Maintenance and regulation of the pH microclimate at the luminal surface of the distal colon of guinea-pig.

1. 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. 2. 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. 3. 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. 4. 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. 5. The apical K+-H+-ATPase in the distal colon of guinea-pig has little influence on pHs in the presence of physiological buffer concentrations. 6. Our findings indicate that bicarbonate plays a major role in maintaining the pH microclimate at the colonic surface.

Effect of SCFA on intracellular pH and intracellular pH regulation of guinea-pig caecal and colonic enterocytes and of HT29-19a monolayers.

The response of the intracellular pH (pHi, measured with BCECF) of the caecal and distal colonic epithelium of guinea pig and of monolayers of HT29 clone 19a cells on the addition of short-chain fatty acids (SCFA) was assessed. Addition of SCFA to the luminal side of these cells had no major effect on pHi, independent of whether the apical Na+/H+ exchange or the apical K+/H+ ATPase was inhibited or not. Addition of SCFA to the serosal side, on the other hand, caused a marked decrease of pHi, followed by an effective regulation back to basal values, and after removal of the acid, the cells became alkalinized. Intracellular pH is mainly regulated by mechanisms in the basolateral membrane. The basolateral Na+/H+ exchanger and the Cl-/HCO3- exchanger were mainly responsible for pHi regulation. Inhibition studies are consistent with a NHE-1 type Na+/H+ exchanger in the basolateral membranes. The apical Na+/H+ exchanger of caecal enterocytes and in HT29 cells, and the apical K+/H+ ATPase in the apical membrane of the distal colon have no or little influence on pHi regulation. The comparison shows that the HT29-19a cell line is an adequate model for studying pHi phenomena of hind gut epithelial cells.

Surface pH of the distal colonic epithelium of guinea-pigs.

A method for the continuous measurement of proton activity at the luminal surface of the gastro intestinal mucosa was developed. The pH-sensitive fluorescent dye 5-N-hexadecanoyl-amino fluorescein (HAF) was used to monitor the surface pH (pHs). HAF integrates in the apical membrane of the colonic cells because of its amphophilic character. By excitation at two different wavelengths, the ratio of the two fluorescence intensities corresponds to the pHs. pHs in the presence of bicarbonate is relatively independent of the luminal pH. In bicarbonate-free, HEPES-buffered solutions, such a constant "microclimate" was not present. Addition of 113 mmol.l-1 butyrate to the luminal solution caused an increase of the surface pH by 0.14 +/- 0.07 (n = 18) pH units in bicarbonate-containing medium. This increase was not observed in bicarbonate-free medium, indicating that the increase in pHs is due to a gain of bicarbonate during butyrate absorption.