Journal
EXPERIMENTAL PHYSIOLOGY
Volume 95, Issue 9, Pages 926-937Publisher
WILEY-BLACKWELL
DOI: 10.1113/expphysiol.2010.053967
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Funding
- NIH [R01-GM078502, NS046653]
- National Kidney Foundation
- Emory URC
- Korean Government [KRF-2007-357-C00121]
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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 14C-methylammonium/methylammonia (14C-MA) was determined. In CO(2)/HCO(3)--free solution, cells at normal pH briefly accumulated 14C-MA over 7 min and reached a plateau. In CO(2)/HCO(3)- solution, however, cells markedly accumulated 14C-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 14C-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 14C-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.
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