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

Normal human colonic luminal (NH4+) concentration ([NH4+]) ranges from similar to 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 mu M 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 mu M DIDS, 100 mu M 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 mu M 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.