Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Syed J. Khundmiri, Melissa A. Metzler, Mohamed Ameen, Vishal Amin, Madhavi J. Rane, Nicholas A Delamere

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

Cardiotonic glycosides, like ouabain, inhibit Na+-K +-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser473 phosphorylation, as evidenced by an increase in phospho-Akt Ser473 band density. Ouabain-stimulated Akt Ser 473 phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser473 phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser473 phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in 86Rb uptake but did not significantly alter Na+-K+-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na+-K+-ATPase-mediated ion transport.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume291
Issue number6
DOIs
StatePublished - Dec 2006
Externally publishedYes

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Opossums
Proto-Oncogene Proteins c-akt
Proximal Kidney Tubule
Phosphorylation
Cell proliferation
Ouabain
Cell Proliferation
Calcium
1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole
Adenosine Triphosphatases
Phosphatidylinositol 3-Kinase
Cardiotonic Agents
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Fura-2
Egtazic Acid
Ion Transport
Cell growth
Calcium Channels
Programmable logic controllers
Glycosides

Keywords

  • Na-K-ATPase
  • Opossum kidney cells

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells. / Khundmiri, Syed J.; Metzler, Melissa A.; Ameen, Mohamed; Amin, Vishal; Rane, Madhavi J.; Delamere, Nicholas A.

In: American Journal of Physiology - Cell Physiology, Vol. 291, No. 6, 12.2006.

Research output: Contribution to journalArticle

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