title: Structural determinants for the ouabain-stimulated increase in Na-K ATPase activity.
abstract: Recent studies suggest that at low concentrations, ouabain increases Na-K ATPase and NHE1 activity and activates the Src signaling cascade in proximal tubule cells. Our laboratory demonstrated that low concentrations of ouabain increase blood pressure in rats. We hypothesize that ouabain-induced increase in blood pressure and Na-K ATPase activity requires NHE1 activity and association. To test this hypothesis we treated rats with ouabain (1μgkg body wt(-1)day(-1)) for 9days in the presence or absence of the NHE1 inhibitor, zoniporide. Ouabain stimulated a significant increase in blood pressure which was prevented by zoniporide. Using NHE1-expressing Human Kidney cells 2 (HK2), 8 (HK8) and 11 (HK11) and Mouse Kidney cells from Wild type (WT) and NHE1 knock-out mice (SWE) cell lines, we show that ouabain stimulated Na-K ATPase activity and surface expression in a Src-dependent manner in NHE1-expressing cells but not in NHE1-deplete cells. Zoniporide prevented ouabain-induced stimulation of (86)Rb uptake in the NHE1-expressing cells. FRET and TIRF microscopy showed that ouabain increased association between GFP-NHE1 and mCherry-Na-K ATPase transfected into NHE1-deficient SWE cells. Mutational analysis demonstrated that the caveolin binding motif (CBM) of Na-K ATPase α1 is required for translocation of both Na-K ATPase α1 and NHE1 to the basolateral membrane. Mutations in activity or scaffold domains of NHE1 resulted in loss of ouabain-mediated regulation of Na-K ATPase. These results support that NHE1 is required for the ouabain-induced increase in blood pressure, and that the caveolin binding motif of Na-K ATPase α1 as well as the activity and scaffolding domains of NHE1 are required for their functional association.
authors.affiliation: Robley Rex Veterans Affairs Medical Center, Louisville, KY, USA; Department of Medicine/Kidney Disease Program, University of Louisville, Louisville, KY, USA; Department of Physiology, University of Louisville, Louisville, KY, USA. Electronic address: syed.khundmiri@louisville.edu.
authors.forename: Syed J
authors.initials: SJ
authors.lastname: Khundmiri
authors.affiliation: Department of Medicine/Kidney Disease Program, University of Louisville, Louisville, KY, USA; Department of Physiology, University of Louisville, Louisville, KY, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Salyer
authors.affiliation: Department of Medicine/Kidney Disease Program, University of Louisville, Louisville, KY, USA.
authors.forename: Brandon
authors.initials: B
authors.lastname: Farmer
authors.affiliation: Department of Physiology, University of Louisville, Louisville, KY, USA.
authors.forename: Natia
authors.initials: N
authors.lastname: Qipshidze-Kelm
authors.affiliation: Department of Medicine/Kidney Disease Program, University of Louisville, Louisville, KY, USA; Department of Physiology, University of Louisville, Louisville, KY, USA.
authors.forename: Rebecca D
authors.initials: RD
authors.lastname: Murray
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY, USA.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Clark
authors.affiliation: Department of Physiology, University of Toledo College of Medicine, Toledo, OH, USA.
authors.forename: Zijian
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, Texas Tech Univ. Health Sciences Center, Lubbock, TX, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Pressley
authors.affiliation: Robley Rex Veterans Affairs Medical Center, Louisville, KY, USA; Department of Medicine/Kidney Disease Program, University of Louisville, Louisville, KY, USA; Department of Physiology, University of Louisville, Louisville, KY, USA.
authors.forename: Eleanor D
authors.initials: ED
authors.lastname: Lederer
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019921:Biotinylation; D001794:Blood Pressure / Q000187:drug effects; D015153:Blotting, Western; D002316:Cardiotonic Agents / Q000494:pharmacology*; D027682:Cation Transport Proteins / Q000502:physiology*; D051242:Caveolin 1 / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006868:Hydrolysis; D007124:Immunoenzyme Techniques; D017136:Ion Transport / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D010042:Ouabain / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D002316:Cardiotonic Agents; D027682:Cation Transport Proteins; D051242:Caveolin 1; C490997:Slc9a1 protein, mouse; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D010042:Ouabain; D000255:Adenosine Triphosphate; D019061:src-Family Kinases; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Blood pressure; NHE1; Na–K ATPase; Ouabain; Src kinase; TIRF microscopy