Abstract

A failure in optimal bicarbonate supply, as seen in the airways of cystic fibrosis patients, produces severe lung disease due to abnormal mucus maturation and acidic pH of airway surface liquid in the airways. The membrane proteins known to be responsible for bicarbonate movement across airway epithelium includes CFTR and TMEM16A channels in the apical membrane and a series of transporters of the SLC4 family in both apical and basolateral membranes. While most of these exchangers work in an electroneutral fashion, the SCLA4A4 or NBCe1 Sodium/Bicarbonate cotransporter moves a ratio of 2-3 bicarbonate and 1 sodium ion into the cell. We aim to test if the NBCe1 cotransporter is necessary for electrogenic bicarbonate transport in mouse airways. All animal procedures were performed according to institutional regulations for animal welfare. Ussing chamber experiments of freshly isolated mouse trachea showed that roughly half of the UTP-evoked anionic current is lost when bicarbonate is replaced with HEPES in the bath solution (-105±11 vs -47±3 µA cm-2 for control and HEPES buffer respectively). Short-circuit current is further decreased when cellular production of HCO3- is inhibited with acetazolamide (32±6 µA cm-2). To further determine if UTP induces HCO3- secretion, acutely isolated airway cells from mouse trachea were loaded with BCECF to track pH changes. UTP induced rapid acidification of airway cells that was abolished in CO2/HCO3- free bath solution but not in low chloride solution. Using a specific NBCe1 inhibitor (S0859) we observed that UTP-evoked short-circuit currents were diminished (62±13 µA cm-2) suggesting NBCe1 involvement in anionic secretion. Preliminary Ussing chamber experiments in Slca4a4-/- mice showed a significant reduction in UTP-evoked anionic secretion and histological examination of tissues obtained from 6 day old and 3 week old Slca4a4-/- animals showed signs of lung emphysema, mucus accumulation and goblet cell hyperplasia in bronchi. In summary, our electrophysiological and imaging data demonstrate that bicarbonate secretion is dependent of NBCe1 cotransporter function in mouse airways. Genetic silencing of NBCe1 produce a severe lung phenotype with features often observed in cystic fibrosis human patients.