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 (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.
Liu
Jiang
J
Department of Medicine, Medical College of Ohio, Toledo, Ohio 43614-5089, USA.
Liang
Man
M
Liu
Lijun
L
Malhotra
Deepak
D
Xie
Zijian
Z
Shapiro
Joseph I
JI
D000818:Animals; D001483:Base Sequence; D051242:Caveolin 1; D022461:Caveolins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects*; D066246:ErbB Receptors / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism; D018374:LLC-PK1 Cells; D010042:Ouabain / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D012413:Rubidium / Q000493:pharmacokinetics; D015398:Signal Transduction / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
D051242:Caveolin 1; D022461:Caveolins; D034741:RNA, Small Interfering; D010042:Ouabain; D002784:Cholesterol; D066246:ErbB Receptors; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium