title: Low dose ouabain stimulates NaK ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells.
abstract: Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of NaK 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 NaK 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 (1μg/kg body wt./day) for 9days and increased angiotensin II levels in cell culture media after 24h treatment with ouabain in human (HKC11), mouse (MRPT), and human adrenal cells. Ouabain 10pM stimulated NKA-mediated86Rb 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.
authors: Department of Physiology, University of Louisville, KY, USA.
authors: Corey J
authors: CJ
authors: Ketchem
authors: Department of Physiology, University of Louisville, KY, USA.
authors: Clayton D
authors: CD
authors: Conner
authors: Department of Physiology, University of Louisville, KY, USA; Department of Medicine, University of Louisville, KY, USA.
authors: Rebecca D
authors: RD
authors: Murray
authors: Department of Medicine, University of Louisville, KY, USA.
authors: Madalyn
authors: M
authors: DuPlessis
authors: Department of Physiology, University of Louisville, KY, USA; Department of Medicine, University of Louisville, KY, USA; Robley Rex VA Medical Center, Louisville, KY, USA.
authors: Eleanor D
authors: ED
authors: Lederer
authors: Department of Medicine, University of Louisville, KY, USA.
authors: Daniel
authors: D
authors: Wilkey
authors: Department of Medicine, University of Louisville, KY, USA. Electronic address: michael.merchant@louisville.edu.
authors: Michael
authors: M
authors: Merchant
authors: Department of Physiology and Biophysics, Howard University College of Medicine, Washington, DC, USA. Electronic address: syed.khundmiri@howard.edu.
authors: Syed J
authors: SJ
authors: Khundmiri
mesh_terms: D000804:Angiotensin II / Q000378:metabolism; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000808:Angiotensinogen / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology* / Q000633:toxicity; D006973:Hypertension / Q000139:chemically induced / Q000201:enzymology; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D010042:Ouabain / Q000494:pharmacology* / Q000633:toxicity; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017207:Rats, Sprague-Dawley; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C534931:AGT protein, human; C544065:AGTR1 protein, human; D047228:Angiotensin II Type 1 Receptor Blockers; D020536:Enzyme Activators; D044140:Receptor, Angiotensin, Type 1; D000808:Angiotensinogen; D000804:Angiotensin II; D010042:Ouabain; C488034:ACE protein, human; D007703:Peptidyl-Dipeptidase A; C499076:ATP1A1 protein, human; C499075:Atp1a1 protein, rat; C497344:Atp1a1 protein, mouse; D000254:Sodium-Potassium-Exchanging ATPase
keywords: AT1R; FRET-TIRF microscopy; Human kidney cells; NaK ATPase; Ouabain; Proteomics