Functional Effect of the p22 phox −930 A/G Polymorphism on p22 phox Expression and NADPH Oxidase Activity in Hypertension

Abstract

Oxidative stress induced by superoxide is implicated in hypertension. NADPH oxidase is the main source of superoxide in phagocytic and vascular cells, and the p22 phox subunit is involved in NADPH oxidase activation. Recently we reported an association of −930 A/G polymorphism in the human p22 phox gene promoter with hypertension. This study was designed to investigate the functional role of this polymorphism in hypertension. We thus investigated the relationships between the −930 A/G polymorphism and p22 phox expression and NADPH oxidase–mediated superoxide production in phagocytic cells from 70 patients with essential hypertension and 70 normotensive controls. Genotyping of the polymorphism was performed by restriction fragment length polymorphism. NADPH oxidase activity was determined by chemiluminescence assays, and p22 phox mRNA and protein expression was measured by Northern and Western blotting, respectively. Compared with hypertensive subjects with the AA/AG genotype, hypertensive subjects with the GG genotype exhibited increased ( P <0.05) phagocytic p22 phox mRNA (1.26±0.06 arbitrary unit [AU] versus 0.99±0.03 AU) and protein levels (0.58±0.05 AU versus 0.34±0.04 AU) and enhanced NADPH oxidase activity (1998±181 counts/s versus 1322±112 counts/s). No differences in these parameters were observed among genotypes in normotensive cells. Transfection experiments on vascular smooth muscle cells showed that the A -to- G substitution of this polymorphism produced an increased reporter gene expression in hypertensive cells. Nitric oxide production, as assessed by measurement of serum nitric oxide metabolites, was lower in GG hypertensive subjects than in AA/AG hypertensive subjects. In conclusion, these results suggest that hypertensive subjects carrying the GG genotype of the p22 phox −930 A/G polymorphism are highly exposed to NADPH oxidase-mediated oxidative stress.