Oxidant stress decreases Na+/H+ antiport activity in bovine pulmonary artery endothelial cells

Abstract

We determined the effects of oxidant stress by the use of tert-butyl hydroperoxide (t-BOOH) on Na+/H+ exchange in pulmonary artery endothelial cells. Monolayers were exposed to the hydroperoxide, followed by measurement of intracellular pH and the rate of recovery from acidosis by utilizing the pH-sensitive probe 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein. t-BOOH (0.4 mM) decreased the rate of acid recovery after a 2-h exposure without evidence of overt cytotoxicity (51Cr-release assay). Glutathione repletion (N-acetyl-L-cysteine) abolished the effect of the hydroperoxide. Lowering intracellular glutathione with buthionine sulfoximine decreased the acid recovery rate at a dose of t-BOOH (0.04 mM) that was not normally associated with a change in this parameter. Preincubation with vitamin E had no protective effect. Dithiothreitol abolished the effect of the hydroperoxide, suggesting oxidation of protein sulfhydryl groups as a mechanism for the altered kinetics of acid recovery. There was no difference in cell buffering capacity between control and treated monolayers. The findings suggest that the decrease in Na+/H+ antiport activity in this model of oxidant stress represents an early perturbation of membrane function and illustrate the role of the glutathione redox system in maintaining the functional integrity of the Na+/H+ antiport in these cells.