The objective of the present application is to elucidate the role of the phosphoinositide (PI) second messenger system in subcellular actions of estrogen (E2) by evaluating two related hypotheses developed form previous investigations on the E2-dependent facilitation of the rodent feminine sexual behavior, lordosis, by alpha1-adrenergic agonist, which are representatives of a large group of lordosis-facilitating agents. The hypotheses are: 1) in E3-primed rates, alpha1-agonists facilitate lordosis by causing an excitation of neurons i the ventromedial hypothalamus (VMH) through an activation of the PI pathway, and 2) EC can induce isozyme of certain key elements of PI system to alter the course of PI turnover, and thereby enable the E2-dependent facilitation to occur. To test hypothesis one, single-unit recording and whole-cell voltage clamp in VMH slices in vitro and parallel behavioral studies will be conducted to see whether activation or inactivation of certain key PI elements will mimic or block the actions of alpha1-agonists in stimulating neuronal activity, as well as facilitating lordosis, and to identify the key PI elements(s) crucial for lordosis facilitation. The effects of E2 on the PI pathway will be investigated by autoradiographic analyses of quantitative and qualitative aspects of the expression of a membrane-bound, key PI element, [3H]cytidine diphosphate diacylglycerol ([3H]CDP-DAG) in VMH neurons, and by Western blot to assess the activation of protein kinase C.
The aim i s to determine whether:A, E2 affects the basal and agonist-evoked production of [3H]CDP-DAG;B, the E2 effect is quantitative, qualitative (changes in patterns of labelled neurons), or both; and C, the E2 effect can be abolished by inactivating upstream PI elements identified to be crucial for lordosis facilitation. The qualitative effect of E2 is indicated by the finding that E2 induces a previously non-existing form of phospholipase C isozyme in the VMH. Estrogen may also induce new isozyme of other key elements of the PI pathway. The proposed investigation will provide, at least, insights into the mechanisms b which E2 alters the PI pathway to regulate cellular function and behavior. Such insights might suggest ways to manipulate the PI pathway to modulate the actions of E2 in regulating neuroendocrine and reproductive function, inducing damage of brain tissue, inducing of cancers, etc., issues of obvious clinical significance.
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