Hypoxia and β2-Agonists Regulate Cell Surface Expression of the Epithelial Sodium Channel in Native Alveolar Epithelial Cells

Abstract

Alveolar hypoxia may impair sodium-dependent alveolar fluid transport and induce pulmonary edema in rat and human lung, an effect that can be prevented by the inhalation of ␤ 2 -agonists. To investigate the mechanism of ␤ 2 -agonist-mediated stimulation of sodium transport under conditions of moderate hypoxia, we examined the effect of terbutaline on epithelial sodium channel (ENaC) expression and activity in cultured rat alveolar epithelial type II cells exposed to 3% O 2 for 24 h. Hypoxia reduced transepithelial sodium current and amiloridesensitive sodium channel activity without decreasing ENaC subunit mRNA or protein levels. The functional decrease was associated with reduced abundance of ENaC subunits (especially ␤ and ␥) in the apical membrane of hypoxic cells, as quantified by biotinylation. cAMP stimulation with terbutaline reversed the hypoxia-induced decrease in transepithelial sodium transport by stimulating sodium channel activity and markedly increased the abundance of ␤-and ␥-ENaC in the plasma membrane of hypoxic cells. The effect of terbutaline was prevented by brefeldin A, a blocker of anterograde transport. These novel results establish that hypoxiainduced inhibition of amiloride-sensitive sodium channel activity is mediated by decreased apical expression of ENaC subunits and that ␤ 2 -agonists reverse this effect by enhancing the insertion of ENaC subunits into the membrane of hypoxic alveolar epithelial cells.

Differential Current Decay Profiles of Epithelial Sodium Channel Subunit Combinations in Polarized Renal Epithelial Cells

“…Moreover, we and others have observed changes in specific subpopulations of subunits in response to hormonal regulation of transport, both in whole cells and at the apical membrane (6,9,20,21). These observations are inconsistent with the idea that cell surface ENaC subunits in these cells are assembled exclusively into ␣␤␥ holochannels whose trafficking and turnover occurs in a congruent manner.…”

“…However, given recent studies linking proteolytic cleavage with ENaC activity, quantitative changes in biochemical levels of ENaC subunits may not reflect changes in current. Moreover, we and others have observed noncoordinate changes in the levels of cell surface ␣-, ␤-, and ␥-ENaC in response to some physiologic stimuli (6,9,20). It is clear that individual ENaC subunits as well as combinations of subunits can traffic to the plasma membrane; however, the contribution of these subunits to ENaC activity is unclear.…”

ERK1/2 Controls Na,K-ATPase Activity and Transepithelial Sodium Transport in the Principal Cell of the Cortical Collecting Duct of the Mouse Kidney

“…Cell Surface Biotinylation of Na,K-ATPase-Cell surface biotinylation was performed according to the protocol described by Planes et al, with minor modifications (22). mpkCCD c14 cells grown on Transwell filters (2 filters/condition) were placed on ice and washed three times with ice-cold phosphate-buffered saline solution complemented with 0.1 mM CaCl 2 and 1 mM MgCl 2 (Buffer F).…”

Gene regulation in the adaptive process to hypoxia in lung epithelial cells

“…In addition to the potential role for an O 2 -responsive element in the ENaC promoter, it is also possible that there are other genes that are O 2 responsive and alter ENaC expression through their expressed protein or metabolic products. During less severe hypoxia or short hypoxic exposure times, the decrease in sodium transport was rather related to reduced sodium protein expression in the apical and basolateral membranes, whereas the mRNA transcripts of ENaC and Na-K-ATPase subunits and intracellular pool of sodium proteins were unchanged (40,61). Several studies in alveolar epithelial cells indicate that hypoxia alters the trafficking of sodium proteins between the cytosol and the membrane.…”

“…In A549 cells, hypoxia (1.5% O 2 for 60 min) decreased Na-KATPase activity by promoting the endocytosis of ␣ 1 and ␤ 1 Na-K-ATPase protein subunits (14). Planès et al (40) reported that during moderate hypoxia (3% O 2 for 18 h), the decrease in sodium channels in the apical membrane was restored by ␤ 2 -agonists, which favored the trafficking of proteins from the cytosol to the membrane. The mechanisms whereby hypoxia promotes endocytosis of sodium proteins are not fully elucidated.…”