The fetus has the remarkable ability to adapt to conditions of chronic hypoxia over the course of gestation. One of the key factors mediating this adaptation is cortisol. Regulation of fetal cortisol is necessary for normal fetal growth and development and response to subsequent stressors. Understanding the mechanisms of fetal adaptation to chronic stress is also important in regard to effective use of antenatal glucocorticoid therapy. Our studies have shown that in late gestation, basal plasma cortisol levels are normal in the long-term hypoxic (LTH) sheep fetus, while expression of key adrenal steroidogenic enzymes (P450 cholesterol side chain cleavage [CYP11A] and P450 17alpha-hydroxylase [CYP17])is suppressed. Paradoxically, basal plasma adrenocorticotropin (ACTH) concentrations are elevated and, in response to a secondary stressor, cortisol production is enhanced compared to normoxic controls. Basal plasma concentrations of the ACTH precursors proopiomelanocortin (POMC) and 22 kDa proACTH (the major intermediate in processing of POMC to ACTH) are also elevated. We have further evidence that processing of POMC to ACTH in the anterior pituitary is enhanced during acute stress, leading to elevated plasma ACTH in LTH fetuses. Physiological concentrations of ACTH precursors have been demonstrated to be potent inhibitors of fetal cortisol production. Thus, it is apparent that the fetal hypothalamic-pituitary-adrenocortical (HPA) axis has acclimatized to LTH at all levels. However, the mechanisms driving this adaptation remain to be elucidated. The proposed studies will extend our previous observations on HPA function in the LTH fetus and examine specific mechanisms involved not only in maintaining basal glucocorticoid secretion but also in responding to secondary stressors. The proposed studies will test the general hypothesis that adaptive changes in the fetal HPA axis allow the fetus to acclimatize to the stress of long-term hypoxia while augmenting the ability to respond to a secondary, acute stressor. Specific Aim 1. To elucidate the mechanisms of enhanced hypothalamic drive (i.e., augmented corticotrope function) in LTH fetuses. These studies are designed to test the hypothesis that: LTH results in enhanced hypothalamic paraventricular nuclei (PVN) expression of cortiocotropin releasing factor (CRF) and/or arginine vasopressin (AVP) and/or increased pituitary responsiveness to these ACTH secretagogues resulting in the increased anterior pituitary corticotrope function observed in response to LTH. Specific Aim 2. To determine the molecular and cellular mechanisms by which the adrenal cortex has acclimatized to LTH that results in the observed suppressed adrenal expression of key rate limiting enzymes in glucocorticoid synthesis under basal conditions. Research to date supports that expression of CYP11A and CYP17 is regulated via interplay between the stimulatory ACTH-cAMP and inhibitory ERK pathways. Thus, these studies will test the hypothesis that LTH adrenals are more sensitive to inhibitory effects of the ACTH precursors POMC and 22kD pro ACTH, interfering with ACTH signaling, resulting in suppressed basal CYP expression. We also hypothesize that the extracellular signal-regulated kinase (ERK) pathway is enhanced in the adrenal cortex of the LTH fetus, leading to enhanced phosphorylation of steroidogenic factor-1 (SF-1) which, in turn, suppresses the expression of the key rate limiting genes regulating glucocorticoid production. Specific Aim 3. To elucidate the intracellular and molecular mechanisms for enhanced glucocorticoid secretion in response to an acute secondary stressor following LTH despite decreased CYP11A and CYP17 expression. These studies are designed to test the hypothesis that the enhanced cortisol output following a secondary stressor in LTH fetuses as compared to controls, is the result of enhanced ACTH stimulation of cAMP and protein kinase A, increasing activation of the steroidogenic acute regulatory protein (STAR). Specific Aim 4. To determine the mechanisms of potential changes in adrenal blood flow during a secondary stressor in LTH animals. These studies will test the hypothesis that: the enhanced cortisol output in LTH fetuses compared to controls following a secondary stressor is the result of enhanced adrenal blood flow and that such changes are mediated, at least in part, by nitric oxide.