PH REGULATION IN HT29 COLON-CARCINOMA CELLS

The pH regulation in HT29 colon carcinoma cells has been investigated using the pH-sensitive fluorescent cent indicator 2',7'-biscarboxyethyl- 5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pH(i)) was 7.21 +/- 0.07 (n = 22) in HCO3--containing and 7.21 +/- 0.09 (n = 12) in HCO3--free solution. HOE-694 (10 mu mol/l), a potent inhibitor of the Na+/H+ exchanger, did not affect control pH(i). As a means to acidify cells we used the NH4+/NH3 (20 mmol/l) prepulse technique. The mean peak acidification was 0.37 +/- 0.07 pH units (n = 6). In HCO3--free solutions recovery from acid load was completely blocked by HOE-694 (1 mu mol/l), whereas in HCO3--containing solutions a combination of HOE-694 and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na+-dependent in HCO3--containing and HCO3--free solutions. Removal of external Cl- caused a rapid, DIDS-blockable alkalinization of 0.33 +/- 0.03 H units (n = 15) and of 0.20 +/- 0.006 pH units (n = 5), when external Na+ was removed together with Cl-. This alkalinization was faster in HCO3--containing than in HCO3--free solutions. The present observations demonstrate three distinct mechanisms of pH(i) regulation in HT29 cells: (a) a Na+/H+ exchanger, (b) a HCO3-/Cl- exchanger and (c) a Na+-dependent HCO3- transporter, probably the Na+-HCO3-/Cl- antiporter. Under HCO3--free conditions the Na+/H+ exchanger fully accounts for recovery from acid load, whereas in HCO3--containing solutions this is accomplished by the Na+/H+ exchanger and a Na+-dependent mechanism, which imports HCO3-. Recovery from alkaline load is caused by the HCO3-/Cl- exchanger.