Key points• The mechanisms of anion and fluid transport by airway submucosal glands are not well understood and may differ from those in surface epithelium.• The Calu-3 cell line is often used as a model for submucosal gland serous cells and has cAMP-stimulated fluid secretion; however, it does not actively transport chloride under short-circuit conditions.• In this study we show that fluid secretion requires chloride, bicarbonate and sodium, that chloride is the predominant anion in Calu-3 secretions, and that a large fraction of the basolateral chloride loading during cAMP stimulation occurs by Cl − /HCO 3 − exchange.• The results suggest a novel cellular model for anion and fluid secretion by Calu-3 and submucosal gland acinar cells Abstract Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl − and HCO 3 − secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (I sc ) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (I eq ) calculated under open-circuit conditions. I sc was equivalent to the HCO 3 − net flux measured using the pH-stat technique, whereas I eq was the sum of the Cl − and HCO 3 − net fluxes. I eq and HCO 3 − fluxes were increased by bafilomycin and ZnCl 2 , suggesting that some secreted HCO 3 − is neutralized by parallel electrogenic H + secretion. I eq and fluid secretion were dependent on the presence of both Na + and HCO 3 − . The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of I eq and HCO 3 − secretion, suggesting that HCO 3 − transport under these conditions requires catalysed synthesis of carbonic acid. Cl − was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50-70% of Cl − and fluid transport was bumetanide-insensitive, suggesting basolateral Cl − loading by a sodium-potassium-chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO 3 − gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO 3 with Cl − , and the resulting HCO 3 − -dependent Cl − transport provides an osmotic driving force for fluid secretion.

Bicarbonate plays an important role in airway host defense, however, its transport mechanisms remain uncertain. Here we examined the relative contributions of the anion channel CFTR (cystic fibrosis transmembrane conductance regulator, ABCC7) and the anion exchanger pendrin (SLC26A4) to HCO 3 − secretion by the human airway cell line Calu‐3. Pendrin and CFTR were both detected in parental Calu‐3 cells, although mRNA and protein expression appeared higher for CFTR than for pendrin. Targeting pendrin transcripts with lentiviral shRNA reduced pendrin detection by immunofluorescence staining but did not alter the rates of HCO 3 − or fluid secretion, HCO 3 − transport under pH‐stat conditions, or net HCO 3 − flux across basolaterally permeabilized monolayers. Intracellular pH varied with step changes in apical Cl− and HCO 3 − concentrations in control and pendrin knockdown Calu‐3 cells, but not in CFTR deficient cells. Exposure to the proinflammatory cytokine IL‐4, which strongly upregulates pendrin expression in airway surface epithelia, had little effect on Calu‐3 pendrin expression and did not alter fluid or HCO 3 − secretion. Similar results were obtained using air–liquid interface and submerged cultures, although CFTR and pendrin mRNA expression were both lower when cells were cultured under submerged conditions. While the conclusions cannot be extrapolated to other airway epithelia, the present results demonstrate that most HCO 3 − secretion by Calu‐3 cells is mediated by CFTR.

Anion transport drives fluid into the airways and is essential for humidifying inspired air and supplying surface liquid for mucociliary transport. Despite the importance of airway secretion in diseases such as cystic fibrosis, the cellular mechanisms remain poorly understood, in part due to the small size and complicated structure of the submucosal glands that produce most of the fluid. The Calu-3 human lung adenocarcinoma cell line has become a popular model for studying airway secretion because it can be cultured as a flat sheet, expresses the cystic fibrosis transmembrane conductance regulator and several acinar cell markers, forms polarized monolayers with tight junctions, has robust cAMP-stimulated anion transport, and responds to secretagogues that regulate the glands in vivo. However, some properties of Calu-3 cells are less consistent with those of native tissue. In particular, Calu-3 monolayers do not secrete chloride when stimulated by forskolin under short-circuit conditions. Bicarbonate ions are thought to carry the short-circuit current (I(sc)) and the drive secretion of alkaline fluid, in contrast to the neutral pH secretions that are produced by submucosal glands. Calu-3 cells also have abnormal chromosomes and characteristics of both serous and mucus cells. In this article, we discuss Calu-3 as a model in light of our ongoing studies, which suggest that Calu-3 monolayers resemble submucosal glands more closely than was previously thought. For example, we find that net HCO(3)(-) flux fully accounts for I(sc) as previously suggested but Cl(-) is the main anion transported under physiological conditions. A novel, HCO(3)(-) -dependent mechanism of Cl(-) transport is emerging which may explain secretion by Calu-3 and perhaps other epithelial cells.

Key points• The companion paper provided evidence for basolateral anion exchange during cAMP stimulation of chloride and fluid secretion by Calu-3 monolayers; however, the molecular basis of this transport was not identified.• To test the role of AE2, an anion exchanger expressed at the basolateral membrane of Calu-3 and many other epithelial cells, we used lentivirus-mediated RNA interference to generate a stable Calu-3 AE2 knock-down cell line and characterized its fluid and anion transport properties.• AE2 knock-down suppressed fluid secretion and increased the fraction of cAMP-stimulated anion secretion that was sensitive to bumetanide inhibition.• Basolateral Cl − /HCO 3 − exchange was nearly abolished in AE2 knock-down cells.• We conclude that AE2 is active during forskolin-stimulated fluid secretion and mediates chloride uptake and bicarbonate recycling at the basolateral membrane.Abstract Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl − /HCO 3 − exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short-circuit current (I sc ). We have studied the role of AE2 in Cl − and fluid secretion by the airway epithelial cell line Calu-3. After confirming expression of its mRNA and protein, a knock-down cell line called AE2-KD was generated by lentivirus-mediated RNA interference in which AE2 mRNA and protein levels were reduced ≥90%. Suppressing AE2 increased the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) by ∼70% without affecting the levels of NKCC1 (Na. cAMP agonists stimulated fluid secretion by parental Calu-3 and scrambled shRNA cells >6.5-fold. In AE2-KD cells this response was reduced by ∼70%, and the secreted fluid exhibited elevated pH and [HCO 3 − ] as compared with the control lines. Unstimulated equivalent short-circuit current (I eq ) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both I eq and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl for basolateral Cl − loading during cAMP-stimulated secretion of Cl − and fluid by Calu-3 cells, and help explain the large bumetanide-insensitive component of fluid secretion reported previously in airway submucosal glands and some other epithelia.

Interest in precision medicine has grown in recent years due to the variable clinical benefit provided by some medications, their cost, and by new opportunities to tailor therapies to individual patients. In cystic fibrosis it may soon be possible to test several corrector drugs that improve the folding and functional expression of mutant cystic fibrosis transmembrane conductance regulator (CFTR) prospectively using cells from a patient to find the one that is best for that individual. Patient-to-patient variation in cell culture responses to correctors and the reproducibility of those responses has not been studied quantitatively. We measured the functional correction provided by lumacaftor (VX-809) using bronchial epithelial cells from 20 patients homozygous for the F508del-CFTR mutation. Significant differences were observed between individuals, supporting the utility of prospective testing. However, when correction of F508del-CFTR was measured repeatedly using cell aliquots from the same individuals, a design effect was observed that would impact statistical tests of significance. The results suggest that the sample size obtained from power calculations should be increased to compensate for group sampling when CFTR corrector drugs are compared in vitro for precision medicine.