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

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 alpha-ENaC subunit degradation, whereas concurrent CSE exposure in the presence of Nox inhibitor, Fulvene 5, resulted in normal proteolytic degradation of alpha-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.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Ethanol alters alveolar fluid balance via Nadph oxidase (NOX) signaling to epithelial sodium channels (ENaC) in the lung.

Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS). However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC) via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH) would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91(phox) subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a 'pro-injury' environment in the alcohol lung

Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells

Amiloride-sensitive epithelial Na(+) channels (ENaC) regulate fluid balance in the alveoli and are regulated by oxidative stress. Since glutathione (GSH) is the predominant antioxidant in the lungs, we proposed that changes in glutathione redox potential (E(h)) would alter cell signaling and have an effect on ENaC open probability (P(o)). In the present study, we used single channel patch-clamp recordings to examine the effect of oxidative stress, via direct application of glutathione disulfide (GSSG), on ENaC activity. We found a linear decrease in ENaC activity as the GSH/GSSG E(h) became less negative (n = 21; P < 0.05). Treatment of 400 μM GSSG to the cell bath significantly decreased ENaC P(o) from 0.39 ± 0.06 to 0.13 ± 0.05 (n = 8; P < 0.05). Likewise, back-filling recording electrodes with 400 μM GSSG reduced ENaC P(o) from 0.32 ± 0.08 to 0.17 ± 0.05 (n = 10; P < 0.05), thus implicating GSSG as an important regulatory factor. Biochemical assays indicated that oxidizing potentials promote S-glutathionylation of ENaC and irreversible oxidation of cysteine residues with N-ethylmaleimide blocked the effects of GSSG on ENaC P(o). Additionally, real-time imaging studies showed that GSSG impairs alveolar fluid clearance in vivo as opposed to GSH, which did not impair clearance. Taken together, these data show that glutathione E(h) is an important determinant of alveolar fluid clearance in vivo

Acute EtOH treatment increases ROS production.

<p>A) Lung tissue preparation was obtained from 3 month old Sprague Dawley rats and treated acutely (for 1 hour) with various concentrations of EtOH. Amplex Red labeling indicates that 0.16% EtOH significantly increases H₂O₂ production. n = 9 observations from 3 animals. DHE labeling (B) and quantification (C) confirms that 0.16% EtOH significantly increases H₂O₂ production in lung slice preparations. n = 6 observations from 3 animals.</p