ROLE AND REGULATION OF NITRIC OXIDE IN THE UTERUS

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Nitric oxide, the chemical identity for endothelium derived relaxing factor (EDRF), is a biological messenger molecule with potent smooth muscle relaxant activity. Due to its extremely short half life, the effects of nitric oxide are auto- and paracrine in nature, and are mediated through the activation of a soluble guanylyl cyclase and the generation of 3',5'-cyclic guanosine monophosphate (cGMP) and several other mechanisms that are mediated directly by NO, i.e., opening of K/Ca channels. Nitric oxide synthase has been shown to be present in several other tissues including the uterus. Uterine nitric oxide synthase activity is increased during pregnancy, and decreases prior to parturition. This proposal addresses the role of nitric oxide in the maintenance of uterine quiescence during pregnancy. We hypothesize that nitric oxide is produced in the uterus (endometrium and myometrium), acts locally in myometrium, and plays an important physiological role in the maintenance of uterine quiescence by decreasing intracellular calcium through cGMP-dependent and cGMP-independent mechanisms. We further hypothesize that nitric oxide synthase activity and mass are increased during pregnancy by estrogen and progesterone. We will test these hypotheses through the following specific aims. 1) Specific Aim I: Study the role of Nitric Oxide and the mechanisms by which it modulates myometrial contractility; 2) Specific Aim II: Characterize nitric oxide synthase activity in the pregnant sheep uterus; and, 3) Special Aim III: Investigate the factors that regulate the expression and activity of myometrial and endometrial nitric oxide synthase in the uterus. Sheep will be used to study the role of endogenous nitric oxide in the regulation of myometrial contractility during pregnancy. We will study the effects of changing endogenous nitric oxide production on spontaneous and oxytocin induced myometrial contractility. Concentrations of cGMP in plasma obtained during the in vivo contractility studies, and tissue and incubation media of in vitro studies, will be used to determine if nitric oxide effects on the uterus are associated with changes in this second messenger. The relative contribution of cGMP to the effects of NO will be determined by studying the effects of cGMP analogs and NO on in vitro relaxation and intracellular calcium. We will study the roles of estrogen and progesterone as regulators of nitric oxide synthase by measuring specific enzyme activity using 14C-labelled substrate, and immunoblotting in uterine tissues. Knowledge gained in the understanding of the cellular events associated with NO actions on myometrium, and the regulation of NOS activity and enzyme mass, will provide new insight on the mechanisms that ensure myometrial quiescence until the time of parturition.