Low dose ouabain stimulates Na—K ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells

Corey J. Ketchem, Clayton D. Conner, Rebecca D. Murray, Madalyn DuPlessis, Eleanor D. Lederer, Daniel Wilkey, Michael Merchant, Syed J. Khundmiri

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of [Formula presented] ATPase activity and activation of the Src signaling cascade in NHE1-dependent manner. Proteomic analysis of human kidney proximal tubule cells (HKC11) suggested that the Angiotensin II type 1 receptor (AT1R) as an ouabain-associating protein. We hypothesize that ouabain-induced stimulation of [Formula presented] ATPase activity is mediated through AT1R. To test this hypothesis, we examined the effect of ouabain on renal cell angiotensin II production, the effect of AT1R inhibition on ouabain-stimulated NKA activity, and the effect of ouabain on NKA-AT1R association. Ouabain increased plasma angiotensin II levels in rats treated with ouabain (1 μg/kg body wt./day) for 9 days and increased angiotensin II levels in cell culture media after 24 h treatment with ouabain in human (HKC11), mouse (MRPT), and human adrenal cells. Ouabain 10 pM stimulated NKA-mediated 86Rb uptake and phosphorylation of EGFR, Src, and ERK1/2. These effects were prevented by the AT1R receptor blocker candesartan. FRET and TIRF microscopy using Bodipy-labeled ouabain and mCherry-NKA or mCherry-AT1R demonstrated association of ouabain with AT1R and NKA. Further our FRET and TIRF studies demonstrated increased association between AT1R and NKA upon treatment with low dose ouabain. We conclude that ouabain stimulates NKA in renal proximal tubule cells through an angiotensin/AT1R-dependent mechanism and that this pathway contributes to cardiac glycoside associated hypertension.

Original languageEnglish (US)
Pages (from-to)2624-2636
Number of pages13
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1863
Issue number11
DOIs
StatePublished - Nov 1 2016

Keywords

  • AT1R
  • FRET-TIRF microscopy
  • Human kidney cells
  • NaK ATPase
  • Ouabain
  • Proteomics

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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