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

From the Department of Medicine (Q.H., Y.X., J.L.Z., R.C.Z.), Division of Cardiology, Johns Hopkins Bayview Medical Center, Johns Hopkins University School of Medicine, Baltimore, Md, and the Département d'Anesthésie-Réanimation Hôpital Lariboisière (S.C.), Paris, France.

Correspondence to Roy C. Ziegelstein, MD, Department of Medicine, Division of Cardiology, Johns Hopkins Bayview Medical Center, 4940 Eastern Ave, Baltimore, MD 21224-2780. E-mail rziegels{at}welchlink.welch.jhu.edu

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 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 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.

Key Words: pH_i • endothelium • Na⁺/H⁺ exchange • free radical

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