Abstract
HCO3− secretion in distal airways is critical for airway mucosal defense. HCO3−/H+ transport across the apical membrane of airway surface epithelial cells was studied by measuring intracellular pH in luminally microperfused freshly dissected mice bronchioles. Functional studies demonstrated that CFTR, ENaC, Cl−–HCO3− exchange, Na+-H+ exchange, and Na+–HCO3− cotransport are involved in apical HCO3−/H+ transport. RT-PCR of isolated bronchioles detected fragments from Cftr, α, β, γ subunits of ENaC, Ae2, Ae3, NBCe1, NBCe2, NBCn1, NDCBE, NBCn2, Nhe1, Nhe2, Nhe4, Nhe5, Slc26a4, Slc26a6, and Slc26a9. We assume that continuous decline of intracellular pH following alkaline load demonstrates time course of HCO3− secretion into the lumen which is perfused with a HCO3−-free solution. Forskolin-stimulated HCO3− secretion was substantially inhibited by luminal application of CFTRinh-172 (5 μM), H2DIDS (200 μM), and amiloride (1 μM). In bronchioles from a cystic fibrosis mouse model, basal and acetylcholine-stimulated HCO3− secretion was substantially impaired, but forskolin transiently accelerated HCO3− secretion of which the magnitude was comparable to wild-type bronchioles. In conclusion, we have characterized apical HCO3−/H+ transport in native bronchioles. We have demonstrated that cAMP-mediated and Ca2+-mediated pathways are involved in HCO3− secretion and that apical HCO3− secretion is largely mediated by CFTR and H2DIDS-sensitive Cl−–HCO3− exchanger, most likely Slc26a9. The impairment of HCO3− secretion in bronchioles from a cystic fibrosis mouse model may be related to the pathogenesis of early lung disease in cystic fibrosis.
Highlights
The airway surface liquid (ASL) is a thin layer of fluid covering the luminal surface of airway epithelium
It is widely accepted that the volume/depth of periciliary liquid layer (PCL) is determined by C l− secretion via cystic fibrosis transmembrane conductance regulator (CFTR) and C a2+-activated
Some connective tissue was attached to the outside of bronchioles (Fig. 1), surface epithelial cells were successfully loaded with BCECF from the lumen and p Hi was measured as long as 30 min
Summary
The airway surface liquid (ASL) is a thin layer of fluid covering the luminal surface of airway epithelium. It is widely accepted that the volume/depth of PCL is determined by C l− secretion via cystic fibrosis transmembrane conductance regulator (CFTR) and C a2+-activated. Cl− channel (CaCC) and N a+ absorption via epithelial Na+ channel (ENaC) [30, 33]. In proximal airways, Cl− secretion is mostly derived from serous cells of submucosal glands [5, 8, 17]. Submucosal glands are absent [10, 35] and concurrent C l− secretion and Na+ absorption was observed in surface epithelial cells [44]. The initial event of CF lung disease is characterized by low PCL volume, which is thought to be achieved by defective CFTR-mediated C l− secretion and abnormally elevated Na+ absorption via ENaC [33]
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