Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-ζ
Laura A. Dada, … , Alejandro M. Bertorello, Jacob I. Sznajder
Laura A. Dada, … , Alejandro M. Bertorello, Jacob I. Sznajder
Published April 1, 2003
Citation Information: J Clin Invest. 2003;111(7):1057-1064. https://doi.org/10.1172/JCI16826.
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Article Immunology

Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-ζ

Abstract

During ascent to high altitude and pulmonary edema, the alveolar epithelial cells (AEC) are exposed to hypoxic conditions. Hypoxia inhibits alveolar fluid reabsorption and decreases Na,K-ATPase activity in AEC. We report here that exposure of AEC to hypoxia induced a time-dependent decrease of Na,K-ATPase activity and a parallel decrease in the number of Na,K-ATPase α1 subunits at the basolateral membrane (BLM), without changing its total cell protein abundance. These effects were reversible upon reoxygenation and specific, because the plasma membrane protein GLUT1 did not decrease in response to hypoxia. Hypoxia caused an increase in mitochondrial reactive oxygen species (ROS) levels that was inhibited by antioxidants. Antioxidants prevented the hypoxia-mediated decrease in Na,K-ATPase activity and protein abundance at the BLM. Hypoxia-treated AEC deficient in mitochondrial DNA (ρ0 cells) did not have increased levels of ROS, nor was the Na,K-ATPase activity inhibited. Na,K-ATPase α1 subunit was phosphorylated by PKC in hypoxia-treated AEC. In AEC treated with a PKC-ζ antagonist peptide or with the Na,K-ATPase α1 subunit lacking the PKC phosphorylation site (Ser-18), hypoxia failed to decrease Na,K-ATPase abundance and function. Accordingly, we provide evidence that hypoxia decreases Na,K-ATPase activity in AEC by triggering its endocytosis through mitochondrial ROS and PKC-ζ–mediated phosphorylation of the Na,K-ATPase α1 subunit.

Authors

Laura A. Dada, Navdeep S. Chandel, Karen M. Ridge, Carlos Pedemonte, Alejandro M. Bertorello, Jacob I. Sznajder

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Figure 3

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Effects of hypoxia on Na,K-ATPase activity and α1 protein abundance at t...
Effects of hypoxia on Na,K-ATPase activity and α1 protein abundance at the plasma membrane is specific and reversible. (a) Na,K-ATPase abundance at the plasma membrane was examined in A549 cells exposed to 1.5% O2 for 60 minutes and surface biotinylated. Cell lysates (150 μg of protein) were pulled down with streptavidin beads. Western blot was performed using Na,K-ATPase α1 subunit (upper panel) or GLUT1 (lower panel) antibody. (b) Cells were surface labeled with biotin before hypoxia (1.5% O2 for 60 minutes). Cell lysates (150 μg of protein) were pulled down with streptavidin beads, and Na,K-ATPase protein abundance was determined as described in a. (c) A549 cells were exposed to 21% O2 or 1.5% O2 for 60 minutes, and then one group (1.5% O2) was returned to 21% O2 for 2 hours. Na,K-ATPase activity was determined by 86Rb+ uptake and expressed as a percentage of control (21% O2). Bars represent the means ± SD of three experiments performed in duplicate. **P < 0.01. (d) A549 cells were exposed to the same protocol as described in c, and Na,K-ATPase abundance was determined. Each bar represents the mean ± SD of three experiments. A representative Western blot is shown. **P < 0.01. (e) GFP-α1 A549 cells were plated onto glass coverslips and exposed to 21% O2, 1.5% O2, or 1.5% O2 and reoxygenation (reox). Cells were fixed and direct fluorescence was visualized using confocal microscopy. Representative confocal images are shown.