A novel role for carbonic anhydrase: cytoplasmic pH gradient dissipation in mouse small intestinal enterocytes

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Abstract
The spatial and temporal distribution of intracellular H+ ions in response to activation of a proton-coupled dipeptide transporter localized at the apical pole of mouse small intestinal isolated enterocytes was investigated using intracellular carboxy-SNARF-1 fluorescence in combination with whole-cell microspectrofluorimetry or confocal microscopy.In Hepes-buffered Tyrode solution, application of the dipeptide Phe-Ala (10 mM) to a single enterocyte reduced pHi locally in the apical submembranous space. After a short delay (8 s), a fall of pHi occurred more slowly at the basal pole.In the presence of CO2/HCO3−-buffered Tyrode solution, the apical and basal rates of acidification were not significantly different and the time delay was reduced to 1 s or less.Following application of the carbonic anhydrase inhibitor acetazolamide (100 μM) in the presence of CO2/HCO3− buffer, addition of Phe-Ala once again produced a localized apical acidification that took 5 s to reach the basal pole. Basal acidification was slower than at the apical pole.We conclude that acid influx due to proton-coupled dipeptide transport can lead to intracellular pH gradients and that intracellular carbonic anhydrase activity, by facilitating cytoplasmic H+ mobility, limits their magnitude and duration.