TITLE

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

AUTHOR(S)
Jiang Liu; Kesiry, Riad; Iperiyasamy, Sankaridrug M.; Malhotra, Deepak; Zijian Xie; Shapiro, Joseph I.
PUB. DATE
July 2004
SOURCE
Kidney International;Jul2004, Vol. 66 Issue 1, p227
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
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 86Rb 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.
ACCESSION #
13347236

 

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