pH regulation in HT29 colon carcinoma cells

The pH regulation in HT₂₉ colon carcinoma cells has been investigated using the pH-sensitive fluorescent 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 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 HCC₃^–-free solutions recovery from acid load was completely blocked by HOE-694 (1 mol/l), whereas in HCO3₃^–-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 pH 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 regulation in HT₂₉ cells: (a) a Na⁺/H⁺ exchanger, (b) a HCO₃^–/Cl^– exchanger and (c) a Na⁺-dependent HCC₃^–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.This study was supported by DFG Gr 480/10