OXIDANT STRESS ALTERS NA+ PUMP AND NA+-K+-CL- COTRANSPORTER ACTIVITIES IN VASCULAR ENDOTHELIAL-CELLS

We have previously shown that oxidant stress activates Ca2+-dependent K+ efflux in pulmonary vascular endothelial cells. The present study was performed to determine the effect of oxidant stress on Na+ and K+ homeostasis using the radiotracers, Na-22+ and Rb-86+. Cellular ion contents at equilibrium were determined after incubation of cells with tert-butyl hydroperoxide (t-BOOH; 0.4 mM) for various durations. Cell content of Rb-86+ was unchanged through incubation periods of 2 h but was significantly decreased at 3 h, whereas cell content of Na-22+ progressively increased with increasing incubation duration. The effect of t-BOOH on Na+ pump and Na+-K+-Cl- cotransporter activities was examined via measurement of Rb-86+ influx in the absence or presence of ouabain and bumetanide, respectively. Oxidant stress time dependently increased ouabain-sensitive Rb-86+ influx, with little alteration in specific ouabain binding. In contrast, bumetanide-sensitive Rb-86+ influx was decreased by incubation with the oxidant. These findings suggest that the oxidant-induced increase in cellular Na+ content is associated with increased plasmalemmal Na+-K+-adenosinetriphosphatase activity. Furthermore, inward ion movement via the bumetanide-sensitive pathway is decreased, suggesting that oxidant stress inhibits the Na+-K+-Cl- cotransporter.