Mass spectrometric determination of HCO3- permeability and carbonic anhydrase activity in intact guinea-pig colon epithelium.

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RESUMO

1. A mass spectrometric method originally used in red blood cells was applied to suspensions of isolated colonocytes and intact colonic epithelium to measure the exchange of 18O between HCO3-, CO2 and H2O to determine intracellular carbonic anhydrase activity (Ai) and membrane bicarbonate permeability (P). 2. In suspensions of isolated guinea-pig colon epithelial cells, colonocytes, we found significantly higher values of Ai and P for cells derived from the proximal colon than for cells from the distal colon. In the case of Ai, this confirms earlier reports. 3. When the 18O exchange process was observed across the mucosal (apical) side of intact colon mucosa, the estimated values of Ai were identical to those obtained for isolated colonocytes, for both the proximal and the distal part of the colon. This is considered to be strong evidence that this method can be applied to a layer of intact epithelium as well as to cell suspensions. 4. The values of P obtained from the apical side of intact colon mucosa were 6 times higher than those estimated from measurements with isolated colonocytes. This indicates that the basolateral membrane of colon epithelium, which participates in the 18O exchange process in isolated colonocytes but not in the 18O exchange process across the apical side of intact mucosa, has a markedly lower bicarbonate permeability than the apical membrane. 5. When the 18O exchange process was observed across the serosal (basolateral) side of intact colon mucosa, the P values, as expected, were low compared with the apical side of intact mucosa. However, rather unexpectedly, the Ai 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 into the epithelial cells, thus causing an apparent fall in Ai. 6. Ai decreased and P increased with increasing temperature, as expected, when studied on the mucosal side of intact colon. This provides additional support for the validity of the method.

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