Hydrogen peroxide decreases pH(i) in human aortic endothelial cells by inhibiting Na+/H+ exchange

Abstract

Postischemic endothelial dysfunction may occur as a result of the effects of endogenous oxidants like hydrogen peroxide. Since endothelium- dependent vasodilator function may be affected by pH(i), the effect of hydrogen peroxide on endothelial pH(i) was examined. Hydrogen peroxide (100 μmol/L for 10 minutes) decreased phi from 7.24 ± 0.01 to 7.02 ± 0.02 and inhibited recovery from an ammonium chloride-induced intracellular acid load in carboxy SNARF 1 (c-SNARF 1)-loaded human aortic endothelial cells in bicarbonate-free solution. Prior inhibition of Na+/H+ exchange with 5-(N- ethyl-N-isopropyl)amiloride (10 μmol/L), by removal of extracellular Na+, or by glycolytic inhibition with iodoacetic acid blocked the subsequent effect of hydrogen peroxide on pH(i). A 2-minute exposure to 100 μmol/L H2O2 decreased intracellular ATP levels by ≃40%; this was prevented by 3- aminobenzamide and nicotinamide (1 mmol/L each), inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase. Both 3-aminobenzamide and nicotinamide significantly inhibited the hydrogen peroxide-induced intracellular acidification and the effect of hydrogen peroxide on recovery from an intracellular acid load. Hydrogen peroxide decreases phi in human endothelial cells by inhibiting Na+/H+ exchange. This appears to be mediated by activation of the DNA repair enzyme poly(ADP-ribose) polymerase and subsequent depletion of intracellular ATP. Since a decrease in phi in this range may alter the activity of NO synthase or affect the synthesis of vasodilator prostaglandins, the effect of hydrogen peroxide on the endothelial Na+/H+ exchanger may be important in the pathogenesis of postischemic endothelial dysfunction.