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Cigarette smoke extract is a Nox agonist and regulates ENaC in alveolar type 2 cells

1 College of Nursing, The University of Arizona, 1305 N. Martin, Tucson, AZ, USA
2 Division of Translational and Regenerative Medicine, College of Medicine, The University of Arizona, 1501 N. Campbell Ave., Tucson, AZ, USA
3 Department of Physiology and Functional Genomics, College of Medicine, University of Florida, 135 Center Drive, Gainesville, FL, USA
4 Department of Internal Medicine, Division of Pulmonary Medicine, School of Medicine, University of Utah, Salt Lake City, UT, USA

There is considerable evidence that cigarette smoking is the primary etiology of chronic obstructive pulmonary disease (COPD), and that oxidative stress occurs in COPD with the family of tissue nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes playing a significant role in lung pathogenesis. The purpose of this study was to determine the effects of cigarette smoke extract (CSE) on Nox signaling to epithelial sodium channels (ENaCs). Pre-treatment with diphenyleneiodonium (DPI), a pan-Nox inhibitor, prevented stimulatory effects of CSE on ENaC activity; open probability (Po) changed from 0.36 ± 0.09 to 0.11 ± 0.02; n = 10, p = 0.01 following CSE and DPI exposure. Likewise, Fulvene-5 (which inhibits Nox2 and Nox4 isoforms) decreased the number of ENaC per patch (from 2.75 ± 0.25 to 1 ± 0.5, n = 9, p = 0.002) and open probability (0.18 ± 0.08 to 0.02 ± 0.08, p = 0.04). Cycloheximide chase assays show that CSE exposure prevented α-ENaC subunit degradation, whereas concurrent CSE exposure in the presence of Nox inhibitor, Fulvene 5, resulted in normal proteolytic degradation of α-ENaC protein in primary isolated lung cells. In vivo, co-instillation of CSE and Nox inhibitor promoted alveolar flooding in C57Bl6 mice compared to accelerated rates of fluid clearance observed in CSE alone instilled lungs. Real-time PCR indicates that mRNA levels of Nox2 were unaffected by CSE treatment while Nox4 transcript levels significantly increased 3.5 fold in response to CSE. Data indicate that CSE is an agonist of Nox4 enzymatic activity, and that CSE-mediated Nox4 plays an important role in altering lung ENaC activity.
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Keywords Nox2; Nox4; COPD; lung injury; cell signaling

Citation: Charles A. Downs, Abdel A. Alli, Nicholle M. Johnson, My N. Helms. Cigarette smoke extract is a Nox agonist and regulates ENaC in alveolar type 2 cells. AIMS Molecular Science, 2016, 3(3): 439-453. doi: 10.3934/molsci.2016.3.439

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Copyright Info: 2016, My N. Helms, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (http://creativecommons.org/licenses/by/4.0)

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