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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Effect of PKC activation and Ser-18 mutation on Na,K-ATPase activity and...
Effect of PKC activation and Ser-18 mutation on Na,K-ATPase activity and abundance in A549 cells. (a) Cells were preincubated with 10 μM Bis or vehicle for 30 minutes, exposed to 1.5% O2 or H2O2 (100 μM), and then surface labeled with biotin. Western blots are representative of three experiments. (b) A549 cells were preincubated with PKC-β, -δ, or -ζ peptide antagonists (0.1 μM) or with a scramble peptide for 15 minutes and exposed to 1.5% O2 for 60 minutes. Cells were surface labeled with biotin as described above. A representative autoradiogram is shown (n = 3). (c) GFP α1 A549 cells were exposed to 1.5% O2 for 20 minutes, and Na,K-ATPase GFP α1 subunit was immunoprecipitated using a polyclonal GFP antibody. An in vitro phosphorylation reaction with the immunoprecipitated Na,K-ATPase, purified PKC, and [γ-32P]ATP was conducted. A representative autoradiogram of the α1 subunit phosphorylation from one of three experiments is shown with the corresponding immunoblot performed with a monoclonal anti-GFP antibody. (d) Na,K-ATPase activity and α1 subunit abundance in cell lysates and BLM lysates from S18A-A549 cells submitted to hypoxia for 60 minutes. Each bar represents the mean ± SD of four different experiments. Corresponding representative Western blots are also shown for each experimental condition.