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MUCOSAL BIOLOGY

Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent

¹Epithelial Pathobiology Group, Department of Surgery, ²Department of Molecular and Cellular Physiology, ³Department of Medicine, and ⁴Veterans Affairs Medical Center, University of Cincinnati, Cincinnati, Ohio

Submitted 1 October 2004 ; accepted in final form 25 June 2005

Normal human colonic luminal (NH₄⁺) concentration ([NH₄⁺]) ranges from 10 to 100 mM. However, the nature of the effects of NH₄⁺ on transport, as well as NH₄⁺ transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH₄⁺ on cAMP-stimulated Cl^– secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH₄⁺ had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH₄⁺ reduced current within 5 min to 61 ± 4% in the presence of 25 mM HCO₃^–. Current inhibition was not simply due to an increase in extracellular K⁺-like cations, in that the current magnitude was 95 ± 5% with 10 mM apical K⁺ and 46 ± 3% with 10 mM apical NH₄⁺ relative to that with 5 mM apical K⁺. We previously demonstrated that inhibition of Cl^– secretion by basolateral NH₄⁺ occurs in HCO₃^–-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH₄⁺ inhibition of current in HCO₃^– buffer did not show anomalous mole fraction behavior and followed the absolute [NH₄⁺] in K⁺-NH₄⁺ mixtures, where K⁺ concentration + [NH₄⁺] = 10 mM. The apical NH₄⁺ inhibitory effect was not prevented by 100 µM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH₄⁺ inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 µM DIDS, 100 µM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 µM niflumic acid, suggesting a role for NH₄⁺ action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO₃^– dependence of apical NH₄⁺ inhibition of secretion is due to the action of NH₄⁺ on an apical anion exchanger.

CFTR; SLC26A3; SLC26A6; downregulated in adenoma; putative anion transporter 1

This article has been cited by other articles:

				R. T. Worrell, L. Merk, and J. B. Matthews Ammonium transport in the colonic crypt cell line, T84: role for Rhesus glycoproteins and NKCC1 Am J Physiol Gastrointest Liver Physiol, February 1, 2008; 294(2): G429 - G440. [Abstract] [Full Text] [PDF]