The overall objective of the proposed research is to determine the mechanism for increased ACTH secretion during pregnancy. This increase is necessary for normal maternal and fetal homeostasis, and occurs without symptoms associated with increased steroid action, such as hypertension. We have hypothesized that action of corticosteroids at mineralocorticoid receptors (MR) leads to reduced MR action in pregnancy, and that progesterone mediates this effect as a competitive antagonist at MR. The studies done thus far have supported this hypothesis, showing changes in hippocampal MR consistent with reduced activation and the presence of antagonist activity, during progesterone treatment or pregnancy. The proposed studies will extend these studies to test the following hypotheses: 1) Progesterone treatment in vivo blocks MR effects on 5HT1A expression in hippocampal neurons, resulting in increased plasma ACTH levels. The magnitude of the effects on both 5HT1A expression and ACTH is related to the levels of cortisol relative to progesterone. 2) Progesterone treatment in vivo has selective effects on neurons in hippocampal regions expressing mineralocorticoid receptors (MR) but not progesterone receptors (PR) (including CA1 and dentate gyrus). 3) The increase in ACTH caused by pregnancy or progesterone treatment is caused by an increase in serotonin effects in the brain; 4) The interaction between cortisol and progesterone in hippocampal cells in vitro involves competition at MR, producing opposing effects on 5HT1A mRNA and on the hyperpolarization response to 5HT (serotonin); and 5) Progesterone inhibits activation of MR, resulting in reduced MR binding to specific response elements, including those in the 5HT1A promoter. The first 3 aims will be studied in experiments in sheep. The sheep will be studied either after chronic progesterone treatment, ovariectomy or during pregnancy; MR binding and 5HT1A mRNA, as well as MR, GR and PR mRNA will be measured in hippocampus, hypothalamus and medulla. To test whether changes in 5HT1A occur in the same types of cells in hippocmapus as express MR (but not PR), mRNA will also be determined by in situ hybridization. To test the role of 5HT1A during pregnancy, the ability of a 5HT1A agonist to inhibit the ACTH levels in pregnancy and in progesterone-treated ewes will also be determined. The final 2 aims will be tested in cultures of hippocampal neurons. The effect of progesterone on MR binding, 5HT1A mRNA and electrical responses to a 5HT agonst will be tested; single cell PCR will also be used to determine if cells contain MR, but not PR. The action of progesterone at MR will also be tested by using MR-selective antagonist and agonist, and GR-PR antagonist. Finally, the ability of progesterone to inhibit MR activation will be tested in gel-shift and super-shift assays using specific response elements in the 5HT1A promoter, including a nGRE and the SP-1 site.
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