Cytoprotective chloride channels as a molecular target for stress-related factors

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In this study, we tried to clarify the molecular basis of cytoprotective Cl^- channel in the basolateral membrane of gastric parietal cells and the novel inhibitory mechanism of the channel by oxidative stress. The following new results were obtained.1. An expression of mRNA coding CLCA1 was examined to verify the possibility that the CLCA1 mediates cytoprotective Cl^- channels. Although the cytoprotective channels were activated by the elevation of [Ca^<2+>]_i as reported for CLCA1, no significant signal of CLCA1 mRNA was observed in rabbit gastric parietal cells.2. CLC-5 Cl^- channels were found to be expressed in rabbit and hog gastric parietal cells. The distribution of CLC-5 protein was similar to that of H^+,K^+-ATPase in the parietal cells. Interestingly, immunoprecipitation of H^+,K^+-ATPase caused coprecipitation of CLC-5 in the gastric tubulovesicles.3. Effects of interleukin-1β(IL-1β) on the cytoprotective Cl^- channels were investigated. In the whole-cell patch-clamp receding, the Cl^- channel activity was inhibited by IL-1β. The IL-1β-induced inhibition of the Cl^- channel was abolished by anti-IL-1β antibody and recombinant IL-1 receptor antagonist. In the dihydrofluorescein diacetate-loaded parietal cells, IL-1β stimulated the production of oxygen radicals. Y-27632, a specific Rho-kinase inhibitor, significantly inhibited the IL-1β-induced effects on the channel activity and production of oxygen radicals. These results indicate that IL-1β inhibits the cytoprotective Cl^- channel via the Rho/Rho-kinase-dependent production of O_2^-. This inhibition of the Cl^- channel by oxidative stress may be related with the IL-1β-induced cell injury in the stomach.4. The Cl^- channels activated by the NO/cGMP pathway were found in isolated colonic mucosa.