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

• Type of Material: Article
• Authors: Soojung Lee, Emory University; Hye Jeong Lee, Emory University; Han Soo Yang, Emory University; Ian M. Thornell, University of Alabama at Birmingham; Mark O. Bevensee, University of Alabama at Birmingham; Inyeong Choi, Emory University
• Language: English
• Date: 2010-09
• Publisher: Wiley: 12 months
• Publication Version: Final Published Version
• Copyright Statement: © 2010 The Authors.
• Final Published Version (URL): http://ep.physoc.org/content/95/9/926.long
• Title of Journal or Parent Work: Experimental Physiology
• ISSN: 0958-0670
• Volume: 95
• Issue: 9
• Start Page: 926
• End Page: 937
• Grant/Funding Information: SL Lee was supported by a Korea Research Foundation grant funded by the Korean Government (KRF-2007-357-C00121); National Institute of General Medical Sciences : NIGMS; Supported by the NIH grant R01-GM078502 and NS046653 (M.B.); National Institute of Neurological Disorders and Stroke : NINDS
• Keywords: bicarbonate transporter; thick ascending limb; Xenopus oocytes; voltage-clamp
• Research Categories: Biophysics, General; Biology, Physiology

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.