ER stress induction increases NADPH oxidase and reduces eNOS activity in endothelial cells

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Cardiovascular diseases are associated with endoplasmic reticulum (ER) stress. We have demonstrated that induction of ER stress in C57BL/6J, by the injection of tunicamycin, impairs vascular endothelium‐dependent relaxation (unpublished data).In this study, we aimed to determine the mechanism by which ER stress reduces nitric oxide synthase activity.We incubated mice primary coronary arterioles endothelial cells with tunicamycin (1 ug/mL) for 6 h in the presence or absence of two ER stress inhibitors (Tudca, 500 ug/mL and PBA, 10 mM). Tunycamicin increased the expression of phosphorylated PERK and eIF2alpha, CHOP and ATF6, which were reduced with ER stress inhibitors. The induction of ER stress enhanced NADPH oxidase activity and Nox1/Nox4 mRNA levels associated with an increase of reactive oxygen species, which were reduced by ER stress inhibitors. Phosphorylated eNOS expression was reduced with tunicamycin and rescued by ER stress inhibition. The transfection of endothelial cells with a reporter plasmid containing the eNOS promoter revealed that ER stress induction reduced eNOS promoter activity by 50%, which was rescued by ER stress inhibition.ConclusionER stress enhances oxidative stress and reduces eNOS promoter activity and phosphorylated eNOS, which are responsible for endothelial dysfunction.