The purpose of the research is to study the cellular and molecular mechanisms of adaptation to stress with emphasis on the regulation of the various components of hypothalamic pituitary adrenal (HPA) axis. This includes the expression of hypothalamic corticotropin releasing hormone (CRH) and vasopressin (VP), pituitary CRH receptors and V1b VP receptors, and adrenal steroidogenesis. In situ hybridization studies using intronic and exonic probes demonstrated that CRH and VP co-expressed in the same neuron undergo differential regulation by stress and exposure to glucocorticoids. During the past year, the development of hypothalamic organotypic cultures made it possible to study changes in CRH and VP transcription in vitro. It was shown that cAMP dependent pathways stimulate VP transcription directly in vasopressinergic neurons of the PVN, supraoptic and suprachiasmatic nuclei (SCN). Basal levels of VP transcription in vitro show circadian variation in the SCN, which depends on cAMP-dependent neurotransmission within the nucleus. The regulation of CRH and VP transcription by glucocorticoids, cAMP and other messenger systems is under current investigation using these hypothalamic cultures, as well as promoter reporter gene systems transfected to hypothalamic cell lines expressing the peptides.Responsiveness of the HPA axis to stress also depends on the levels of CRH and VP receptors in the pituitary corticotroph. Type 1CRH receptor (CRHR1) downregulation during either stimulation of the HPA axis or glucocorticoid administration is associated with high CRHR1 mRNA levels suggesting that regulation occurs at translational and post-translational sites. Using reporter genes, in vitro translation and Western blot analyses, it was demonstrated that a shot open reading frame (ORF) in the 5' untranslated region of the CRHR1 mRNA inhibits translation of the main ORF. The role of cytosolic binding proteins regulating this process is under current investigation. Specific CRHR1 antibodies were used to demonstrate that CRHR1 protein increases after activation of the HPA axis by adrenalectomy, while it decreases after glucocorticoid administration. The transcriptional regulation and tissue specific expression of the V1b VP receptor is also under investigation using a recently isolated genomic clone of the V1b VP receptor.
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