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 the hypothalamic pituitary adrenal (HPA) axis. This includes the expression of hypothalamic corticotropin releasing hormone (CRH) and vasopressin (VP), pituitary CRH and V1b VP receptors, and adrenal steroidogenesis. At the hypothalamic level, in vivo and in vitro studies have shown that the increases in CRH transcription during stress are transient even if the stimulus is sustained. Using adrenalectomized rats with constant levels of corticosterone replacement, we have shown that termination of CRH transcription is independent of the increases in plasma glucocorticoids in response to stress. We also demonstrated that termination of CRH transcription is associated with increased expression of inducible cAMP early repressor (ICER), a repressor isoform of cAMP responsive element modulator (CREM), co-localized in CRH cells of the PVN. This paralleled formation of ICER-CRH CRE complexes in the electromobility gel shift assay (EMSA) suggesting that endogenous levels of ICER can interact with the CRH CRE. EMSA using hypothalamic nuclear extracts of control and stressed rats and CRH CRE radiolabeled oligonuclotides showed several shifted bands, of which the two lower bands were supershifted with CREM antibody. Chromatin immunoprecipitation assays revealed stress-inducible binding of CREM to the CRH promoter CRE and decreases of Pol II association with the CRH promoter at 3 h when CRH transcription has declined. These data indicates that induction of ICER contributes to the limitation of CRH transcription during stress. The role of oxytocin in the hyporesponsiveness of the HPA axis to stress during late pregnancy and lactation was studied by examining the effects of intracerebroventricular (icv) oxytocin infusion in ovariectomized rats receiving low doses of estradiol replacement with or without progesterone. In the absence of progesterone, central oxytocin infusion inhibited corticosterone responses to restraint stress but had no effect on plasma ACTH and CRH mRNA levels in the PVN. However, in the presence of progesterone, central oxytocin administration increased the magnitude and duration of corticosterone and ACTH responses and potentiated the stimulatory effect of stress on CRH mRNA levels. The study shows that central oxytocin can either inhibit or enhance HPA axis activity depending on the levels of circulating sex steroids. These findings may be relevant to the pathogenesis of psychiatric disorders associated with reproduction such as post partum depression and premenstrual syndrome. Extension of studies on immune-neuroendocrine interactions showed that alterations in fluid homeostasis secondary to endotoxemia are due primarily to renal insensitivity to VP rather than defective hypothalamic VP secretion. These studies revealed marked and sustained decreases in VP V2 receptors and aquaporin 2 in kidney medulla following LPS induced endotoxemia, and that these alterations lead to impaired capacity to concentrate urine. Chronic immune challenge also resulted in changes in sympathoadrenal and renin-angiotensin-aldosterone responses to novel stressors. Repeated treatment of rats with increasing doses of lipopolysaccharide (LPS) resulted in a decrease of plasma epinephrine and aldosterone as well as in renin activity responses compared to those after acute administration. Repeated LPS administration was associated with decreased plasma aldosterone responses to a different stressor (immobilization) in spite of preserved or even elevated responses of plasma renin activity and catecholamines. Studies using in situ hybridization and dispersed adrenal glomerulosa cells demonstrated that decreased aldosterone responses are the result of decrease in aldosterone synthase expression and activity. These alterations may contribute to deficient cardiovascular adaptation during chronic inflammatory states. Transcriptional and post-transcriptional mechanisms regulating the number of CRH and VP receptors in the pituitary play an important role in the control of HPA axis activity. An essential element regulating V1b receptor transcription is a GAGA box located in the proximal promoter, which binds a protein complex found in pituitary nuclear extracts. Stress causes transient increases in GAGA binding activity and studies in the hypothalamic cell line H32 which expresses endogenous V1b receptors, showed that VP increases GAGA binding activity through transactivation of the epidermal growth factor (EGF) receptor (EGF-R) and the mitogen activated protein (MAP) kinase pathway. Incubation of H32 cells or primary anterior pituitary cultures with VP led to transient phosphorylation of extracellular signal-regulated protein kinase (pERK) (2.5 min, declining by 10 min), an effect which was prevented by the epidermal growth factor (EGF) receptor inhibitor, AG1478. ERK phosphorylation by VP was preceded (1 min) by serine-phosphorylation and association to the EGFR of the adapter protein Shc, and phosphorylation of tyrosine 1173 (Tyr1173) of the EGF-R. Transactivation of the EGFR and MAPK by VP was not mediated by either protein kinase C (PKC)-induced Src-phosphorylation, or metalloprotease-induced cleavage of an EGF-like peptide, and it was unaffected by Pertussis toxin or a dominant negative for G-protein beta-gamma subunits. Transfection of a PKC alpha dominant negative prevented VP-induced EGFR (Tyr1173) phosphorylation and its association to the adapter protein Shc, and inhibited ERK phosphorylation at 2.5 min. In contrast, transfection of a PKC beta1 dominant negative prevented the reduction of VP-stimulated Tyr 1173 and EKK phosphorylation at 10 min, indicating that PKC beta1 is responsible for the decline in pERK. The use of specific VP receptor subtype analogs in H32 cells, primary pituitary cell cultures or transfected CHO cells revealed identical mechanisms of MAPK activation by V1a or V1b receptors. However, the effect was cell specific since both receptor subtypes transactivated MAP kinase independently of the EGF receptor. These data suggest that while MAP kinase transactivation by VP depends on PKC alpha-induced phosphorylation of Shc and its association with the EGFR, PKC beta-1 mediates the declining phase. Activation of GAGA binding by VP-induced EGFR transactivation may mediate stimulation of V1b receptor transcription and contribute to the pituitary V1b receptor upregulation observed during stress. These effects of VP have important functional implications not only in the regulation of the VP receptor but also potentially mediating mitogenic and trophic actions of VP in the brain and pituitary corticotroph. On the regulation of the type-1 CRH receptor (CRHR1), experiments were performed to determine the involvement of RNA binding proteins on the regulation of CRHR1 protein synthesis. Gel shift assays using transcribed RNA from the CRHR1 5?UTR and cytosolic extracts from anterior pituitaries from adrenalectomized rats (condition associated with high CRHR1 mRNA and binding downregulation), revealed an increase in RNA binding proteins. This was specific for CRHR1 since binding to the type-1 angiotensin receptor was unchanged. Competition studies and UV cross-linking analysis suggest that formation of CRF-R1 and AT1aR 5'UTR RNA protein complexes require at least some proteins that are common to both receptor mRNAs. These data show that alterations of the hypothalamic-pituitary-adrenal axis specifically regulate mRNA binding proteins that interact with the 5'UTR of the CRF-R1, suggesting an involvement in translational regulation of CRF-R1 mRNA.
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