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.

SUBMITTER: Lee S 

PROVIDER: S-EPMC2923243 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature