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Ouabain Induces Endocytosis of Plasmalemmal Na/K-ATPase in LLC-PK1 Cells by a Clathrin-dependent Mechanism
Pharmaceutical Science and Research
  • Jiang Liu, Marshall University
  • Riad Kesiry
  • Sankaridrug M. Periyasamy
  • Deppak Malhorta
  • Zijian Xie
  • Joseph I. Shapiro
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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.

The version of record is available from the publisher at Copyright © 2004 by the International Society of Nephrology. All rights reserved.

Citation Information
Liu, J., Kesiry, R., Periyasamy, S. M., Malhotra, D., Xie, Z., & Shapiro, J. I. (2004). Ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-dependent mechanism. Kidney International, 66, 227-241.