Acute Effects of Cigarette Smoke Extract on Alveolar Epithelial Sodium Channel Activity and Lung Fluid Clearance

Abstract

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).

Receptor for Advanced Glycation End-Products Regulates Lung Fluid Balance via Protein Kinase C–gp91phox Signaling to Epithelial Sodium Channels

“…This has significant implications for a variety of acute and chronic lung diseases in which lung fluid balance is altered. We and others have evaluated the effects of cigarette smoke on ion transport in airway epithelial cells, and we have reported an increase in ENaC activity when alveolar epithelial cells are challenged with an aqueous form of cigarette smoke (19,39). In addition, studies show an increase in the expression of RAGE protein in the airway and distal lung of smokers and those with COPD (11,12).…”

“…We have previously reported the effects of a variety of agonists on the biophysical properties of ENaC-in particular, the number of inserted channels and the molecular characteristics of these channels (19,28,29,37,38). ENaC channels are classified based on their selectivity for Na Figures 3D and 4C).…”

“…Recently, we have shown that cigarette smoke extract increases ENaC activity in T1 and T2 cells, which resulted in altered lung fluid volumes (19). This implicates ENaC dysfunction in smokingrelated lung diseases, such as COPD.…”

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

“…Cells were treated with GSH or GSSG as indicated for 1 h before detection of reactive oxygen species (ROS). Amplex Red was used to determine H2O2 levels in the cell culture supernatant as previously described (12,16). Isolated rat T2 cells were seeded to tissue culture plates and exposed to mixtures of GSH/GSSG corresponding to specific redox potentials (verified by HPLC).…”

“…However, the role of reactive oxygen species generation and resident antioxidants in edematous lung injury and their interplay with fluid regulation remains unclear. Since the redox state of the lung can be significantly impacted by the environment (12,15,16), it is important to examine the effect of prooxidants and antioxidants in the lung.…”