Ouabain-induced Endocytosis of the Plasmalemmal Na/K-ATPase in LLC-PK1 Cells Requires Caveolin-1
Background: We have demonstrated that ouabain causes dose- and time-dependent decreases in 86Rb 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 -cyclodextrin reduced ouabain-induced accumulation of Na/K-ATPase -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 -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 -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 -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.
Liu, J., Liang M., Liu, L., Malhotra, D., Xie, Z., & Shapiro, J. I. (2005). Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1. Kidney International, 67, 1844-1854.