Department of Pediatrics, Emory University, Atlanta, Georgia, USA.
Am J Physiol Lung Cell Mol Physiol. 2010 Apr;298(4):L509-20. doi: 10.1152/ajplung.00230.2009. Epub 2010 Jan 22.
We examine whether alveolar cells can control release of O(2)(-) through regulated NADPH oxidase (NOX) 2 (NOX2) activity to maintain lung fluid homeostasis. Using FACS to purify alveolar epithelial cells, we show that type 1 cells robustly express each of the critical NOX components that catalyze the production of O(2)(-) (NOX2 or gp91(phox), p22(phox), p67(phox), p47(phox), and p40(phox) subunits) as well as Rac1 at substantially higher levels than type 2 cells. Immunohistochemical labeling of lung tissue shows that Rac1 expression is cytoplasmic and resides near the apical surface of type 1 cells, whereas NOX2 coimmunoprecipitates with epithelial sodium channel (ENaC). Since Rac1 is a known regulator of NOX2, and hence O(2)(-) release, we tested whether inhibition or activation of Rac1 influenced ENaC activity. Indeed, 1 microM NSC23766 inhibition of Rac1 decreased O(2)(-) output in lung cells and significantly decreased ENaC activity from 0.87 +/- 0.16 to 0.52 +/- 0.16 [mean number of channels (N) and single-channel open probability (P(o)) (NP(o)) +/- SE, n = 6; P < 0.05] in type 2 cells. NSC23766 (10 microM) decreased ENaC NP(o) from 1.16 +/- 0.27 to 0.38 +/- 0.10 (n = 6 in type 1 cells). Conversely, 10 ng/ml EGF (a known stimulator of both Rac1 and O(2)(-) release) increased ENaC NP(o) values in both type 1 and 2 cells. NP(o) values increased from 0.48 +/- 0.21 to 0.91 +/- 0.28 in type 2 cells (P < 0.05; n = 10). In type 1 cells, ENaC activity also significantly increased from 0.40 +/- 0.15 to 0.60 +/- 0.23 following EGF treatment (n = 7). Sequestering O(2)(-) using 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) compound prevented EGF activation of ENaC in both type 1 and 2 cells. In conclusion, we report that Rac1-mediated NOX2 activity is an important component in O(2)(-) regulation of ENaC.
我们研究肺泡细胞是否可以通过调节 NADPH 氧化酶 (NOX) 2 (NOX2) 活性来控制 O(2)(-)的释放，以维持肺液的动态平衡。我们使用 FACS 纯化肺泡上皮细胞，结果显示，I 型细胞强烈表达催化 O(2)(-)生成的关键 NOX 成分（NOX2 或 gp91(phox)、p22(phox)、p67(phox)、p47(phox)和 p40(phox)亚基）以及 Rac1，其表达水平明显高于 II 型细胞。对肺组织的免疫组织化学标记表明，Rac1 表达为细胞质，位于 I 型细胞的顶表面附近，而 NOX2 与上皮钠通道 (ENaC) 共免疫沉淀。由于 Rac1 是已知的 NOX2 的调节剂，因此也是 O(2)(-)释放的调节剂，我们测试了 Rac1 的抑制或激活是否会影响 ENaC 活性。事实上，1 microM NSC23766 抑制 Rac1 会降低肺细胞中的 O(2)(-)输出，并显著降低 II 型细胞中的 ENaC 活性，从 0.87 +/- 0.16 降至 0.52 +/- 0.16 [平均通道数 (N)和单通道开放概率 (P(o)) (NP(o)) +/- SE，n = 6；P < 0.05]。NSC23766（10 microM）使 I 型细胞中的 ENaC NP(o)从 1.16 +/- 0.27 降至 0.38 +/- 0.10（n = 6）。相反，10 ng/ml EGF（已知同时刺激 Rac1 和 O(2)(-)释放）增加了 I 型和 II 型细胞中的 ENaC NP(o)值。在 II 型细胞中，NP(o)值从 0.48 +/- 0.21 增加到 0.91 +/- 0.28（P < 0.05；n = 10）。在 I 型细胞中，EGF 处理后 ENaC 活性也显著从 0.40 +/- 0.15 增加到 0.60 +/- 0.23（n = 7）。使用 2,2,6,6-四甲基哌啶-N-氧自由基 (TEMPO) 化合物螯合 O(2)(-)可防止 EGF 激活 I 型和 II 型细胞中的 ENaC。总之，我们报告 Rac1 介导的 NOX2 活性是 O(2)(-)调节 ENaC 的重要组成部分。
