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


CONCLUSIONS
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.
Liu
Jiang
J
The Department of Medicine, Medical College of Ohio, Toledo, Ohio, USA.
Kesiry
Riad
R
Periyasamy
Sankaridrug M
SM
Malhotra
Deepak
D
Xie
Zijian
Z
Shapiro
Joseph I
JI
D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002966:Clathrin / Q000502:physiology*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D018374:LLC-PK1 Cells; D008564:Membrane Potentials; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine; D019061:src-Family Kinases / Q000502:physiology
D033962:Adaptor Protein Complex 2; D002966:Clathrin; D007527:Isoenzymes; D010042:Ouabain; D019869:Phosphatidylinositol 3-Kinases; D000254:Sodium-Potassium-Exchanging ATPase; D019061:src-Family Kinases