The ovarian hormones estradiol (E) and progesterone (P) act in the hypothalamus and preoptic area (HPOA) to stimulate the preovulatory release of pituitary gonadotropins and coordinate the expression of reproductive mating behavior, namely lordosis by the female through the action of norepinephrine (NE). The goal of the proposed research is to examine the molecular mechanisms by which estradiol and progesterone modulate signal transduction on alpha1-and beta-adrenoceptors in the HPOA and to relate these to the expression of reproductive behavior.
Specific Aim 1 will test the hypothesis that estradiol elevates alpha1B-adrenoceptors in populations of HPOA neurons that express ER. Immunocytochemical approaches will be employed to determine: 1) whether E increases alpha1B-adrenoceptor protein in regions of the HPOA that also express ER; 2) whether A1B - adrenoceptors and ER are colocalized in some or all of these neurons; and 3) whether hypothalamic neurons expressing alpha1B-adrenoceptors project to the midbrain central gray (MCG).
Specific aim 2 will test the hypothesis that in the HPOA of E-primed females P will switch alpha1 adrenoceptor signaling from activation of phospholipase C to calcium- dependent activation of the nitric oxide (NO)/soluble guanylyl cyclase pathway. Support for this hypothesis is derived from the observation that alpha1-adrenergic activation of NO synthesis influences the preovulatory release of LH and because expression of reproductive behavior in E+P treated female rats is inhibited by NO synthetase inhibitors and inhibitors of soluble guanylyl cyclase.
Specific aim 3 will test the hypothesis that E increases the expression of one or more protein kinase C (PKC) isoenzymes in the HPOA. PKC is a major downstream mediator of alpha1-adrenergic signal transduction; hence, induction of PKC could further amplify alpha1-adrenergic signaling in the HPOA. Experiments will utilize assays of PKC catalytic activity as well as phorbol ester binding and Western Blots to identify the separate isoenzymes.
Specific Aim 4 will test the hypothesis that E regulates molecules involved in adrenergic receptor-G protein coupling. Molecular biological and immunological methods will be used to determine the effects of E on: 1) mRNA and protein levels of b-adrenergic receptor kinase 1 and 2 (b-ARK1, and b-ARK2) and 2) the mRNA and protein levels of b-arrestin1 and b-arrestin2. These are significant questions because b-ARKs and b-arrestins impede the interactions of b-adrenergic, a2-adrenergic and u-opioid receptors with G proteins, and they find that E treatment decreases the function of all three of these receptors in the HPOA with measurably downregulating the receptors.
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