Determination of Intracellular Carbonic Anhydrase Activity and Bicarbonate Permeability in Intact Colon Epithelia by Observation of the 18O-labelling of Co2

Abstract

We applied a mass spectrometric method, developed by Itada and Forster [1] for erythrocyte suspensions, which observes the exchange of 18O between HCO3 −, CO2 and H2O, to determine intracellular carbonic anhydrase activity, A i , and bicarbonate permeability, P, in intact guinea pig colon epithelium. To study the validity of the results with intact distal and proximal colon epithelia, we compared them with measurements of Ai and P in suspensions of isolated epithelial cells from the distal and proximal guinea pig colon. In both parts of the colon there is good agreement between the Ai values of colonocytes in suspension and in the intact colon epithelium. In addition we tested the temperature dependence of Ai and P, the oxygen supply to the colon during the measurements, and the intactness of the epithelial cells by trypan blue staining. We conclude that it is valid to use the method of Itada and Forster [1] with intact colon epithelial sheets. This new application allows us to study separately the apical and serosal membrane of the epithelial cells. We used the method to determine whether there is a short chain fatty acid – bicarbonate exchanger in the apical or serosal membrane of proximal and distal colon epithelium. For this purpose we measured the membrane bicarbonate permeability in the presence and absence of 25 mM propionate in the reaction solution. Only in the apical membrane of the proximal colon we find a higher P in presence of propionate than in its absence, showing that there the transport of short chain fatty acids is coupled to that of bicarbonate.