Preventing endotoxin-stimulated alveolar macrophages from decreasing epithelium Na+ channel (ENaC) mRNA levels and activity

The acute respiratory distress syndrome is characterized by impairment of t he alveolar-capillary barrier. Our laboratory has shown that distal lung ep ithelial cell (DLEC) amiloride-sensitive Na+ transport is impaired by in vi tro coculture with endotoxin (lipopolysaccharide)-stimulated alveolar macro phages (AM) through an L-areinine-dependent mechanism. To investigate the e ffect of this model on mRNA levels of the rat epithelial Na+ channel mature fetal rat DLEC monolayers were incubated for 16 h with rat AM (1 X 10(7)) and lipopolysaccharide (10 mu g/mL), or the cell-free supernatant of lipopo lysaccharide-stimulated rat AM. Such exposure resulted in a profound decrea se in mRNA expression for all subunits (alpha, beta, and gamma) of the mt e pithelial Na+ channel, without affecting 18S RNA levels. This effect was pr evented by the antioxidant N-acetylcysteine. In separate experiments, confl uent DLEC monolayers were: exposed to lipopolysaccharide-stimulated AM supe rnatant for 16 h with or without N-acetylcysteine and DTT and studied in Us sing chambers. As previously demonstrated in our laboratory, AM supernatant resulted in a significant (p < 0.05) impairment of DLEC Na+ transport, as reflected by a decrease in the amiloride-sensitive component of short-circu it current (control, 3.96 +/- 0.18 mu A/cm(2) vesus supernatant, 2.34 +/- 0 .56 mu A/cm(2); p < 0.05). This effect was significantly reversed by N-acet ylcysteine (3.55 +/- 0.48 mu A/cm(2)), but not by DTT (1.87 +/- 0.21 mu A/c m(2)). N-acetylcysteine, but not DTT, increased DLEC thiol levels. These st udies elucidate mechanisms by which activated BM impair alveolar epithelial barrier function in an b vitro model of acute lung injury.