Background: Proteolytic degradation of epithelial sodium channels (ENaC) assists in regulating net salt and water balance in lung epithelia. Results: H 2 O 2 increases surface expression of ␣-ENaC, transepithelial Na transport, and alveolar fluid clearance via redoxsensitive Nedd8. Conclusion: Redox-sensitive Nedd8 is involved in the ubiquitination of lung ENaC. Significance: Understanding ROS-mediated signaling of lung ENaC is crucial for understanding pulmonary physiology and pathology.

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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 gp91phox 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.

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Cav2.1 channels regulate Ca2+ signaling and excitability of cerebellar Purkinje neurons. These channels undergo a dual feedback regulation by incoming Ca2+ ions, Ca2+-dependent facilitation and inactivation. Endogenous Ca2+-buffering proteins, such as parvalbumin (PV) and calbindin D-28k (CB), are highly expressed in Purkinje neurons and therefore may influence Cav2.1 regulation by Ca2+. To test this, we compared Cav2.1 properties in dissociated Purkinje neurons from wild-type (WT) mice and those lacking both PV and CB (PV/CB−/−). Unexpectedly, P-type currents in WT and PV/CB−/− neurons differed in a way that was inconsistent with a role of PV and CB in acute modulation of Ca2+ feedback to Cav2.1. Cav2.1 currents in PV/CB−/− neurons exhibited increased voltage-dependent inactivation, which could be traced to decreased expression of the auxiliary Cavβ2a subunit compared with WT neurons. Although Cav2.1 channels are required for normal pacemaking of Purkinje neurons, spontaneous action potentials were not different in WT and PV/CB−/− neurons. Increased inactivation due to molecular switching of Cav2.1 β-subunits may preserve normal activity-dependent Ca2+ signals in the absence of Ca2+-buffering proteins in PV/CB−/− Purkinje neurons.

The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91 phox signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 6 3.03 mg versus 2.48 6 0.53 mg), lower sRAGE (1,205 6 292 pg/ml versus 1,910 6 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 6 14 ml versus 133 6 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 6 0.08 to 0.83 6 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 6 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 6 0.05 to 0.53 6 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 6 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47 phox and increased gp91 phox -dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91 phox abrogated the response.Keywords: acute respiratory distress syndrome; chronic obstructive pulmonary disease; pulmonary edema; alveolar microenvironment; lung injury Clinical RelevanceReceptor for advanced glycation end-products (RAGE) plays a critical role in regulating inflammation in the lung and may be a therapeutic target in the treatment of acute and chronic lung diseases. Herein we describe the signal transduction pathway through which RAGE regulates the epithelial sodium channel.

Cigarette smoke contains high levels of reactive species. Moreover, cigarette smoke can induce cellular production of oxidants. The purpose of this study was to determine the effect of cigarette smoke extract (CSE)-derived oxidants on epithelial sodium channel (ENaC) activity in alveolar type 1 (T1) and type 2 (T2) cells and to measure corresponding rates of fluid clearance in mice receiving a tracheal instillation of CSE. Single-channel patch clamp analysis of T1 and T2 cells demonstrate that CSE exposure increases ENaC activity (NPo), measured as the product of the number of channels (N) and a channels open probability (Po), from 0.17 6 0.07 to 0.34 6 0.10 (n ¼ 9; P ¼ 0.04) in T1 cells. In T2 cells, CSE increased NPo from 0.08 6 0.03 to 0.35 6 0.10 (n ¼ 9; P ¼ 0.02). In both cell types, addition of tetramethylpiperidine and glutathione attenuated CSE-induced increases in ENaC NPo. Biotinylation and cycloheximide chase assays indicate that CSE-derived ROS increases channel activity, in part, by maintaining cell surface expression of the a-ENaC subunit. In vivo studies show that tracheal instillation of CSE promoted alveolar fluid clearance after 105 minutes compared with vehicle control (n ¼ 10/ group; P , 0.05).

Downs CA, Kreiner L, Zhao XM, Trac P, Johnson NM, Hansen JM, Brown LA, Helms MN. Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells.