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 ϩ 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 ϩ channel, mature fetal rat DLEC monolayers were incubated for 16 h with rat AM (1 ϫ 10 7 ) and lipopolysaccharide (10 g/mL), or the cell-free supernatant of lipopolysaccharide-stimulated rat AM. Such exposure resulted in a profound decrease in mRNA expression for all subunits (␣, ␤, and ␥) of the rat epithelial Na ϩ 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 Ͻ 0.05) impairment of DLEC Na 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 I sc , short-circuit current LPS, lipopolysaccharide MEM, minimum essential medium NAC, N-acetyl cysteine NAME, N -nitro-L-arginine methyl ester NO, nitric oxide PD, transepithelial potential difference rENaC, rat epithelial sodium channel ROS, reactive oxygen species ARDS, or ALI, represents an important cause of mortality in critically ill patients (1). Both direct lung injury and distant insults (1), most commonly nonpulmonary sepsis, can initiate a process resulting in a fundamental derangement of the intrinsic barrier property of the lung, manifested clinically as highpermeability pulmonary edema, which is one characteristic of ARDS. It is widely held that these devastating changes most commonly arise because of widespread and uncontrolled activation of inflammatory cells, including neutrophils and macrophages, with the release of a wide range of potentially injurious mediators, such as cytokines, proteolytic enzymes, biologically active lipids, and ROS (2). Activation of inflammatory cells could, in turn, have deleterious effects on the ability of alveolar epithelia to clear edema fluid.The failure of the alveolar-capillary membrane, the structure responsible for preserving a fluid-free alveolar space vital for adequate gas exchange, has been the focus of increasing attention in continuing efforts to understand the basic mechanisms

Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A4 through AhR-cAMP-dependent pathway

“…In this study, CysLTR2 and AhR were the only receptors present on the basolateral membrane of the A549 cells, which is consistent with previous findings showing that A549 cells expressed mRNAs of cysLT 2 and AhR only [16][17][18]. The beneficial effects of LXA 4 were reversed by ANF, an AhR antagonist, but not by the cysLT 2 receptor antagonist BAY-u9773.…”

Acute Lung Injury Edema Fluid Decreases Net Fluid Transport across Human Alveolar Epithelial Type II Cells

“…Although we primarily studied the role of the inflammatory cytokines in this study, ALI pulmonary edema fluid contains other biologically active substances such as proteolytic enzymes, lipids, and reactive oxygen species, particularly the NO metabolite peroxynitrite, which can affect not only fluid clearance but other alveolar epithelial type II cell activity such as surfactant homeostasis. In a similar model in the rat, O'Brodovich and co-workers (45)(46)(47)(48) found that endotoxinstimulated alveolar macrophages or the corresponding supernatant decreases ENaC mRNA levels and activity as well as amiloride-sensitive sodium channel activity among distal lung epithelial cells. The inhibitory effect is prevented by the addition of a nitric-oxide synthase inhibitor or an antioxidant.…”

Downregulation of ENaC activity and expression by TNF-α in alveolar epithelial cells

“…Several lines of evidence suggest that bacterial (25,41) and viral infections (40) modulate the amiloride-sensitive Na ϩ transport in lung epithelial cells. In vitro, conditioned medium from LPS-activated macrophages leads to a decrease in amiloride-sensitive current in fetal distal lung epithelial cells (15,22,23) via a nitric oxide-dependent mechanism (15,23).…”