title: Preventing endotoxin-stimulated alveolar macrophages from decreasing epithelium Na+ channel (ENaC) mRNA levels and activity.
abstract: 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 x 10(7)) and lipopolysaccharide (10 microg/mL), or the cell-free supernatant of lipopolysaccharide-stimulated rat AM. Such exposure resulted in a profound decrease in mRNA expression for all subunits (alpha, beta, and gamma) 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 < 0.05) impairment of DLEC Na+ transport, as reflected by a decrease in the amiloride-sensitive component of short-circuit current (control, 3.96 +/- 0.18 microA/cm2 versus supernatant, 2.34 +/- 0.56 microA/cm2; p < 0.05). This effect was significantly reversed by N-acetylcysteine (3.55 +/- 0.48 microA/cm2), but not by DTT (1.87 +/- 0.21 microA/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.
authors.affiliation: Department of Surgery, University of Toronto, Ontario, Canada.
authors.forename: A J
authors.initials: AJ
authors.lastname: Dickie
authors.forename: B
authors.initials: B
authors.lastname: Rafii
authors.forename: J
authors.initials: J
authors.lastname: Piovesan
authors.forename: C
authors.initials: C
authors.lastname: Davreux
authors.forename: J
authors.initials: J
authors.lastname: Ding
authors.forename: A K
authors.initials: AK
authors.lastname: Tanswell
authors.forename: O
authors.initials: O
authors.lastname: Rotstein
authors.forename: H
authors.initials: H
authors.lastname: O'Brodovich
mesh_terms: D000818:Animals; D002450:Cell Communication / Q000502:physiology*; D004731:Endotoxins / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D005260:Female; D008262:Macrophage Activation / Q000187:drug effects / Q000502:physiology; D016676:Macrophages, Alveolar / Q000166:cytology / Q000502:physiology*; D012333:RNA, Messenger / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D015222:Sodium Channels / Q000502:physiology*
chemical_list: D004731:Endotoxins; D012333:RNA, Messenger; D015222:Sodium Channels