CFTR and calcium-activated chloride channels in primary cultures of human airway gland cells of serous or mucous phenotype

Abstract

Using cell culture models, we have investigated the relative importance of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCC) in Cl secretion by mucous and serous cells of human airway glands. In transepithelial recordings in Ussing chambers, the CFTR inhibitor CFTR(inh)-172 abolished 60% of baseline Cl secretion in serous cells and 70% in mucous. Flufenamic acid (FFA), an inhibitor of CaCC, reduced baseline Cl secretion by ∼20% in both cell types. Methacholine and ATP stimulated Cl secretion in both cell types, which was largely blocked by treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and partially by mucosal FFA or CFTR(inh)-172 with the exception of methacholine responses in mucous cells, which were not blocked by FFA and partially (∼60%) by CFTR(inh)-172. The effects of ionomycin on short-circuit current (I(sc)) were less than those of ATP or methacholine. Forskolin stimulated Cl secretion only if Cl in the mucosal medium was replaced by gluconate. In whole cell patch-clamp studies of single isolated cells, cAMP-induced Cl currents were ∼3-fold greater in serous than mucous cells. Ionomycin-induced Cl currents were 13 times (serous) or 26 times (mucous) greater than those generated by cAMP and were blocked by FFA. In serous cells, mRNA for transmembrane protein 16A (TMEM16A) was ∼10 times more abundant than mRNA for CFTR. In mucous cells it was ∼100 times more abundant. We conclude: 1) serous and mucous cells both make significant contributions to gland fluid secretion; 2) baseline Cl secretion in both cell types is mediated predominantly by CFTR, but CaCC becomes increasingly important after mediator-induced elevations of intracellular Ca; and 3) the high CaCC currents seen in patch-clamp studies and the high TMEM16A expression in intact polarized cells sheets are not reflected in transepithelial current recordings.

Epinephrine stimulation of anion secretion in the Calu-3 serous cell model

“…Effectors that are specific for increasing intracellular Ca 2ϩ such as carbachol, histamine, methacholine, and ATP as well as effectors such as forskolin that increase cAMP all increase Cl Ϫ /HCO 3 Ϫ secretion (1,11,21,22,26,36). In this regard, results in the Calu-3 cell line agree with both human and porcine primary cultures (11,40).…”

“…However, in addition to the importance of Ca 2ϩ -activated K ϩ channels, more recent studies have demonstrated the presence of an apical CaCC. TMEM16A is highly expressed in secretory epithelial tissues, where it has been implicated to play a key role in Ca 2ϩ -dependent Cl Ϫ secretion (6,11,28,29,41). Fischer and colleagues (11) identified TMEM16A mRNA in both human serous cells and in the Calu-3 cell line.…”

“…There is a substantial literature showing that CaCCs exist in airway epithelial in general and in Calu-3 cells in particular (11,21,22,(25)(26)(27)(28)38). A prime candidate for the CaCC is the recently elucidated TMEM16A or ANO1 (5,33,41).…”

“…The Calu-3 cells contain CFTR, which exhibits Cl Ϫ /HCO 3 Ϫ secretion when stimulated via the adenylyl cyclase/adenosine 3=,5=-cyclic monophosphate (cAMP)/protein kinase A (PKA) pathway (35). Calu-3 cells also express mRNA and activity consistent with transmembrane protein 16A (TMEM16A) (ANO1), a recently described Ca 2ϩ -activated Cl Ϫ channel (CaCC) (11). Although not as thoroughly studied as CFTR, TMEM16A has been described as the predominant CaCC in airways (26).…”

Cystic fibrosis and the relationship between mucin and chloride secretion by cultures of human airway gland mucous cells

“…There is evidence for fluid secretion by mucous cells of native glands (67). Finally, mucous cultures contain both CFTR and calcium-activated chloride channels as revealed by patch clamping and Ussing chamber studies (22). Therefore, it is possible that alterations in secretion of Cl Ϫ (or mucins) by mucous cells may contribute to the abnormally concentrated gland secretions in CF.…”