DETERMINATION OF INTRACELLULAR CARBONIC-ANHYDRASE ACTIVITY AND BICARBONATE PERMEABILITY IN INTACT COLON EPITHELIA BY OBSERVATION OF THE O-18-LABELING OF CO2

We applied a mass spectrometric method, developed by Itada and Forster [1] for erythrocyte suspensions, which observes the exchange of O-18 between HCO3-, CO2 and H2O, to determine intracellular carbonic anhydr ase 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 measuremen ts of A(i) 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 A(i) values of colonocytes in suspensio n and in the intact colon epithelium. In addition we tested the temper ature dependence of A(i) 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 Itad a and Forster [1] with intact colon epithelial sheets. This new applic ation allows us to study separately the apical and serosal membrane of the epithelial cells. We used the method to determine whether there i s a short chain Fatty acid-bicarbonate exchanger in the apical or sero sal 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 api cal membrane of the proximal colon we find a higher P in presence of p ropionate than in its absence, showing that there the transport of sho rt chain Fatty acids is coupled to that of bicarbonate.