Hydrogen peroxide stimulates sodium-potassium pump activity in cultured pulmonary arterial endothelial cells

Abstract

Oxidant injury to pulmonary vascular endothelium is an important factor in the pathogenesis of acute lung injury. Oxidant injury to other cell types has been reported to alter the function of Na-K-adenosinetriphophatase (ATPase) an enzyme important in maintenance of cellular ionic homeostasis and in transport of ions across biological membranes. We investigated the effect of H2O2 (0.001-10 mM) or xanthine (X) (15.2 micrograms/ml) plus xanthine oxidase (XO) (0.0153 U/ml) on the Na-K pump activity of cultured bovine pulmonary arterial endothelial cells (PAECs). We used a functional assay, using 86RbCl as a tracer for K+ and expressing Na-K pump activity as ouabain-inhibitable K+ uptake. Our results demonstrate that H2O2 and X/XO stimulate Na-K pump activity of bovine PAECs, an effect prevented by catalase. In addition, we assessed the affinity, number, and turnover of [3H]ouabain binding sites on intact endothelial monolayers and found that H2O2 increased affinity to [3H]ouabain, decreased the number of binding sites, and increased the rate of pump turnover. Influx of 22Na increased in response to a nonlytic concentration of H2O2. Cell injury, as assessed by 51Cr release, adherent cell number, and phase-microscopic morphology, was not observed after 30-min incubations with the lowest dose (1 mM) of H2O2 effective in stimulating Na-K pump activity, or after incubation with X/XO. Na-K pump inhibition by ouabain significantly increased the 51Cr release caused by H2O2 or by X/XO, suggesting that the increase in Na-K pump activity may be a compensatory response to the cellular alterations produced by H2O2. Incubation with H2O2 decreased cell ATP content, an effect which was not prevented by coincubation with ouabain. In summary, these results show that H2O2 increases Na-K pump activity of PAECs, an effect mediated, at least in part, by increased intracellular [Na] and by an increased rate of pump turnover. It is possible that the increased pump activity may be an early marker of endothelial cell perturbation.

Insulin: new roles for an ancient hormone

“…Furthermore, in both normal subjects and patients with essential hypertension local insulin infusion in physiological amounts potentiates acetylcholine-induced vasodilatation [60]. Both cell types carry insulin receptors as well as Na þ -K þ -ATPase activity in their plasma membrane [61,62], and can synthesise and release NO. Both cell types carry insulin receptors as well as Na þ -K þ -ATPase activity in their plasma membrane [61,62], and can synthesise and release NO.…”

“…The exact site of this action of insulin, whether the endothelial or the smooth muscle cell, is not known with certainty. Both cell types carry insulin receptors as well as Na þ -K þ -ATPase activity in their plasma membrane [61,62], and can synthesise and release NO. In cultured smooth muscle [63] insulin attenuates agonistinduced increases in intracellular calcium concentrations ([Ca 2þ ] i ) thereby causing relaxation.…”

Insulin as a Vascular Hormone: Implications for the Pathophysiology of Cardiovascular Disease

“…Ex vivo studies treating preconstricted preparations of human coronary, pulmonary and radial arteries with insulin have revealed dose‐dependent endothelium‐dependent relaxation; 54 however, it is not known whether the effect of insulin in these models is to stimulate production of a vasodilator substance, such as NO, from the endothelium or to attenuate the efficacy of the experimental vasoconstrictor. It has also been proposed that the mechanism by which insulin stimulates endothelial NO production may be via its action on endothelial Na + /K + ‐ATPase; 55 , 56 as there are no voltage‐gated Ca 2+ channels in endothelial cells, hyperpolarization leads to an increase in intracellular [Ca 2+ ], 57 which may activate the Ca 2+ ‐dependent enzyme endothelial NOS.…”

Oxidant Stress Stimulates Na/K Pump Activity in Bovine Pulmonary Arterial Endothelial Cells

“…7 We found that short-term exposure (30 min) to reagent H202 and xanthine/xanthine oxidase stimulate pulmonary arterial endothelial Na/K pump activity (Fig l). 8 Hydrogen peroxide decreased intracellular ATP con¬ tent; thus, changes in ATP content did not account for increased pump activity.8 Scatchard analysis indicated that the number of endothelial cell 3H-ouabain binding sites was decreased by H202.8 Cell membrane expression of ax Na/K pump subunit, as assessed by Western blots, was not altered by H202.7 Thus, increased numbers of membrane pump sites did not account for oxidant-induced enhance¬ ment of Na/K pump activity.…”