HCO3-dependent pH(i) regulation in tracheal epithelial cells

Regulation of intracellular pR (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 Delta pH/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 pHi 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 mu M SCH28080 (inhibits H,K-ATPase) had a variable inhibitory effect (25-75%), indicating that a gastric-like H,K-ATPase, bur not electrogenic H pump, may contribute in a minor way to the recovery from acidification. Cl-free solution and 500 mu M H2DIDS (dihydro-4,4'-diisothiocyanatastilbene-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 mu M DNDS (4-4 ''-dinitro-2-2'-stilbenedisulfonate blocks the ORCC but not the cystic fibrosis transmembrane conductance regulator, CFTR) had no effect on pHi 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 pHi 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.