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 HCO3-. The aim of this study was to identify the HCO3- transporters present in these cells by studying their role in the regulation of intracellular pH (pHi). Calu-3 cells were grown on coverslips, loaded with the pH-sensitive fluorescent dye BCECF, and their fluorescence intensity monitored as an indication of pHi. Cells were acidified with NH4Cl (25 mm, 1 min) and pHi recovery recorded. In the absence of HCO3-, 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 mm), consistent with the activity of a Na+-H+ exchanger (NHE). In the presence of HCO3- and CO2, 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 µm), 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 pHi recovery rates, suggesting that an electroneutral, DNDS-sensitive, Cl--HCO3- exchanger together with a NHE may be involved in pHi regulation and HCO3- secretion in these cells. RT-PCR detected the expression of the electrogenic Na+-HCO3- cotransporter NBC1 and the Cl--HCO3- exchanger (AE2) but not the electroneutral Na+-HCO3- cotransporter NBCn1.