Book Description
"Transepithelial fluid and ion secretion play a vital role in airway epithelial functions yet the mechanisms which mediate the secretion remain uncertain. In this study, we investigate the potential involvement of two ion exchangers, the basolateral anion exchanger type 2 (AE2, SLC4A2), and the apical anion exchanger pendrin (PDS, SLC26A4) in the secretion process.AE2 carries out Na+-independent Cl-/HCO3- exchange. We have identified the splice variants AE2a and AE2b2 in the Calu-3 cell line, a commonly used model for human airway submucosal gland serous cells. To assess their physiological role, we targeted both variants using lentivirus-mediated RNA interference to generate a stable AE2 knock-down Calu-3 cell line (AE2-KD). Maximally stimulated fluid secretion was reduced in AE2-KD cell monolayers when compared with control monolayers. Moreover, the residual secretion by AE2-KD cells was 4-fold more sensitive to basolateral bumetanide, an inhibitor of the Na+-K+-2Cl cotransporter NKCC1, relative to control cells suggesting a larger fraction of the basolateral Cl- entry was mediated by NKCC1 after knockdown of AE2. Forskolin-stimulated transepithelial currents were also more bumetanide-sensitive in AE2-KD cells. pH-stat measurements of basolateral HCO3- flux in the absorptive direction after permeabilization of the apical membrane indicated that basolateral Cl- /HCO3- exchange in AE2-KD cells was negligible compared to control Calu-3 cells. Thus AE2 appears to be the predominant anion exchanger mediating basolateral HCO3- flux in these cells. Altogether, these results demonstrate that AE2-mediated Cl-/HCO3- exchange contributes to basolateral Cl- entry and plays an important role in anion and fluid secretion by Calu-3 airway epithelial cells.In the next chapter, we focused on the mechanism of HCO3- exchange efflux at the apical membrane of human airway cells. A survey of known exchanger mRNA transcripts revealed several members of the SLC26A family. To assess the potential contribution of electroneutral apical Cl-/ HCO3-exchange mediated by pendrin (PDS, SLC26A4) to HCO3- secretion, we used lentivirus-mediated shRNA to specifically silence pendrin expression in Calu-3 cells and generate a stable pendrin knockdown cell line (PDS-KD). Apical Cl-/ HCO3- exchange assays in Ussing chamber did not support a direct role of PDS in HCO3- secretion, and fluid secretion was also unaffected in PDS-KD Calu-3 cells. We conclude from these studies that pendrin plays little if any role in HCO3- secretion by Calu-3 cells and that most HCO3- exits through CFTR channels.Pendrin is strongly implicated in inflammation, therefore we examined its role in airway epithelial cells under inflammatory conditions. T helper type 2 (Th2) and airway epithelial cells have been shown previously to produce proinflammatory cytokines such as IL-4/ IL-13 which dramatically upregulate pendrin mRNA and protein expression in human bronchial epithelial cell cultures. To test if pendrin upregulation elevates bicarbonate efflux, chronic acid loading and inflammatory signaling, Calu-3 and primary nasal cells were treated for 48h with IL-4. Pendrin expression was measured using quantitative real-time PCR, and fluid and anion transport by the cells were investigated using pH-stat and fluorescence imaging assays. IL-4 failed to increase pendrin expression or HCO3- secretion in Calu-3 cells but dramatically elevated pendrin levels in human primary nasal cells, and this was associated with increased transport of fluid and HCO3-. The responses to IL-4 in nasal cells were attenuated by si-RNA that specifically targets pendrin and were almost abolished by a CFTR inhibitor. These results suggest that increases in HCO3- and fluid secretion induced by IL-4 in primary nasal cells are dependent on both pendrin and CFTR.In summary, these studies clarify the roles of apical and basolateral anion exchangers in secretion by airway epithelia." --