Basolateral chloride loading by the anion exchanger type 2: role in fluid secretion by the human airway epithelial cell line Calu-3

Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl-/HCO3- exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short-circuit current (I-sc). We have studied the role of AE2 in Cl- and fluid secretion by the airway epithelial cell line Calu-3. After confirming expression of its mRNA and protein, a knock-down cell line called AE2-KD was generated by lentivirus-mediated RNA interference in which AE2 mRNA and protein levels were reduced >= 90%. Suppressing AE2 increased the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) by similar to 70% without affecting the levels of NKCC1 (Na+-K+-2Cl(-) cotransporter) or NBCe1 (Na+-nHCO(3)(-) cotransporter). cAMP agonists stimulated fluid secretion by parental Calu-3 and scrambled shRNA cells >6.5-fold. In AE2-KD cells this response was reduced by similar to 70%, and the secreted fluid exhibited elevated pH and [HCO3-] as compared with the control lines. Unstimulated equivalent short-circuit current (I-eq) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both I-eq and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl-/HCO3- exchange measured by basolateral pH-stat in cells with permeabilized apical membranes was abolished in AE2-KD monolayers, and the intracellular alkalinization resulting from basolateral Cl- removal was reduced by similar to 80% in AE2-KD cells. These results identify AE2 as a major pathway for basolateral Cl- loading during cAMP-stimulated secretion of Cl- and fluid by Calu-3 cells, and help explain the large bumetanide-insensitive component of fluid secretion reported previously in airway submucosal glands and some other epithelia.