The acute respiratory distress syndrome is characterized by impairment of the alveolar-capillary barrier. Our laboratory has shown that distal lung epithelial cell (DLEC) amiloride-sensitive Na+ transport is impaired by in vitro coculture with endotoxin (lipopolysaccharide)-stimulated alveolar macrophages (AM) through an l-arginine-dependent mechanism. To investigate the effect 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 × 107) and lipopolysaccharide (10 μg/mL), or the cell-free supernatant of lipopolysaccharide-stimulated rat AM. Such exposure resulted in a profound decrease in mRNA expression for all subunits (α, β, and γ) of the rat epithelial Na+ channel, without affecting 18S RNA levels. This effect was prevented by the antioxidant N-acetylcysteine. In separate experiments, confluent DLEC monolayers were exposed to lipopolysaccharide-stimulated AM supernatant for 16 h with or without N-acetylcysteine and DTT and studied in Ussing chambers. As previously demonstrated in our laboratory, AM supernatant resulted in a significant (p + transport, as reflected by a decrease in the amiloride-sensitive component of short-circuit current (control, 3.96 ± 0.18 μA/cm2versus supernatant, 2.34 ± 0.56 μA/cm2;p N-acetylcysteine (3.55 ± 0.48 μA/cm2), but not by DTT (1.87 ± 0.21 μA/cm2). N-acetylcysteine, but not DTT, increased DLEC thiol levels. These studies elucidate mechanisms by which activated AM impair alveolar epithelial barrier function in an in vitro model of acute lung injury.Abbreviations: AM, alveolar macrophages; ARDS, adult respiratory distress syndrome; ALI, acute lung injury; DLEC, distal lung epithelial cells; ENaC, epithelial sodium channel; FBS, fetal bovine serum; GSH, glutathione; Isc, short-circuit current; LPS, lipopolysaccharide; MEM, minimum essential medium; NAC, N-acetyl cysteine; NAME, Nω- nitro-l-arginine methyl ester; NO, nitric oxide; PD, transepithelial potential difference; rENaC, rat epithelial sodium channel; ROS, reactive oxygen species
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