MASS-SPECTROMETRIC DETERMINATION OF HCO3- PERMEABILITY AND CARBONIC-ANHYDRASE ACTIVITY IN INTACT GUINEA-PIG COLON EPITHELIUM

1. A mass spectrometric method originally used in red blood cells was applied to suspensions of isolated colonocytes and intact colonic epit helium to measure the exchange of O-18 between HCO3-, CO2 and H2O to d etermine intracellular carbonic anhydrase activity (A(i)) and membrane bicarbonate permeability (P). 2. In suspensions of isolated guinea-pi g colon epithelial cells, colonocytes, we found significantly higher v alues of A(i) and P for cells derived from the proximal colon than for cells from the distal colon. In the case of A(i), this confirms earli er reports. 3. When the O-18 exchange process was observed across the mucosal (apical) side of intact colon mucosa, the estimated values of A(i) were identical to those obtained for isolated colonocytes, for bo th the proximal and the distal part of the colon. This is considered t o be strong evidence that this method can be applied to a layer of int act epithelium as well as to cell suspensions. 4. The values of P obta ined from the apical side of intact colon mucosa were 6 times higher t han those estimated from measurements with isolated colonocytes. This indicates that the basolateral membrane of colon epithelium, which par ticipates in the O-18 exchange process in isolated colonocytes but not in the O-18 exchange process across the apical side of intact mucosa, has a markedly lower bicarbonate permeability than the apical membran e. 5. When the O-18 exchange process was observed across the serosal ( basolateral) side of intact colon mucosa, the P values, as expected, w ere low compared with the apical side of intact mucosa. However, rathe r unexpectedly, the A, values derived from these measurements were 2-3 times lower than those obtained with isolated colonocytes. It appears possible that the latter finding is an artifact due to the submucosal tissue markedly slowing down CO2 diffusion from the bathing medium in to the epithelial cells, thus causing an apparent fall in A(i). 6. A(i ) decreased and P increased with increasing temperature, as expected, when studied on the mucosal side of intact colon. This provides additi onal support for the validity of the method.