PROJECT SUMMARY Reduced nitric oxide (NO) availability is a common pathophysiological feature of many cardiovascular diseases. While NO donors are used, significant issues limit their utility, including the development of nitrate tolerance and endothelial dysfunction. Although intermittent nitrate therapy is common practice, this strategy has limitations and is not considered a solution to nitrate tolerance issues. Extensive intravenous use of sodium nitroprusside is associated with a risk for cyanide poisoning. There is thus a strong need to identify new approaches to promote endogenous NO production. The stromal interaction molecule 1 (STIM1), a Ca2+-sensing protein in the endoplasmic reticulum (ER), is a critical determinant of store-operated Ca2+ entry, which in endothelial cells (ECs) is required for sustained endogenous NO production. Vascular smooth muscle-specific STIM1 deletion blunted hypertension and cardiac hypertrophy caused by angiotensin II. In contrast, endothelial STIM1 is predictably important for vasorelaxation, such that EC-specific STIM1 deletion attenuates endothelium- dependent vasodilation and increases blood pressure. However, no strategy currently exists to directly target endothelial STIM1 for clinical benefit. In this application, we propose to develop a novel vasorelaxing peptide (EFG2) that acts by promoting endogenous NO production and has several unique properties that are of potential clinical benefit. Our peptide is derived from the G protein-coupled estrogen receptor, is designed to be specific for the endothelium, and directly targets STIM1 to trigger endothelial Ca2+ entry without depleting ER Ca2+ store, thereby stimulating endogenous NO production while protecting ER function. Our objectives in this proposal are to delineate the mechanisms of action of EFG2 in endothelial cells, assess its effects on several aspects of EC function, begin testing its potential as a vasorelaxing agent using a model of hypertension, and through these studies, provide training for students in the graduate and medical programs at DMU. Three specific aims are proposed. AIM 1 will delineate the mechanisms of action of EFG2 in endothelial cells, by testing its ability to activate the prototypical ICRAC current and document its target domains on STIM1 that are responsible for its effect to trigger endothelial Ca2+ entry. AIM 2 will assess the effects of EFG2 on several aspects of EC function, by testing the hypotheses that EFG2 promotes eNOS activity, reduces inflammation-induced expression of vascular adhesion molecules and smooth muscle proliferation, but does not cause ER stress. AIM 3 will test the hypothesis that EFG2 reduces blood pressure in hypertensive rats in a NO-dependent fashion. Completion of the studies will introduce a mechanistically novel vasorelaxing peptide with several unique features that are of potential clinical benefit, provide quality student training, and enhance overall research outcomes of Des Moines U...