title: Role of anion exchangers in Cl- and HCO3- secretion by the human airway epithelial cell line Calu-3.
abstract: Despite the importance of airway surface liquid pH in the lung's defenses against infection, the mechanism of airway HCO3- secretion remains unclear. Our aim was to assess the contribution of apical and basolateral Cl-/HCO3- exchangers to Cl- and HCO3- transport in the Calu-3 cell line, derived from human airway submucosal glands. Changes in intracellular pH (pHi) were measured following substitution of Cl- with gluconate. Apical Cl- substitution led to an alkalinization in forskolin-stimulated cells, indicative of Cl-/HCO3- exchange. This was unaffected by the anion exchange inhibitor DIDS but inhibited by the CFTR blocker CFTRinh-172, suggesting that the HCO3- influx might occur via CFTR, rather than a solute carrier family 26 (SLC26) exchanger, as recently proposed. The anion selectivity of the recovery process more closely resembled that of CFTR than an SLC26 exchanger, and quantitative RT-PCR showed only low levels of SLC26 exchanger transcripts relative to CFTR and anion exchanger 2 (AE2). For pHi to rise to observed values (∼7.8) through HCO3- entry via CFTR, the apical membrane potential must reverse to at least +20 mV following Cl- substitution; this was confirmed by perforated-patch recordings. Substitution of basolateral Cl- evoked a DIDS-sensitive alkalinization, attributed to Cl-/HCO3- exchange via AE2. This appeared to be abolished in forskolin-stimulated cells but was unmasked by blocking apical efflux of HCO3- via CFTR. We conclude that Calu-3 cells secrete HCO3- predominantly via CFTR, and, contrary to previous reports, the basolateral anion exchanger AE2 remains active during stimulation, providing an important pathway for basolateral Cl- uptake.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom;
authors.forename: Dusik
authors.initials: D
authors.lastname: Kim
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom;
authors.forename: Juyeon
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Oral Biology, Semmelweis University, Budapest, Hungary; and.
authors.forename: Beáta
authors.initials: B
authors.lastname: Burghardt
authors.affiliation: Faculty of Medicine and Human Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Len
authors.initials: L
authors.lastname: Best
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom; martin.steward@manchester.ac.uk.
authors.forename: Martin C
authors.initials: MC
authors.lastname: Steward
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001639:Bicarbonates / Q000378:metabolism*; D002460:Cell Line; D027963:Chloride-Bicarbonate Antiporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism
chemical_list: D001639:Bicarbonates; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
keywords: Cl−/HCO3− exchange; SLC26A4 (pendrin); airway surface liquid; anion exchanger 2; bicarbonate; cystic fibrosis; cystic fibrosis transmembrane conductance regulator; intracellular pH