Jv. Meharg et al., HYDROGEN-PEROXIDE STIMULATES SODIUM-POTASSIUM PUMP ACTIVITY IN CULTURED PULMONARY ARTERIAL ENDOTHELIAL-CELLS, The American journal of physiology, 265(6), 1993, pp. 120000613-120000621
Oxidant injury to pulmonary vascular endothelium is an important facto
r in the pathogenesis of acute lung injury. Oxidant injury to other ce
ll types has been reported to alter the function of Na-K-adenosinetrip
hosphatase (ATPase) an enzyme important in maintenance of cellular ion
ic homeostasis and in transport of ions across biological membranes. W
e investigated the effect of H2O2 (0.001-10 mM) or xanthine (X) (15.2
mug/ml) plus xanthine oxidase (XO) (0.0153 U/ml) on the Na-K pump acti
vity of cultured bovine pulmonary arterial endothelial cells (PAECs).
We used a functional assay, using (RbCl)-Rb-86 as a tracer for K+ and
expressing Na-K pump activity as ouabain-inhibitable K+ uptake. Our re
sults demonstrate that H2O2 and X/XO stimulate Na-K pump activity of b
ovine PAECs, an effect prevented by catalase. In addition, we assessed
the affinity, number, and turnover of [H-3]ouabain binding sites on i
ntact endothelial monolayers and found that H2O2 increased affinity to
[H-3]ouabain, decreased the number of binding sites, and increased th
e rate of pump turnover. Influx of Na-22 increased in response to a no
nlytic concentration of H2O2. Cell injury, as assessed by Cr-51 releas
e, adherent cell number, and phase-microscopic morphology, was not obs
erved after 30-min incubations with the lowest dose (1 mM) of H2O2 eff
ective in stimulating Na-K pump activity, or after incubation with X/X
O. Na-K pump inhibition by ouabain significantly increased the Cr-51 r
elease caused by H2O2 or by X/XO, suggesting that the increase in Na-K
pump activity may be a compensatory response to the cellular alterati
ons produced by H2O2. Incubation with H2O2 decreased cell ATP content,
an effect which was not prevented by coincubation with ouabain. In su
mmary, these results show that H2O2 increases Na-K pump activity of PA
ECs, an effect mediated, at least in part, by increased intracellular
[Na] and by an increased rate of pump turnover. It is possible that th
e increased pump activity may be an early marker of endothelial cell p
erturbation.