Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3

Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl^- and HCO₃^- 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 HCO₃^- net flux measured using the pH-stat technique, whereas I_eq was the sum of the Cl^- and HCO₃^- net fluxes. I_eq and HCO₃^- fluxes were increased by bafilomycin and ZnCl₂, suggesting that some secreted HCO₃^- is neutralized by parallel electrogenic H⁺ secretion. I_eq and fluid secretion were dependent on the presence of both Na⁺ and HCO₃^-. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of I_eq and HCO₃^- secretion, suggesting that HCO₃^- 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 HCO₃^- 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 HCO₃^- secretion was increased by bilateral Cl^- removal and therefore did not require apical Cl^-/HCO₃^- exchange. The results suggest a model in which most HCO₃^- is recycled basolaterally by exchange with Cl^-, and the resulting HCO₃^--dependent Cl^- transport provides an osmotic driving force for fluid secretion.