Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent.
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.
Worrell
Roger T
RT
Epithelial Pathobioloby Group, Dept. of Surgery, University of Cincinnati, OH 45219, USA. Roger.Worrell@uc.edu
Best
Alison
A
Crawford
Oscar R
OR
Xu
Jie
J
Soleimani
Manoocher
M
Matthews
Jeffrey B
JB
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000187:drug effects / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002460:Cell Line; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D003106:Colon / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D026901:Membrane Transport Proteins / Q000378:metabolism; D018721:Muscarinic Agonists / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013267:Stilbenes / Q000494:pharmacology; D000076988:Sulfate Transporters
D017920:Antiporters; D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D018118:Chloride Channels; D026901:Membrane Transport Proteins; D018721:Muscarinic Agonists; D000644:Quaternary Ammonium Compounds; C439529:SLC26A6 protein, human; C489861:Slc26a3 protein, rat; D013267:Stilbenes; D000076988:Sulfate Transporters; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D002217:Carbachol; D000242:Cyclic AMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid