Role of CFTR and chloride/bicarbonate exchangers in airway epithelial bicarbonate secretion

HCO3 secretion plays a vital role in regulating the pH and mucus viscosity of airway surface liquid to facilitate airway mucociliary clearance of inhaled pathogen. In cystic fibrosis (CF), reduced HCO3 secretion contributes to defective mucociliary clearance which predisposes the lungs to bacterial infection. Calu-3 cells are used as a model of human submucosal gland serous cells which are involved in CFTR (cystic fibrosis transmembrane conductance regulator)-dependent HCO3 secretion, a process that appears to involve functional interactions with both apical and basolateral Cl/HCO3 anion exchangers (AE), but through regulatory pathways that are not well understood. The aim of this thesis was to investigate the signalling mechanisms that regulate CFTR-dependent AE activity in Calu-3 cells. Under resting conditions, Calu-3 cells showed a DIDS-sensitive Cl and HCO3dependent basolateral anion exchange activity consistent with AE2 (SLC4A2) expression. However, apical AE activity was not detected. Increasing cytosolic Ca, or removal of extracellular Ca, had no effect on basolateral AE activity. In contrast, lowering cytosolic Ca with BAPTA-AM, or inhibiting calmodulin (CaM), reduced basolateral AE activity. Furthermore, an intact actin cytoskeleton, as well as active dynamin, were essential for maintaining basolateral AE activity, possibly via supply of new proteins to the basolateral membrane. Inhibiting CK2 or protein phosphatase 1 (PP1) abolished basolateral AE activity, and CK2 inhibition was linked to CaM. This suggests that AE activity was maintained through a novel CaM-dependent mechanism involving phosphorylation/dephosphorylation by CK2/PP1. In support of this, transient transfection of HEK293 cells with mouse AE2, with and without CK2 cotransfection, clearly demonstrated CK2-dependent AE2 activity. Stimulation of Calu-3 cells with cAMP agonists both activated an apical anion exchanger via a PKA and Epac-dependent mechanism, and inhibited the basolateral anion exchanger, but through a PKA and Epac-independent mechanism. Blocking CFTR with GlyH-101 caused an apparent inhibition of apical AE activity, but addition of basolateral DIDS restored apical activity, suggesting that a basolateral HCO3 transporter was activated when CFTR was inhibited. Removal of extracellular Ca partially reduced the cAMP-induced inhibition of the basolateral AE activity, but had no effect on cAMP-stimulated apical AE activity. Moreover, increasing cytosolic Ca, or lowering cytosolic Ca with BAPTA-AM, markedly reduced cAMPstimulated apical AE activity, but it had no effect on cAMP-induced inhibition of the basolateral anion exchanger. Actin-cytoskeleton disruption had no effect on apical AE activity but dynamin inhibition caused a significant decrease. A similar decrease in apical AE activity was observed when CK2 was inhibited, but in contrast to the basolateral anion exchanger, this appeared to be via a CaM-independent mechanism. Inhibiting CK2, however, had no effect on the cAMP-induced inhibition of the basolateral AE activity, suggesting that CK2 regulation of Calu-3 anion exchangers is through cAMP-independent mechanisms. These findings provide new insights into the signalling pathways that regulate both the apical and basolateral anion exchangers in Calu-3 cells and help define their respective roles in airway HCO3 secretion. The results could potentially open up new avenues for modulating AE activity which could be beneficial in HCO3 secretory diseases such as CF.