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

• Published in: American journal of physiology: Gastrointestinal and liver physiology 2005-10, Vol.289 (4), p.G768-G778
• Main Authors: Worrell, Roger T, Best, Alison, Crawford, Oscar R, Xu, Jie, Soleimani, Manoocher, Matthews, Jeffrey B
• Format: Article
• Language: English
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; Animals; Antiporters - drug effects; Antiporters - metabolism; Bicarbonates - metabolism; Blotting, Northern; Blotting, Western; Carbachol - pharmacology; Carbonic Anhydrase Inhibitors - pharmacology; Cell Line; Chloride Channels - antagonists & inhibitors; Colon - drug effects; Colon - metabolism; Cyclic AMP - antagonists & inhibitors; Cyclic AMP - pharmacology; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Humans; Immunohistochemistry; Membrane Transport Proteins - metabolism; Muscarinic Agonists - pharmacology; Quaternary Ammonium Compounds - pharmacology; Rats; Reverse Transcriptase Polymerase Chain Reaction; Stilbenes - pharmacology
• ISSN: 0193-1857; 1522-1547
• DOI: 10.1152/ajpgi.00451.2004

Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). 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 NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) 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 HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is due to the action of NH(4)(+) on an apical anion exchanger.