In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. Thus, defining the mechanisms of adenosine generation is critical for elucidating the role of this nucleoside in airway homeostasis. In this study, we identified the source of adenosine on the mucosal surface of human airway epithelia. Polarized primary cultures of human nasal or bronchial epithelial cells were assayed for transepithelial transport, cytosolic and cell surface adenosine production. Ussing chamber experiments indicated that serosal 1 M ). In bronchial cultures and tissues, ecto 5-NT accounted for >80% of total activity toward 0.01 mM AMP, compared with <15% for 5 mM AMP. The proximal airway AP isoform was identified as nonspecific AP (NS AP) by levamisole sensitivity and mRNA expression. The two ectoenzymes presented opposite airway distributions, ecto 5-NT and NS AP mRNA dominating in higher and lower airways, respectively. Collectively, these experiments support a major role for extracellular nucleotide catalysis and for ecto 5-NT and NS AP in the regulation of adenosine concentrations on airway surfaces.
Mucociliary clearance (MCC)1 constitutes an essential component of airway defense against the development of infectious lung diseases (1). Several epithelial functions involved in MCC are regulated by extracellular nucleotides. For instance, P2Y 2 receptor activation by ATP or UTP-stimulated Ca 2ϩ -dependent Cl Ϫ channels (I CA ) (2, 3), cilia beating frequency (CBF) (4, 5), and mucin secretion from goblet cells and submucosal glands (6, 7). Two members of the P2X receptor subfamily were recently identified in human airway epithelial cultures: P2X 4 and P2X 5 (8). These ATP-gated cationic channels increased Ca 2ϩ -dependent Cl Ϫ secretion (8) and CBF (9) in airway epithelia. Interestingly, extracellular adenosine was found to be responsible for regulating the post-peak sustained phase of increased CBF induced by ATP on human nasal explants (4). In a human bronchial cell line lacking P2Y 2 receptors (Calu-3; Ref. 10), the channel activity of the cystic fibrosis transmembrane regulator was inhibited by 8-p-sulfophenyltheophylline, a nonspecific blocker of cell surface adenosine receptors (11). Subsequently, adenosine was shown to regulate CBF (4, 5) and ion transport (12-15) through activation of cell surface A 2B receptors.The importance of adenosine receptor-mediated regulation of MCC remains unclear because of the lack of information on endogenous sources of extracellular adenosine on the mucosal surface of airway epithelia. Adenosine could originate from the interstitial compartment and penetrate airway epithelial tight junctions to reach the lumen. The nucleoside could also be generated intracellularly by the cytosolic AMP-specific 5Ј-nucleotidase (CN-I) (16,17) and reach the mucosal surface through nucleoside transporters (for review see Ref. 18). Alternatively, ATP release and cell surface conversion into adenosine has...