HCO₃-dependent pH_i regulation in tracheal epithelial cells

Abstract

Regulation of intracellular pH (pH_i) was studied in cultured bovine tracheal epithelial cells using microspectrofluorimetry of the fluorescent indicator 2′, 7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The cells, which were grown on coverslips and superfused in a chamber on the stage of a microscope, were acidified by NH₄Cl-prepulses, and pH_i recovery was measured (in ΔpH/min) at approximately pH_i 6.7. In HCO₃-free solutions the recovery rate was 0.14 pH/min, and addition of amiloride or Na-free solution reduced this rate to 0.02–0.03 pH/min. In HCO₃/CO₂-buffered Ringer’s, the rate of recovery was 0.32 pH/min, and amiloride or Na-free reduced the rate to 0.08–0.10 pH/min. This residual Na-independent and HCO₃-dependent pH_i recovery was studied by using inhibitors of HCO₃ and H transporters. Bafilomycin (inhibits H-ATPases) at 100 nM did not significantly affect pH_i recovery, while 100 μM SCH28080 (inhibits H,K-ATPase) had a variable inhibitory effect (25–75%), indicating that a gastric-like H,K-ATPase, but not electrogenic H pump, may contribute in a minor way to the recovery from acidification. Cl-free solution and 500 μM H₂DIDS (dihydro-4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, blocks anion exchange and the outwardly rectifying Cl channel, ORCC), both blocked apparent anion exchange activity, but had no effect on the recovery; 100 μM DNDS (4-4″-dinitro-2-2′-stilbenedisulfonate blocks the ORCC but not the cystic fibrosis transmembrane conductance regulator, CFTR) had no effect on pH_i recovery; DPC (diphenylamine carboxylate, blocks the CFTR and the ORCC) caused a complete and reversible inhibition of the recovery. When [K] was increased ten fold to depolarize the cell’s membrane potential, the magnitude of the pH_i recovery (though not the rate) was enhanced. Thus, the HCO₃-dependent, Na-and Cl-independent, DPC-blockable pH_i recovery may be largely due to an influx of HCO₃ via CFTR Cl channels. Under physiological conditions, when the electrochemical gradient for HCO₃ is likely to be outwardly rather than inwardly directed, the CFTR (or another HCO₃-permeable channel) may mediate HCO₃ secretion and contribute to regulation of pH of the periciliary fluid.