The risk of myocardial infarction and stroke increases dramatically in women after menopause. Loss of endogenous estrogen associated with menopause contributes to this increased cardiovascular risk. An important, recently established vasoprotective function of estrogen is enhancement of endothelial nitric oxide (NO) secretion which promotes vasodilation and antagonizes thrombosis. NO secreted by the endothelium is regulated through Ca2+ activation of endothelial NO synthase (eNOS). The objective of my research is to elucidate the cellular and molecular mechanisms whereby estrogen and raloxifene, a selective estrogen receptor modulator, enhance release of endothelial vasodilatory factors (EDRF) including NO, prostacyclin (PGI2), and endothelium-dependent hyperpolarizing factors (EDHF). I propose that after estrogen and raloxifene bind to their receptors, they alter gene expression and/or ion transport mechanisms raising [Ca2+]i through enhanced Ca2+ influx and/or decreased Ca2+ extrusion in endothelial cells. Aims: I will test which combination of the plausible target molecules (e.g., eNOS, EDHF) are affected by estrogen and raloxifene treatment. In addition, I will evaluate whether activation of non-genomic receptors by physiologically relevant concentrations of estrogen modulates calcium-dependent eNOS in endothelial cells. The knowledge gained is expected to provide new insight into the role of estrogen and raloxifene therapy in cardiovascular protection. Experiments will be performed in intact valvular endothelium and aortic rings taken from different groups of rats as well as in endothelial cells derived from human umbilical veins (HUVECs). EDRF release will be measured using Bioassay. Bioassay allows us to identify the nature of EDRF and its cellular action(s) in our preparation. The role of intracellular Ca2+ in stimulation of EDRF secretion will be determined using ratiometric fura- 2 fluorimetry. The extent of altered gene expression will be assessed by RT-PCR, Western blots, and immunohistochemistry. Understanding the mechanisms underlying the cardioprotective action of estrogen is expected to contribute significantly to the development of new therapeutic strategies, whereby the beneficial cardioprotective effects of synthetic estrogen analogues might be separated from the undesirable components of estrogen activity. The enhanced insight into these protective mechanisms is expected to eventually also be beneficial for the male population.