The role of SLC26 transporters in airway epithelial cell function

Abstract

HC0₃⁻ plays a vital role in the airways, as mucus viscosity and ciliary beat have both been shown to be dependent upon the pH of the airway surface liquid. In cystic fibrosis (CF), reduced HC0₃⁻ and fluid secretion produce dehydrated mucus, which impairs airway defence and predisposes the airways to bacterial infection. Calu-3 cells are used as a model of the serous cells of human tracheobronchial submucosal glands which are involved in CFTR (cystic fibrosis transmembrane conductance regulator)dependent HC03⁻ secretion. CFTR is widely regarded as the sole mediator of apical C1⁻ and HC0₃⁻ secretion in Calu-3 cells and human serous airway cells. However, the discovery ofSLC26 C1⁻/HC0₃⁻ exchange activity linked to the expression of CFTR in tracheal epithelial cells, as well as a functional interaction between apically co-localised SLC26 exchangers and CFTR in mediating HC03- secretion in many HC0₃⁻ secreting epithelia including the pancreas and gastrointestinal tract, has led to some doubt over this hypothesis. The aim of this work was to investigate the potential role of SLC26 anion exchange (AE) in HC0₃⁻ secretion from Calu-3 cells and its dependence on CFTR. AE activity was assessed by real time measurements of intracellular pH (pHi) using the pH-sensitive dye BCECF-AM, from cells grown as monolayers on semi-permeable supports. Calu-3 cells under non-stimulated conditions displayed Na⁺-dependent basolateral C1⁻/HC0₃⁻ exchange, which was abolished by H₂DIDS. No apical AE activity could be detected under resting conditions. Stimulation of cells with a cAMP agonist (forskolin, adenosine, VIP) produced a switch in AE activity, activating an apical AE and completely inhibiting the basolateral AE. The cAMP-activated apical AE activity was Na⁺-independent, H₂-DIDS-insensitive and could transport a range of monovalent anions in exchange for HC0₃⁻ with the selectivity profile: Iodide=Br->Cr=Formate =N0₃⁻=SCN-. The profile of this anion exchanger corresponds with Pendrin (SLC26A4), which quantitative RT-PCR analysis showed to be expressed in these Calu-3 cells. Consistent with this, Pendrin knockdown (KD) Calu-3 cells had a reduced rate of AE activity, compared to wild-type (WT) cells. Protein phosphatase 1 inhibition by okadaic acid activated an apical AE under non-stimulated conditions with a similar profile to Pendrin, which CFTR inhibitor studies demonstrated to be CPTR-independent. Interestingly, fluid secretion studies established that Calu-3 cells produced a more alkali fluid when treated with okadaic acid, consistent with enhanced HC0₃⁻ secretion. The role of CFTR in C1⁻HC0₃⁻ AE activity was assessed using CFTR inhibitors and CFTR KD Calu-3 cells. Although apical AE activity in WT Calu-3 cells could be abolished by inhibiting CFTR (with either CFTRinh-172 or GlyH-lOl), it could be restored by the addition of basolateral H₂-DIDS, suggesting that the CFTR inhibition reveals a H₂-DIDS-sensitive basolateral transport process which masks the effects of apical AE on pHi, rather than abolishing apical AE activity itself. In CFTR KD Calu-3 cells the rate of apical AE activity was significantly decreased compared to WT cells. These results suggest a role for CFTR in regulating apical C1⁻HC0₃⁻ exchange activity and/or contributing to C1⁻dependent HCO₃⁻ transport. Similarly cAMP inhibition of the basolateral AE was also found to be mediated via a CFTR-dependent mechanism, highlighting the importance of CFTR in the cAMP-dependent switch in Calu-3 AE activity. Such a switch in AE activity by cAMP, would favour bicarbonate secretion, consistent with the finding that cAMP enhances HC0₃⁻-dependent fluid secretion from Calu-3 cells. Apical AE activity was also observed in human bronchial epithelial cells (HBECs), which could not be blocked by GlyH-101, but was absent in CF HBECs.