(Scanned from the applicant's description) Nitric oxide (NO), produced by endothelial NO synthase (eNOS), plays a key role in pulmonary vasodilation at birth and in the genesis of persistent pulmonary hypertension of the newborn (PPHN). Studies in fetal sheep have shown that the hormone estrogen, which rises markedly in the fetal blood during parturition, causes pulmonary vasodilation due to nongenomic effects on NO production. Estrogen also reverses the vascular abnormalities in a lamb model of PPHN. We have shown in ovine fetal pulmonary artery endothelial cells (PAEC) that estradiol (E2) cause eNOS activation through novel nongenomic actions of estrogen receptor (ER) a and calcium-mediated signaling. The OBJECTIVE of this proposal is to determine the molecular mechanisms by which E2 causes nongenomic eNOS activation in fetal PAEC.
Four Aims will be addressed in primary and immortalized PAEC, and transfected COS-7 cells. We have new evidence that a subset of ERa is localized to PAEC plasma membrane (PM) where they modulate eNOS activity.
AIM 1 is to determine the mechanisms underlying ERa PM function, testing the hypotheses that E2 causes internalization of PM ERa, that processes needed for genomic ERa function are not required, and that specific ERa domains are involved in PM function. We also have preliminary evidence that fetal PAEC express ERb, and that eNOS stimulation by E2 Is greater after ERb overexpression.
AIM 2 is to determine the role of ERb in eNOS activation, testing the hypotheses that a subset of PM ERb is capable of nongenomic function, and that neither ERa nor dimerization are needed for PM ERb action. In further initial studies, E2 activation of eNOS was blocked by pertussis toxin.
AIM 3 is to determine the role of G proteins in eNOS activation by E2, testing the hypotheses that G proteins are critically involved, that downstream signaling is mediated by Gai, and that E2 causes ERa-Gai interaction on PM. Finally, in recent studies we have detected ERa protein in PAEC caveolae, which compartmentalize signaling molecules on the PM including eNOS, and have found that E2 causes potent, ER-dependent eNOS activation in isolated caveolae membranes.
AIM 4 is to characterize an E2-eNOS signaling module in PAEC caveolae, testing the hypotheses that known signaling proteins are coupled in caveolae, and that a caveolae-associated calcium pool is released by E2 to activate eNOS. Unknown components will be identified by ERa immunoprecipitation and microsequencing and yeast two-hybrid screening. These studies will fill major gaps in our understanding of NO production in the developing lung as well as our knowledge of nongenomic E2 actions in the coronary and uterine circulation and in nonvascular cells. Ultimately, we may be able to take greater therapeutic advantage of the effects of E2 on the pulmonary and also coronary and uterine circulations, thereby optimizing the vascular health of both the fetus and the mother.
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