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The fluorescent dye 5-N-hexadecanoyl-aminofluorescein (HAF) was used to study the mechanisms involved in maintaining a relatively constant luminal surface pH (pHs) in the distal colon of the guinea-pig. The fatty acyl chain of the HAF molecule inserts into the apical membrane of epithelial cells. This allows a continuous measurement of the surface pH for several hours.
The localization of HAF was confirmed by confocal laser-scanning microscopy and by using monoclonal antibodies against fluorescein. The insertion of HAF into the apical membrane of the colonocytes did not change the transepithelial conductance or the short-circuit current of the epithelium.
With the HAF method a pH microclimate was confirmed at the colonic surface. Although the pH of the bulk luminal solution was decreased in bicarbonate-containing solution from 7·4 to 6·4 the pHs changed only in the range 7·54-6·98.
In the absence of bicarbonate pHs almost followed changes of bulk luminal pH. In the presence of bicarbonate there was a decrease in pHs after removal of chloride from the luminal side and an increase in pHs after addition of butyrate to the luminal solution. This suggests the involvement of a bicarbonate-anion exchange in bicarbonate secretion: a Cl--HCO3- as well as a short-chain fatty acid--HCO3- exchange.
The apical K+-H+-ATPase in the distal colon of guinea-pig has little influence on pHs in the presence of physiological buffer concentrations.
Our findings indicate that bicarbonate plays a major role in maintaining the pH microclimate at the colonic surface.
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