Rac1-mediated NADPH oxidase release of O2- regulates epithelial sodium channel activity in the alveolar epithelium

Takemura Y, Goodson P, Bao HF, Jain L, Helms MN. Rac1-mediated NADPH oxidase release of O-2(-) regulates epithelial sodium channel activity in the alveolar epithelium. Am J Physiol Lung Cell Mol Physiol 298: L509-L520, 2010. First published January 22, 2010; doi:10.1152/ajplung.00230.2009.-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 mu M 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) (NPo) +/- SE, n = 6; P < 0.05] in type 2 cells. NSC23766 ( 10 mu M) decreased ENaC NPo 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 NPo values in both type 1 and 2 cells. NPo 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.