Study of the O-GlcNAc/eNOS pathway in the reduced vascular reactivity to phenylephrine observed in late pregnancy in spontaneously hypertensive rats (SHR)

Abstract

The hypertensive syndromes are the principal causes of maternal mortality. We are studing the physiological effect of pregnancy on blood pressure, for better understanding of the changes observed in hypertension associated with pregnancy. Studies demonstrated that in normotensive and hypertensive rats (SHR) there is a significant reduction in blood pressure in the end of pregnancy, which would be directly associated with reduction in sympathetic activity in mesenteric perivascular beds or other vessels. Furthermore, reduced pressor responses to vasoconstrictors such as phenylephrine, vasopressin, serotonin and endothelin were observed in mesenteric beds of normotensive or hypertensive rats (SHR) at the end of pregnancy. These changes have been attributed to a major participation of the endothelium derived relaxing factors (EDFRs) especially to nitric oxide (NO) on the vascular smooth muscle. Some studies found that greater synthesis of NO occur in vessel during pregnancy. Recently, we demonstrated an increased activity/phosphorylation of eNOS by PI3-K/Akt pathway in aortas of hypertensive pregnant (Zancheta et al., 2015). However, it has been suggested that O-GlcNAc of the eNOS is involved in the reduction of eNOS activity. Our hypothesis is that O-GlcNAc of eNOS is reduced in aortas of pregnant rats. The objective of this study is evaluate the participation of O-GlcNAc of eNOS in endothelial modulation of reduced aorta reactivity to phenylephrine, and possible changes in the expression of proteins involved in O-GlcNAc of eNOS through biomolecular assays (Western Blotting).To achieve these objectives will be performed in vitro experiments to evaluate the effect of Pugnac, inhibitor of O-GlcNAc, in the aorta of normotensive and hypertensive pregnant rats (SHR) to phenylephrine (PE, 1nM to 10 mM). We will performe biomolecular assays (Western Blott) to evaluate the expression of proteins involved in the O-GlcNAc in aortas from normotensive and hypertensive pregnant rats. Primary antibodies are used: O-GlcNAc (sc-59623, Santa Cruz) diluted 1: 1000 and O-GlcNAc transferase (sc-74547, Santa Cruz) diluted 1: 1000. The expression of ²-actin (1: 8000) will be used for normalization of the results. The results will be compared between aortas from normotensive and hypertensive, by two-way analysis of variance (ANOVA) followed by Tukey test. The differences between the results will be considered significant when p <0.05.(AU)