THE COLON-CARCINOMA CELL-LINE CACO-2 CONTAINS AN H+/K+-ATPASE THAT CONTRIBUTES TO INTRACELLULAR PH REGULATION

The presence of an H+/K+-ATPase and its contribution to the regulation of intracellular pH (pH(i)) was investigated in Caco-2 cells. The H+/K+-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H+/K+-ATPase. Cell pH was determined using the pH-sensitive dye 2',7'-bis(carboxyethyl)-carboxyfluorescein. Control pH(i), measured in HCO3--free medium, was 7.62+/-0.03 (n = 27) when cells were cultured for 14 days and decreased to 7.40+/-0.03 (n = 18) after 35 days in culture. Recovery of pH(i) following a NH4+/NH3 pulse could be reduced by either 100 muM SCH 28080 or 1 mM amiloride, or by removing extracellular Na+. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H+/K +-ATPase and a Na+/H+ exchanger in regulating pH(i). Recovery rates at pH(i) 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na+/H+ exchange to the recovery. Recovery of pH(i) was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethylmaleimide inhibited a vacuolar type of H+-ATPase, since bafilomycin A1 had no effect on pH(i) recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pH(i), which is most conveniently studied after 28 days in culture, when the relative contribution of a Na+/H+ exchanger to pH(i) regulation is decreased.