Nebivolol inhibits superoxide formation by NADPH oxidase and endothelial dysfunction in angiotensin II-treated rats

Nebivolol is a beta(1)-receptor antagonist with vasodilator and antioxidant properties. Because the vascular NADPH Oxidase is an important superoxide source, we studied the effect of nebivolol on endothelial function and NADPH Oxidase activity and expression in the well-characterized model of angiotensin II-induced hypertension. Angiotensin II infusion (1 mg/kg per day for 7 days) caused endothelial dysfunction in male Wistar rats and increased vascular superoxide as detected by lucigenin-derived chemiluminescence, as well as dihydroethidine staining. Vascular NADPH Oxidase activity, as well as expression at the mRNA and protein level, were markedly upregulated, as well as NOS III uncoupled, as evidenced by NO Synthase III inhibitor experiments and dihydroethidine staining and by markedly decreased hemoglobin-NO concentrations. Treatment with the beta-receptor blocker nebivolol but not metoprolol (10 mg/kg per day for each drug) normalized endothelial function, reduced superoxide formation, increased NO bioavailability, and inhibited upregulation of the activity and expression of the vascular NADPH Oxidase, as well as membrane association of NADPH Oxidase subunits (Rac1 and p67(phox)). In addition, NOS III uncoupling was prevented. In vitro treatment with nebivolol but not atenolol or metoprolol induced a dissociation of p67(phox) and Rac1, as well as an inhibition of NADPH Oxidase activity assessed in heart membranes from angiotensin II-infused Animals, as well as in homogenates of NOX1 and cytosolic subunit-transfected and phorbol ester-stimulated HEK293 cells. These findings indicate that nebivolol interferes with the assembly of NADPH Oxidase. Thus, inhibitory effects of this beta-blocker on vascular NADPH Oxidase may explain, at least in part, its beneficial effect on endothelial function in angiotensin II-induced hypertension.