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. 2005 May;67(5):1844-54.
doi: 10.1111/j.1523-1755.2005.00283.x.

Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1

Jiang Liu  1 , Man Liang, Lijun Liu, Deepak Malhotra, Zijian Xie, Joseph I Shapiro
Affiliations
Free article

Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1

Jiang Liu et al. Kidney Int. 2005 May.
Free article

Abstract

Background: We have demonstrated that ouabain causes dose- and time-dependent decreases in (86)Rb uptake in pig renal proximal tubule cell line (LLC-PK1) cells; and ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells in a clathrin-dependent pathway. Our data also suggest a role of endocytosis in both ouabain-induced signal transduction and proximal tubule sodium handling. The present study addresses the molecular mechanisms involved in this process.

Methods: Studies were performed with cultured LLC-PK1 and a stable-expressed caveolin-1 knockdown LLC-PK1 cell line by SiRNA method.

Results: In wild-type LLC-PK1 cells, depletion of cholesterol by methyl beta-cyclodextrin reduced ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coated vesicles, as well as in endosomes. Depletion of cholesterol also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. In LLC-PK1 cells expressing mock-vehicle and caveolin-1 siRNA, depletion of caveolin-1 abolished ouabain-induced decrease in Rb uptake and decrease in the plasmalemmal Na/K-ATPase content. Depletion of caveolin-1 also significantly reduced the ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coat vesicles, as well as early and late endosomes. In addition, depletion of caveolin-1 also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. These data suggest that caveolae are involved in ouabain-induced endocytosis and signal transduction by initiating assembly of signaling cascades through the caveolar Na/K-ATPase and/or the interaction with clathrin-mediated endocytosis of the Na/K-ATPase.

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