Charles A, Dawicki D D, Oldmixon E, Kuhn C, Cutaia M, Rounds S
Department of Medicine, Brown University School of Medicine, Rhode Island 02908, USA.
J Lab Clin Med. 1997 Aug;130(2):157-68. doi: 10.1016/s0022-2143(97)90092-5.
The Na/K pump is critically important in maintenance of cell homeostasis in the face of injury. Little is known about the regulation of endothelial cell Na/K-pump activity. We previously reported that short-term (30-minute) oxidant-induced endothelial cell perturbation increased Na/K-pump activity in intact monolayers of bovine pulmonary artery endothelial cells (BPAECs). In this study we investigated the mechanism of oxidant-induced increases in endothelial Na/K-pump activity, focusing on short-term modulation of alpha1-pump subunit. By using immunofluorescence microscopy and confocal scanning laser microscopy, we found alpha1 subunit on both apical and basal aspects of BPAECs without polarized distribution. Short-term (30-minute) incubation of PAEC monolayers with H2O2 (1 mmol/L) did not change the relative amounts of alpha1 subunit in membrane fractions, as assessed by immunoblotting. Phosphorylation of the alpha1 subunit also was not affected by H2O2 treatment. Because protein kinases have been reported to alter Na/K-pump activity in several tissues and because H2O2 has been reported to increase PKC activity of endothelial cells, we determined the effects of inhibition and activation of protein kinase C (PKC) on Na/K-pump activity quantitated as ouabain-inhibitable uptake of 86Rb. We also determined the effects of PKC activation and inhibition on H2O2-induced increases in Na/K-pump activity. Inhibitors of PKC increased Na/K-pump activity over a 30-minute period in intact monolayers. Inhibition or depletion of PKC did not prevent H2O2-induced increases in pump activity. These results indicate that PKC is an endogenous regulator of pulmonary artery endothelial cell Na/K-pump activity but that the effects of H2O2 are not mediated by activation of PKC or by changes in the expression or phosphorylation of alpha1 subunit.
面对损伤时，钠钾泵对于维持细胞内稳态至关重要。目前对于内皮细胞钠钾泵活性的调节知之甚少。我们之前报道过，短期（30分钟）氧化剂诱导的内皮细胞扰动会增加完整的牛肺动脉内皮细胞（BPAECs）单层中的钠钾泵活性。在本研究中，我们研究了氧化剂诱导内皮钠钾泵活性增加的机制，重点关注α1泵亚基的短期调节。通过免疫荧光显微镜和共聚焦扫描激光显微镜，我们发现BPAECs的顶端和基底表面均有α1亚基，且无极化分布。用H2O2（1 mmol/L）对PAEC单层进行短期（30分钟）孵育，通过免疫印迹评估，膜组分中α1亚基的相对含量并未改变。H2O2处理也不影响α1亚基的磷酸化。因为已有报道称蛋白激酶会改变多个组织中的钠钾泵活性，且有报道称H2O2会增加内皮细胞的蛋白激酶C（PKC）活性，所以我们测定了抑制和激活蛋白激酶C（PKC）对以哇巴因抑制的86Rb摄取量来定量的钠钾泵活性的影响。我们还测定了PKC激活和抑制对H2O2诱导的钠钾泵活性增加的影响。PKC抑制剂在30分钟内增加了完整单层中的钠钾泵活性。PKC的抑制或耗竭并不能阻止H2O2诱导的泵活性增加。这些结果表明，PKC是肺动脉内皮细胞钠钾泵活性的内源性调节因子，但H2O2的作用并非由PKC的激活或α1亚基的表达或磷酸化变化介导。
