Regulation of intracellular pH in Calu‐3 human airway cells

The Calu‐3 human cell line exhibits features of submucosal gland serous cells and secretes HCO 3 − . The aim of this study was to identify the HCO 3 − transporters present in these cells by studying their role in the regulation of intracellular pH (pH i ). Calu‐3 cells were grown on coverslips, loaded with the pH‐sensitive fluorescent dye BCECF, and their fluorescence intensity monitored as an indication of pH i . Cells were acidified with NH 4 Cl (25 m m , 1 min) and pH i recovery recorded. In the absence of HCO 3 − , initial recovery was 0.208 ± 0.016 pH units min −1 (n = 37). This was almost abolished by removal of extracellular Na + and by amiloride (1 m m ), consistent with the activity of a Na + ‐H + exchanger (NHE). In the presence of HCO 3 − and CO 2 , recovery (0.156 ± 0.018 pH units min −1 ) was abolished (reduced by 91.8 ± 6.7 %, n = 7) by removal of Na + but only attenuated (by 63.3 ± 5.8 %, n = 9) by amiloride. 4,4‐Dinitrostilbene‐2,2‐disulfonic acid (DNDS) inhibited recovery by 45.8 ± 5.0 % (n = 7). The amiloride‐insensitive recovery was insensitive to changes in membrane potential, as confirmed by direct microelectrode measurements, brought about by changing extracellular [K + ] in the presence of either valinomycin or the K + channel opener 1‐EBIO. In addition, forskolin (10 μ m ), which activates the cystic fibrosis transmembrane conductance regulator Cl − conductance in these cells and depolarises the cell membrane, had no effect on recovery. Removal of extracellular Cl − trebled pH i recovery rates, suggesting that an electroneutral, DNDS‐sensitive, Cl − ‐HCO 3 − exchanger together with a NHE may be involved in pH i regulation and HCO 3 − secretion in these cells. RT‐PCR detected the expression of the electrogenic Na + ‐HCO 3 − cotransporter NBC1 and the Cl − ‐HCO 3 − exchanger (AE2) but not the electroneutral Na + ‐HCO 3 − cotransporter NBCn1.