title: Rac1-mediated NADPH oxidase release of O2- regulates epithelial sodium channel activity in the alveolar epithelium.
abstract: 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.
authors.affiliation: Department of Pediatrics, Emory University, Atlanta, Georgia, USA.
authors.forename: Yoshizumi
authors.initials: Y
authors.lastname: Takemura
authors.forename: Preston
authors.initials: P
authors.lastname: Goodson
authors.forename: Hui Fang
authors.initials: HF
authors.lastname: Bao
authors.forename: Lucky
authors.initials: L
authors.lastname: Jain
authors.forename: My N
authors.initials: MN
authors.lastname: Helms
mesh_terms: D056809:Alveolar Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D001826:Body Fluids / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D005434:Flow Cytometry; D006706:Homeostasis / Q000187:drug effects; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000634:Aminoquinolines; D053503:Epithelial Sodium Channels; D008562:Membrane Glycoproteins; C487513:NSC 23766; D011743:Pyrimidines; D013481:Superoxides; D004815:Epidermal Growth Factor; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein