title: Sodium-bicarbonate cotransporter NBCn1 in the kidney medullary thick ascending limb cell line is upregulated under acidic conditions and enhances ammonium transport.
abstract: In this study, we examined the effect of bicarbonate transporters on ammonium/ammonia uptake in the medullary thick ascending limb cell line ST-1. Cells were treated with 1 mm ouabain and 0.2 mM bumetanide to minimize carrier-mediated NH(4)(+) transport, and the intracellular accumulation of (14)C-methylammonium/methylammonia ((14)C-MA) was determined. In CO(2)/HCO(3)(-)-free solution, cells at normal pH briefly accumulated (14)C-MA over 7 min and reached a plateau. In CO(2)/HCO(3)(-) solution, however, cells markedly accumulated (14)C-MA over the experimental period of 30 min. This CO(2)/HCO(3)(-)-dependent accumulation was reduced by the bicarbonate transporter blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS; 0.5 mM). Replacing Cl(-) with gluconate reduced the accumulation, but the reduction was more substantial in the presence of DIDS. Incubation of cells at pH 6.8 (adjusted with NaHCO(3) in 5% CO(2)) for 24 h lowered the mean steady-state intracellular pH to 6.96, significantly lower than 7.28 for control cells. The presence of DIDS reduced (14)C-MA accumulation in control conditions but had no effect after acidic incubation. Immunoblotting showed that NBCn1 was upregulated after acidic incubation and in NH(4)Cl-containing media. The Cl(-)-HCO(3)(-) exchanger AE2 was present, but its expression remained unaffected by acidic incubation. Expressed in Xenopus oocytes, NBCn1 increased carrier-mediated (14)C-MA transport, which was abolished by replacing Na(+). Two-electrode voltage clamp of oocytes exhibited negligible current after NH(4)Cl application. These results suggest that DIDS-sensitive HCO(3)(-) extrusion normally governs NH(4)(+)/NH(3) uptake in the medullary thick ascending limb cells. We propose that, in acidic conditions, DIDS-sensitive HCO(3)(-) extrusion is inactivated, while NBCn1 is upregulated to stimulate NH(4)(+) transport.
authors.affiliation: Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Soojung
authors.initials: S
authors.lastname: Lee
authors.forename: Hye Jeong
authors.initials: HJ
authors.lastname: Lee
authors.forename: Han Soo
authors.initials: HS
authors.lastname: Yang
authors.forename: Ian M
authors.initials: IM
authors.lastname: Thornell
authors.forename: Mark O
authors.initials: MO
authors.lastname: Bevensee
authors.forename: Inyeong
authors.initials: I
authors.lastname: Choi
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D027321:Anion Transport Proteins / Q000378:metabolism; D017920:Antiporters / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism*; D001692:Biological Transport; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D005942:Gluconates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007679:Kidney Medulla / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008138:Loop of Henle / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D008744:Methylamines / Q000378:metabolism; D018408:Patch-Clamp Techniques; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D051381:Rats; D064606:SLC4A Proteins; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D014981:Xenopus
chemical_list: D027321:Anion Transport Proteins; D017920:Antiporters; D001639:Bicarbonates; D002712:Chlorides; D005942:Gluconates; D008744:Methylamines; D000644:Quaternary Ammonium Compounds; D064606:SLC4A Proteins; C498902:Slc4a7 protein, rat; D027982:Sodium-Bicarbonate Symporters; D000641:Ammonia; D012964:Sodium; C027451:methylamine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C030691:gluconic acid