitation and autoradiography in cells preloaded with radio-labeledpalmitic acid revealed no radioactivity incorporation into GR, suggestingpalmitoylation had no effect on membrane association of GR. However, mutationsL687-690A, L682A, E680G and K685G, within helix8of the ligand binding domain,prevented membrane and nuclear localization and [3H] dexamethasone binding. TheL687-690A mutation also reduced association of GR with heat shock protein90and transcriptional activity of the receptor. These data demonstrate that the region680-690(helix8), is critical for ligand binding and receptor function, and this novel region inthe ligand binding domain of the GR is essential for biological activity ofglucocorticoid receptor.