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Author Notes:

Address correspondence: Inyeong Choi, Ph.D., Department of Physiology, Emory University School of Medicine, 615 Whitehead Research Bldg. Atlanta, GA 30322. Phone: 404-712-2092; Fax 404-727-2648; ichoi@emory.edu.

* Equal contribution

Subjects:

Research Funding:

National Institute of Neurological Disorders and Stroke : NINDS

National Institute of General Medical Sciences : NIGMS

Supported by the NIH grant R01-GM078502 and NS046653 (M.B.)

SL Lee was supported by a Korea Research Foundation grant funded by the Korean Government (KRF-2007-357-C00121)

Keywords:

  • bicarbonate transporter
  • Xenopus oocytes
  • voltage-clamp
  • thick ascending limb

Bicarbonate Cotransporter NBCn1 in the Kidney Medullary Thick Ascending Limb Cell Line is Upregulated under Acidic Conditions and Enhances Ammonium Transport

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Journal Title:

Experimental Physiology

Volume:

Volume 95, Number 9

Publisher:

, Pages 926-937

Type of Work:

Article | Final Publisher PDF

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 NH4+ transport, and the intracellular accumulation of 14C-methylammonium/methylammonia (MA) was determined. In CO2∕HCO3−-free solution, cells at normal pH briefly accumulated 14C-MA over 7 min and reached a plateau. In CO2∕HCO3− solution, however, cells markedly accumulated 14C-MA over the experiment period of 30 min. This CO2∕HCO3−-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. Incubating cells at pH 6.8 (adjusted with NaHCO3 in 5% CO2) for 24 h lowered the mean steady-state intracellular pH to 6.96, significantly lower than 7.28 for controls. DIDS reduced 14C-MA accumulation in controls but had no effect after acidic incubation. Immunoblot showed that NBCn1 was upregulated after acidic incubation and in NH4Cl-containing media. The Cl/HCO3 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 NH4Cl application. These results suggest that DIDS-sensitive HCO3− extrusion normally governs NH4+/NH3 uptake in the MTAL cells. We propose that, under acidic conditions, DIDS-sensitive HCO3− extrusion is inactivated while NBCn1 is upregulated to stimulate NH4+ transport.

Copyright information:

© 2010 The Authors.

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