Evaluation of post-translational modification mechanisms of endothelial Nitric Oxide Synthase (eNOS) associated with the nitric oxide bioavailability in Spontaneously Hypertensive Rats (SHR) in the end of pregnancy

Abstract

Previous studies from our laboratory suggest that pregnancy is a favorable physiological condition to reduced blood pressure, vascular reactivity to alpha1-adrenergic stimulation and to reestablishment of the endothelial function in Spontaneously Hypertensive Rats (SHR). In pregnant SHR, the increased production and bioavailability of NO, resulting from increased phosphorylation of the PI3K-Akt- eNOS pathway, reestablished endothelium-eNOS-dependent modulation on aortas reactivity to phenylephrine (PE). The objective of this study is to analyze possible alterations in post-translational modification mechanisms associated to increased eNOS activity in blood vessel of pregnant SHR. The glycosylation of eNOS by hexosamine pathway and protein-protein interactions associated to eNOS phosphorylation will be investigate in blood vessel of normotensive pregnant rats and pregnant SHR. Western Blot Assays will performed to analyze following proteins expression: O-GlcNAc transferase, O-GlcNAc, eNOS, p-eNOS, CAV-1, P-CAV-1, Hsp90, p-Hsp90, calmodulin (CaM) as well as their interactions by co-immunoprecipitation method. In functional studies, we will evaluate the participation of pos-translational mechanisms on vascular hiporeactivity to phenilefrine associated to pregnancy. We will evaluate the participation of OGA, by inhibition with PUGNAc (100 mmol/L) and Hsp90, by inhibition with Geldamycin (1 mmol/L) or radicicol (20 nmol/L). The results will be analyzed by ANOVA multivariance test, two-way, and the significative differences will be considered when p <0.05. (AU)