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We have evaluated the effect of lipopolysaccharides (LPS), endotoxins from gram negative bacteria, on sodium-coupled amino acid and phosphate transport by alveolar epithelial type II cells and on their alteration induced by oxidants. Alveolar type II cells were obtained by enzymatic digestion of rat lung and grown for 24 h prior to incubation with LPS and then exposed or not exposed to H2O2 (2.5 mM; 20 min). LPS (10 μg/ml, 24 h) induced a significant increase in the Na-dependent component of alanine and phosphate uptake while they decreased Na, K-ATPase activity measured by ouabain-sensitive 86Rb influx. We showed that this stimulatory effect i) was independent from macrophage products since it was not mimicked either by supernatant of LPS-treated alveolar macrophages or by pretreatment with tumor necrosis factor and/or interleukin 1 and ii) was dependent on protein synthesis since it was abolished by protein synthesis inhibitors cycloheximide and actinomycin D. Moreover, LPS blunted H2O2-induced decrease of Na-dependent alanine and phosphate uptake. This protective effect of LPS against H2O2 injury i) was independent of macrophage products, ii) was abolished by cycloheximide, and iii) was not associated with either changes in extracellular H2O2 clearance or catalase and glutathione peroxidase activities. We conclude that, in alveolar type II cells, LPS stimulate sodium-coupled transport by a process involving protein synthesis and partially prevent H2O2-induced decrease of Na-coupled transport without discernible change in antioxidant activities. © 1995 Wiley-Liss, Inc.

Culture of primary alveolar type II cells has been widely used to investigate the Na+ transport characteristics of alveolar epithelium. However, this model was restricted by early morphological and physiological dedifferentiation in culture. Recently, a cell line has been obtained by transfection of neonatal type II cells with the simian virus SV40 large T antigen gene (SV40-T2). SV40-T2 cells have retained proliferative characteristics of the primary type II cells (Clement et al., 1991, Exp. Cell Res., 196:198-205.) In the present study, we have characterized Na+ transport pathways in SV40-T2 cells. SV40-T2 cells retained most cardinal properties of the original alveolar epithelial cells. Na+ entry occurred, as in primary cultures, through both Na(+)-cotransporters and amiloride-sensitive Na+ channels. SV40-T2 cells expressed Na(+)-phosphate. Na(+)-amino acid and Na(+)-K(+)-Cl cotransports which are quantitatively similar to that of primary cultures. The existence of amiloride-sensitive Na+ channels was supported by molecular and functional data. SV40-T2 expressed the cloned alpha- and gamma-mRNAs for the rat epithelial Na+ channel (rENaC), whereas beta subunit was not detected, and 22Na+ influx was significantly inhibited by 10 microM amiloride. Na+, which enters SV40-T2 cells, is extruded through a Na+, K(+)-ATPase: mRNA for alpha 1 and beta 1 isoforms of Na+, K(+)-ATPase were present and Na+, K(+)-ATPase activity was evidenced either on intact cells by the presence of a ouabain-sensitive component of 86Rb+ influx or on cell homogenates by the measurement of ouabain-inhibitable ATP hydrolysis. These results indicate that SV40-T2 cell line displays most of the Na+ transport characteristics of well-differentiated primary cells in the first days of culture. We conclude that the SV40-T2 cell line provides a model of differentiated alveolar type II cells and may be a powerful tool to study, in vitro, the modulation of Na+ transport in pathophysiological conditions.