Meharg J V, McGowan-Jordan J, Charles A, Parmelee J T, Cutaia M V, Rounds S
Pulmonary Section, Providence Veterans Affairs Medical Center, Rhode Island.
Am J Physiol. 1993 Dec;265(6 Pt 1):L613-21. doi: 10.1152/ajplung.1993.265.6.L613.
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.
肺血管内皮的氧化损伤是急性肺损伤发病机制中的一个重要因素。据报道，其他细胞类型的氧化损伤会改变钠钾 - 三磷酸腺苷酶（ATP酶）的功能，该酶在维持细胞离子稳态以及离子跨生物膜转运中起重要作用。我们研究了过氧化氢（H₂O₂，0.001 - 10 mM）或黄嘌呤（X，15.2微克/毫升）加黄嘌呤氧化酶（XO，0.0153单位/毫升）对培养的牛肺动脉内皮细胞（PAECs）钠钾泵活性的影响。我们采用了一种功能测定法，使用⁸⁶RbCl作为钾离子的示踪剂，并将钠钾泵活性表示为哇巴因抑制的钾离子摄取量。我们的结果表明，H₂O₂和X/XO刺激牛PAECs的钠钾泵活性，而过氧化氢酶可阻止这种效应。此外，我们评估了完整内皮单层上[³H]哇巴因结合位点的亲和力、数量和周转率，发现H₂O₂增加了对[³H]哇巴因的亲和力，减少了结合位点的数量，并提高了泵的周转率。在对非裂解浓度的H₂O₂作出反应时，²²Na的流入增加。在用能有效刺激钠钾泵活性的最低剂量（1 mM）的H₂O₂孵育30分钟后，或在用X/XO孵育后，未观察到通过⁵¹Cr释放、贴壁细胞数量和相差显微镜形态评估的细胞损伤。哇巴因对钠钾泵的抑制显著增加了由H₂O₂或X/XO引起的⁵¹Cr释放，这表明钠钾泵活性的增加可能是对H₂O₂产生的细胞改变的一种代偿反应。用H₂O₂孵育会降低细胞ATP含量，与哇巴因共同孵育并不能阻止这种效应。总之，这些结果表明，H₂O₂增加了PAECs的钠钾泵活性，这种效应至少部分是由细胞内[Na]增加和泵周转率提高介导的。泵活性增加可能是内皮细胞扰动的早期标志物。
