This is a proposal to investigate the role of EDHF, characterized as metabolites of cytochrome P450 (CYP) in the actions of estrogen, especially when NO synthesis is reduced or deficient. My working hypothesis is that when NO synthesis is impaired, estrogen affords a beneficial effect by potentiation of NO-independent vasodilator responses (primarily CYP-mediated arteriolar hyperpolarization) to shear stress, providing for the improved endothelial function in skeletal muscle arterioles of female compared to male mice. To this end, responses to shear stress in skeletal muscle arterioles of eNOS and estrogen receptor (ER) gene-knockout (KO) mice will be studied.
In Specific aim 1, I aim to provide a general overview of the relationships among gender, CYP/EDHF and shear stress-sensitive mechanisms in the regulation of arteriolar function, by studying flow-induced dilation and the mediators responsible for the responses, changes in smooth muscle membrane potential and CYP activity in response to shear stress.
In Specific aim 2, the protocols similar to those described in Specific aim 1 will be performed in arterioles of ovariectomized (OV) and ovariectomized with estrogen replacement eNOS-KO mice, to examine the essential role of estrogen in the gender specific regulation of arteriolar mediation to shear stress.
In Specific aim 3, effects of the in vitro presence of estrogen on the regulation of arteriolar function will be studied by incubation of NO/estrogen deficient vessels from male and OV eNOS-KO mice with physiologic concentrations of estrogen. The underlying mechanisms by which estrogen evokes EDHF-mediation of arteriolar responses will be clarified by using molecular analyses including western blotting and immunohistochemistry, and also by incubation of the vessels with an ER antagonist, transcriptional inhibitors or PI3-Akt-phosphorylation inhibitors. Finally, in Specific aim 4, ER-KO mice will be used to investigate the interactions among NO, EDHF and estrogen. Also, specific roles of ERs in the mediation of arteriolar function via shear stress-sensitive mechanisms will be examined by comparison of the responses of vessels from wild type controls. The knowledge generated by these studies will unravel the roles of estrogen in the regulation of arteriolar function related specifically to the involvement of EDHF-mediated dilator pathways in adaptation to NO deficiency. ? ?
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