C̲ystic f̲ibrosis t̲ransmembrane conductance r̲egulator (CFTR) is the anion channel defective in cystic fibrosis (CF), a lethal hereditary disease. Increasing evidence suggests that CFTR conducts bicarbonate as well. A defective bicarbonate-transporting function of CFTR may underlie the pathogenesis of CF disease. Recently, a novel soluble adenylyc cyclase (sAC) has been cloned. Unlike the conventional transmembrane AC, sAC lacks apparent transmembrane domains and is not activated by G proteins and forskolin. Interestingly, sAC is stimulated by bicarbonate. Therefore, it is of great interest to examine whether bicarbonate-stimulated sAC is functionally coupled to bicarbonate-conducting CFTR to form an autoregulatory mechanism for bicarbonate transport in airway epithelial cells....[ Read more ]

C̲ystic f̲ibrosis t̲ransmembrane conductance r̲egulator (CFTR) is the anion channel defective in cystic fibrosis (CF), a lethal hereditary disease. Increasing evidence suggests that CFTR conducts bicarbonate as well. A defective bicarbonate-transporting function of CFTR may underlie the pathogenesis of CF disease. Recently, a novel soluble adenylyc cyclase (sAC) has been cloned. Unlike the conventional transmembrane AC, sAC lacks apparent transmembrane domains and is not activated by G proteins and forskolin. Interestingly, sAC is stimulated by bicarbonate. Therefore, it is of great interest to examine whether bicarbonate-stimulated sAC is functionally coupled to bicarbonate-conducting CFTR to form an autoregulatory mechanism for bicarbonate transport in airway epithelial cells.

Our studies with RT-PCR and immunoblotting showed that both mRNA and protein of sAC are expressed in Calu-3 cells, a widely used human airway epithelial cell line. We tested whether sAC is functional in Calu-3 cells by cAMP assay. Addition of 40 mM NaHC0₃ increased intracellular cAMP production by 30 %, and the increase was completely blocked by pretreatment of cells with 2-hydroxylestradiol (2-HE), a sAC inhibitor. In contrast, 2-HE had no effect on forskolin-stimulated cAMP increase. These results demonstrated the presence of functional sAC in Calu-3 cells, and validated that 2-HE could serve as a specific sAC inhibitor.

We then moved on to investigate the functional coupling of CFTR and sAC by whole cell voltage clamp studies. Addition of 25 mM NaHC0₃ stimulated whole cell current by 59.0 %. In the presence of 2-HE, no current increase by NaHC0₃ was observed, suggesting that sAC is physiologically coupled to CFTR activity. We further investigated the functional coupling of CFTR and sAC at single channel level. In cell-attached single channel recordings, adding NaHC0₃ in the bath robustly stimulated CFTR channels, and the activation was inhibited by 2-HE. The CFTR activation is not mediated by sodium ion, as the addition of 25 mM NaCl and 35 mM NaN0₃ in the bath had no effect on CFTR. These data further support that bicarbonate-sensitive sAC is functionally coupled to CFTR channels.

Although sAC is predominantly present in cytoplasm, it has been suggested by several studies that sAC could be membrane associated and we also detected a fraction of sAC in the membrane fraction in Calu-3 cells by Western blotting. These observations prompted us to further investigate the possibility that sAC and CFTR are functionally co-localized in the apical membrane of Calu-3 cells. In excised apical membrane patches, addition of 12.5 mM and 25 mM NaHC0₃ robustly activated CFTR by five- and ten-fold, respectively. The activation was abolished by 2-HE, consistent with the involvement of sAC.

The results together suggest that sAC regulates CFTR channel activity and may be functionally co-localized with CFTR in the apical plasma membrane in Calu-3 cells to form a compartmentalized auto-regulatory mechanism for bicarbonate transport in the airway epithelial cells.