Ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-dependent mechanism

Kidney Int. 2004 Jul;66(1):227-41. doi: 10.1111/j.1523-1755.2004.00723.x.


Background: We have demonstrated that ouabain causes dose- and time-dependent decreases in (86)Rb uptake in porcine proximal tubular (LLC-PK1) cells. The present study addresses the molecular mechanisms involved in this process.

Methods: Studies were performed with cultured LLC-PK1 and Src family kinase deficient (SYF) cells.

Results: We found that 50 nmol/L ouabain applied to the basal, but not apical, aspect for 12 hours caused decreases in the plasmalemmal Na/K-ATPase. This loss of plasmalemmal Na/K-ATPase reverses completely within 12 to 24 hours after removal of ouabain. Ouabain also increased the Na/K-ATPase content in both early and late endosomes, activated phosphatidylinositol 3-kinase (PI(3)K), and also caused a translocation of some Na/K-ATPase to the nucleus. Immunofluorescence demonstrated that the Na/K-ATPase colocalized with clathrin both before and after exposure to ouabain, and immunoprecipitation experiments confirmed that ouabain stimulated interactions among the Na/K-ATPase, adaptor protein-2 (AP-2), and clathrin. Potassium (K) depletion, chlorpromazine, or PI(3)K inhibition all significantly attenuated this ouabain-induced endocytosis. Inhibition of the ouabain-activated signaling process through Src by 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) significantly attenuated ouabain-induced endocytosis. Moreover, experiments performed in SYF cells demonstrated that ouabain induced increases in the endocytosis of the Na/K-ATPase when Src was reconstituted (SYF+), but not in the Src-deficient (SYF-) cells.

Conclusion: These data demonstrate that ouabain stimulates a clathrin-dependent endocytosis pathway that translocates the Na/K-ATPase to intracellular compartments, thus suggesting a potential role of endocytosis in ouabain-induced signal transduction as well as proximal tubule sodium handling.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Protein Complex 2 / metabolism
  • Animals
  • Cell Membrane / enzymology*
  • Clathrin / physiology*
  • Clathrin-Coated Vesicles / metabolism
  • Endocytosis / drug effects*
  • Endosomes / metabolism
  • Enzyme Activation / physiology
  • Isoenzymes / metabolism
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / enzymology*
  • Kidney Tubules, Proximal / physiology
  • LLC-PK1 Cells
  • Membrane Potentials
  • Osmolar Concentration
  • Ouabain / pharmacology*
  • Phosphatidylinositol 3-Kinases / metabolism
  • Sodium-Potassium-Exchanging ATPase / antagonists & inhibitors
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Swine
  • src-Family Kinases / physiology


  • Adaptor Protein Complex 2
  • Clathrin
  • Isoenzymes
  • Ouabain
  • src-Family Kinases
  • Sodium-Potassium-Exchanging ATPase