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 (pHi) 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 pHi, measured in HCO(3-)-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 pHi following a NH+4/NH3 pulse could be reduced by either 100 microM 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 pHi. Recovery rates at pHi 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 pHi was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethyl-maleimide inhibited a vacuolar type of H+-ATPase, since bafilomycin A1 had no effect on pHi recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pHi, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na+/H+ exchanger to pHi regulation is decreased.
Abrahamse
S L
SL
Department of Physiology, University of Nijmegen, The Netherlands.
Bindels
R J
RJ
van Os
C H
CH
D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000897:Anti-Ulcer Agents / Q000494:pharmacology; D002277:Carcinoma / Q000201:enzymology*; D003110:Colonic Neoplasms / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects*; D007093:Imidazoles / Q000494:pharmacology; D018942:Macrolides; D012965:Sodium Chloride / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000900:Anti-Bacterial Agents; D000897:Anti-Ulcer Agents; D007093:Imidazoles; D018942:Macrolides; C035235:Sch 28080; D000643:Ammonium Chloride; C057620:bafilomycin A; D012965:Sodium Chloride; D000584:Amiloride; D017506:H(+)-K(+)-Exchanging ATPase; D005033:Ethylmaleimide