PROJECT SUMMARY ABSTRACT Preterm Birth (PTB) is a global problem and preemies are at risk for major disability. At the present time, 10-14% of US births are premature depending on maternal ethnicity. African Americans are disproportionally affected; a recognized health disparity. The regulation of birth timing is unknown and PTB cannot be prevented. Tocolytics currently employed have unwanted effects on mother and fetus limiting dosing and none are FDA approved. Tocolytic approaches to prevent PTB must be based on myometrial relaxation mechanisms known to be unique. This is an urgent and unmet need exacerbated because the regulation of uterine smooth muscle relaxation is disparate from that of other smooth muscles. Activation of cAMP-protein kinase (PKA) by the β2 agonist terbutaline, or blockade of calcium entry by nifedipine is unable to provide meaningful tocolysis, and while nitric oxide relaxes term myometrium, it does so in a cGMP- independent manner. Moreover, nitric oxide-mediated relaxation is blunted in spontaneous preterm labor, a dysfunction correlated with over-expression of S-nitrosoglutathione reductase. There is an unmet need to identify the pathways driving quiescence during human gestation to develop new effective tocolytics. Our central hypothesis is that Pezo1 stretch-activated channels exist in both the microvascular endothelium and the uterine smooth muscle and both receptor pools coordinate gestational relaxation via stretch. Piezo1- mediated nitric oxide generation in the endothelial cell compartment S-nitrosates the K+ channel TREK-1 in the myocyte compartment. A Piezo1 agonist together with blockade of nitric oxide metabolism by inhibition of S- nitrosoglutathione reductase to promote nitrosation, together with small molecule activation of TREK-1 will result in additive potassium efflux that will reverse the dysfunctional relaxation of preterm tissues to NO donors. This supplement project is proposed under the Nevada INBRE grant P20GM103440. The goal of the Nevada INBRE is to enhance biomedical research infrastructure, research competitiveness, training and workforce development throughout the state of Nevada. The specific focus area of the INBRE is growth and differentiation. As a participating NIH IC, the NICHD seeks to fund the development of novel and effective pharmacotherapeutic strategies to improve clinical outcomes for women and mothers. The goals of the Nevada INBRE and the NICHD come together in the setting of fetal development in utero interrupted by spontaneous preterm labor leading to preterm birth of an underdeveloped fetus. The research project described in this application is part of the development of a future NIH COBRE Center program in women’s reproductive health research supported by the Core services of the INBRE at the University of Nevada School of Medicine. Successfully exploring the research described in this supplement request will provide data to support a full NIH R01 application to provide p...