l HBE cells, and we undertook experiments to test the hypothesis that IL-17A would also increase Cl2/HCO32 exchange in these cells. The experimental system we chose to investigate apical Cl2/ HCO32 exchange was one in which Cl2 is rapidly replaced in the mucosal solution by gluconate, an impermeant anion. Cl2 removal from the mucosal bath creates a large concentration gradient for Cl2 from serosal to mucosal across the epithelium. Therefore, in the presence of an apical membrane Cl2/HCO32 MedChemExpress 221244-14-0 exchanger one will see a rise in pHi as HCO32 enters the cell in exchange for Cl2. In the absence of a Cl2/HCO32 exchanger, one would expect to see little or no change in pHi with mucosal Cl2 removal because there is no HCO32 transport pathway. Our data confirm that IL-17A induces a Cl2/HCO32 exchange mechanism at the apical membrane of normal HBE cells that is not present in untreated cells as IL-17A-treated cells but not controls respond to removal of mucosal Cl2 with a rise in pHi. As predicted, this change is reversible with replacement of mucosal Cl2. To test the hypothesis that this pathway required HCO32, we performed the same experiment in the absence of CO2/HCO32 with solutions buffered to pH 7.4 using HEPES. In these experiments, there was no rise in pHi with Cl2 removal, suggesting that HCO32 entry was responsible for the observed rise in pHi. Nonetheless, we observed a small decrease in pHi with Cl2 
replacement. One possible explanation for this change in pHi is that the IL-17A-induced Cl2/HCO32 exchange pathway can also exchange Cl2 for OH2, as has been shown for Pendrin. We note that baseline pH in the CO2/HCO32-free conditions was higher than that in cells in the presence of CO2/HCO32, presumably because lack of CO2 entry into the cells resulted in less H+ generation from carbonic anhydrase. It is also possible, therefore, that we did not see a rise in pHi with Cl2 removal from the mucosal solution because the resistance to HCO32 entry in the presence of elevated pHi was greater than the driving force for Cl2 exit from the cell. A second possibility is that the assay itself failed to detect a rise in pHi because the starting pHi was close to the top of the linear range of detection according to our calibration. However, we note that SNARF dyes demonstrate fluorescence changes at up to pH 9 according to the support material ). B. Consistent with the increase in Pendrin expression, apical membrane Cl2/HCO32 is also increased in CF HBE cells treated with IL-17A..In either case above, our data would underestimate Cl2/HCO32 exchange with Cl2 removal and replacement. Because of these limitations, our data taken together are strongly suggestive of Cl2/ HCO32 exchange, but do not establish an absolute dependence on the presence of HCO32. To test the hypothesis that the IL-17A-induced Cl2/HCO32 exchange pathway was Pendrin, we adapted a previously published method for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19660899 siRNA inhibition of Pendrin expression. We were successful at reducing, but not completely eliminating Pendrin mRNA and protein expression in antiPendrin siRNA-treated cells. This residual expression is likely correlated with the small residual response in pHi to Cl2 removal from the mucosal solution seen in the treated cells. Despite our experimental paradigm, in which we manipulate the direction of Pendrin exchange to transport HCO32 into the IL-17 Induces Pendrin in HBE Cells 7 IL-17 Induces Pendrin in HBE Cells cell, we hypothesize that under physiological conditions in the airw