title: Stimulation of Na,K-ATPase by low potassium requires reactive oxygen species.
abstract: The signaling pathway that transduces the stimulatory effect of low K+ on the biosynthesis of Na,K-ATPase remains largely unknown. The present study was undertaken to examine whether reactive oxygen species (ROS) mediated the effect of low K+ in Madin-Darby canine kidney (MDCK) cells. Low K+ increased ROS activity in a time- and dose-dependent manner, and this effect was abrogated by catalase and N-acetylcysteine (NAC). To determine the role of ROS in low-K+-induced gene expression, the cells were first stably transfected with expression constructs in which the reporter gene chloramphenicol acetyl transferase (CAT) was under the control of the avian Na,K-ATPase alpha-subunit 1.9 kb and 900-bp 5'-flanking regions that have a negative regulatory element. Low K+ increased the CAT expression in both constructs. Catalase or NAC inhibited the effect of low K+. To determine whether the increased CAT activity was mediated through releasing the repressive effect or a direct stimulation of the promoter, the cells were transfected with a CAT expression construct directed by a 96-bp promoter fragment that has no negative regulatory element. Low K+ also augmented the CAT activity expressed by this construct. More importantly, both catalase and NAC abolished the effect of low K+. Moreover, catalase and NAC also inhibited low-K+-induced increases in the Na,K-ATPase alpha1- and beta1-subunit protein abundance and ouabain binding sites. The antioxidants had no significant effect on the basal levels of CAT activity, protein abundance, or ouabain binding sites. In conclusion, low K+ enhances the Na,K-ATPase gene expression by a direct stimulation of the promoter activity, and ROS mediate this stimulation and also low-K+-induced increases in the Na,K-ATPase protein contents and cell surface molecules.
authors.affiliation: Department of Medicine, Uniformed Services University, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA. xiazhou@usuhs.mil
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhou
authors.forename: Wu
authors.initials: W
authors.lastname: Yin
authors.forename: Sonia Q
authors.initials: SQ
authors.lastname: Doi
authors.forename: Shawn W
authors.initials: SW
authors.lastname: Robinson
authors.forename: Kunio
authors.initials: K
authors.lastname: Takeyasu
authors.forename: Xuetao
authors.initials: X
authors.lastname: Fan
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002374:Catalase / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005057:Eukaryotic Cells / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D017930:Genes, Reporter / Q000187:drug effects / Q000235:genetics; D011191:Potassium Deficiency / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D021122:Protein Subunits / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D017382:Reactive Oxygen Species; D002374:Catalase; D000254:Sodium-Potassium-Exchanging ATPase; D000111:Acetylcysteine