title: Long-term regulation of Na+,K+-ATPase in opossum kidney cells by ouabain.
abstract: Na(+),K(+)-ATPase, a basolateral transporter responsible for tubular reabsorption of Na(+) and for providing the driving force for vectorial transport of various solutes and ions, can also act as a signal transducer in response to the interaction with steroid hormones. At nanomolar concentrations ouabain binding to Na(+),K(+)-ATPase activates a signaling cascade that ultimately regulates several membrane transporters including Na(+),K(+)-ATPase. The present study evaluated the long-term effect of ouabain on Na(+),K(+)-ATPase activity (Na(+) transepithelial flux) and expression in opossum kidney (OK) cells with low (40) and high (80) number of passages in culture, which are known to overexpress Na(+),K(+)-ATPase (Silva et al., 2006, J Membr Biol 212, 163-175). Activation of a signal cascade was evaluated by quantification of ERK1/2 phosphorylation by Western blot. Na(+),K(+)-ATPase activity was determined by electrophysiological techniques and expression by Western blot. Incubation of cells with ouabain induced activation of ERK1/2. Long-term incubation with ouabain induced an increase in Na(+) transepithelial flux and Na(+),K(+)-ATPase expression only in OK cells with 80 passages in culture. This increase was prevented by incubation with inhibitors of MEK1/2 and PI-3K. In conclusion, ouabain-activated signaling cascade mediated by both MEK1/2 and PI-3K is responsible for long-term regulation of Na(+) transepithelial flux in epithelial renal cells. OK cell line with high number of passages is suggested to constitute a particular useful model for the understanding of ouabain-mediated regulation of Na(+) transport.
authors.affiliation: Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: E
authors.initials: E
authors.lastname: Silva
authors.forename: P
authors.initials: P
authors.lastname: Soares-da-Silva
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D007668:Kidney / Q000166:cytology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009893:Opossums / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013997:Time Factors
chemical_list: D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010042:Ouabain; D019869:Phosphatidylinositol 3-Kinases; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase