Sodium-bicarbonate cotransporter NBCn1 in the kidney medullary thick ascending limb cell line is upregulated under acidic conditions and enhances ammonium transport.
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.
Lee
Soojung
S
Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Lee
Hye Jeong
HJ
Yang
Han Soo
HS
Thornell
Ian M
IM
Bevensee
Mark O
MO
Choi
Inyeong
I
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
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