Yu Ling, Bao Hui-Fang, Self Julie L, Eaton Douglas C, Helms My N
The Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine, Whitehead Biomedical Research Bldg., 615 Michael St., Atlanta, GA 30322, USA.
Am J Physiol Renal Physiol. 2007 Nov;293(5):F1666-77. doi: 10.1152/ajprenal.00444.2006. Epub 2007 Sep 5.
Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel (ENaC) activity. We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O2*) production in A6 distal nephron cells. Aldosterone (50 nM to 1.5 microM) induced increases in dihydroethidium fluorescence in a dose-dependent manner in confluent A6 epithelial cells. Using single-channel measurements, we showed that sequestering endogenous O2* (with the O2* scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl) significantly decreased ENaC open probability from 0.10 +/- 0.03 to 0.03 +/- 0.01. We also found that increasing endogenous O2* in A6 cells, by applying a superoxide dismutase inhibitor, prevented nitric oxide (NO) inhibition of ENaC activity. ENaC open probability values did not significantly change from control values (0.23 +/- 0.05) after superoxide dismutase and 1.5 microM NO coincubation (0.21 +/- 0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O2* by approximately 30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O2* alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity.
氧自由基在信号转导中起重要作用，并且已被证明会影响上皮钠通道（ENaC）的活性。我们发现，醛固酮作为调节肾脏ENaC活性的主要激素，可增加A6远端肾单位细胞中超氧化物（O2*）的生成。醛固酮（50 nM至1.5 microM）以剂量依赖性方式诱导汇合的A6上皮细胞中二氢乙锭荧光增加。通过单通道测量，我们发现螯合内源性O2*（使用O2清除剂2,2,6,6-四甲基哌啶1-氧基）可使ENaC开放概率从0.10±0.03显著降低至0.03±0.01。我们还发现，通过应用超氧化物歧化酶抑制剂增加A6细胞内源性O2，可防止一氧化氮（NO）对ENaC活性的抑制。超氧化物歧化酶与1.5 microM NO共同孵育后，ENaC开放概率值与对照值（0.23±0.05）相比无显著变化（0.21±0.04）。我们报告，黄嘌呤氧化酶和次黄嘌呤化合物可使O2局部浓度增加约30%；使用这种混合物，在细胞贴附式膜片中可使ENaC通道数量乘以开放概率增加（从0.1增加到0.3）。我们的数据还表明，O2通过一种不依赖于cGMP的机制改变NO活性，因为用ODQ化合物（一种对NO敏感的鸟苷酸环化酶的选择性抑制剂）预处理A6细胞未能阻断2,2,6,6-四甲基哌啶1-氧基对ENaC活性的抑制。
