Glutathione regulation of epithelial sodium channels (ENaC) in alveolar type1 and type 2 cells.

Abstract

We used single channel patch clamp recordings to examine the effect of oxidative stress, via H2O2 accumulation, and alteration of the glutathione redox potential on the activity of ENaC. We found that addition of 20 μM H2O2 significantly increased ENaC open probability (Po) from 0.34+0.16 to 0.470+0.16 in alveolar type 1 (AT1) and 0.17+0.05 to 0.44+0.07 in type 2 (AT2) cells; P<0.05. We also found a linear decrease in ENaC activity as the GSH/GSSG redox potential became less negative (n=19; P<0.05). Sequentially treating cells with H2O2 and then GSH resulted in significant decreases in ENaC activity. Likewise, singular treatment of GSSG significnatly decreased ENaC Po from 0.39 + 0.06 to 0.13 + 0.05 and decreased the number of active channels in the membrane, thus implicating GSSG as an important pro‐injury signaling molecule in edematous lung disorders. Fluorescent maleimide labeling and co‐immunoprecipitation studies show that α‐ENaC subunit is glutathionylated. Real time X‐ray imaging studies show that GSH and GSSG are important determinants of alveolar fluid clearance in vivo. From these results we conclude that ENaC is regulated by changes in the glutathione redox potential. Research was supported by R00HL09226 awarded to MNH; Children's Center for Development in Lung Biology Grant awarded to MNH