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 fluores cent 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 addi tion of amiloride or Na-free solution reduced this rate to 0.02-0.03 p H/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. T his residual Na-independent and HCO3-dependent pHi recovery was studie d by using inhibitors of HCO3 and H transporters, Bafilomycin (inhibit s H-ATPases)at 100 nM did not significantly affect pH(i) recovery, whi le 100 mu M SCH28080 (inhibits H,K-ATPase) had a variable inhibitory e ffect (25-75%), indicating that a gastric-like H,K-ATPase, bur not ele ctrogenic H pump, may contribute in a minor way to tile recovery from acidification. Cl-free solution and 500 mu M H2DIDS ydro-4,4'-diisothi ocyanatastilbene-2,2'-disulfonic acid, blocks anion exchange and the o utwardly rectifying Cl channel, ORCC), both blocked apparent anion exc hange 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 p Hi recovery DPC (diphenylamine carboxylate; blocks the CFTR and the OR CC) 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 channel s. Under physiological conditions, when the electrochemical gradient f or HCO3 is likely to be outwardly rather than inwardly directed, the C FTR (or another HCO3-permeable channel) may mediate HCO3 secretion and contribute to regulation of pH of the periciliary fluid.