title: Low efficiency of functional translation of ouabain-resistant α2-Na(+),K(+)-ATPase mRNA in C7-MDCK epithelial cells.
abstract: Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory β1-β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase. α2R mRNA in transfected cells was ∼8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10 µmol/L ouabain led to complete inhibition of (86)Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of (86)Rb influx in α1R-transfectd cells was observed at 1000 µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3 µmol/L ouabain , α1R-cells survived after 24 h incubation with 1000 µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na(+),K(+)-ATPase subunit mRNA and immunoreactive protein does not contribute to Na(+)/K(+) pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na(+),K(+)-ATPase on Na(+)/K(+) pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.
authors.affiliation: Research Centre, Centre hospitalier de l'Université de Montréal (CRCHUM) - Technopôle Angus, 2901 Rachel East, Montreal, QC H1W 4A4, Canada.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Akimova
authors.forename: James
authors.initials: J
authors.lastname: Van Huysse
authors.forename: Johanne
authors.initials: J
authors.lastname: Tremblay
authors.forename: Sergei N
authors.initials: SN
authors.lastname: Orlov
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism* / Q000502:physiology*; D017136:Ion Transport / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D007527:Isoenzymes / Q000235:genetics; D007668:Kidney / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010042:Ouabain / Q000009:adverse effects*; D011188:Potassium / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D059368:RNA Isoforms / Q000235:genetics*; D012414:Rubidium Radioisotopes / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology*; D014162:Transfection / Q000379:methods
chemical_list: D007527:Isoenzymes; D059368:RNA Isoforms; D012414:Rubidium Radioisotopes; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium