Hydrogen Peroxide Decreases pH i in Human Aortic Endothelial Cells by Inhibiting Na + /H + Exchange

Abstract

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 pH i 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 H 2 O 2 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 pH i 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 pH i 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.