Ecto 5′-Nucleotidase and Nonspecific Alkaline Phosphatase

Abstract

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...

Effect of Zinc in Enteropathogenic Escherichia coli Infection

“…However, analysis of the rabbit intestinal tissues also showed significantly reduced EPEC adherence and reduced villus blunting, effects which did not accord with the known roles of ecto-5Ј-nucleotidase. In addition, zinc was more efficacious in the inhibition of EPEC-induced fluid secretion into intestinal loops than ␣,␤-methylene-ADP (data not shown), even though ␣,␤-methylene-ADP is a more potent inhibitor of ecto-5Ј-nucleotidase than zinc (9,26,33). Both of these findings led us to consider whether zinc might be acting on a target or targets other than ecto-5Ј-nucleotidase.…”

Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia

“…eNPPs and eNTPDases, as well as ecto-alkaline phosphatases (31,45,46), which are inhibited by ␤,␥-methylene-ATP, ebselen, and levamisole, respectively. Consistent with this notion, addition of a mixture of the three inhibitors resulted in a Ͼ50-fold reduction in ATP hydrolysis rates (Fig.…”

Mathematical Model of Nucleotide Regulation on Airway Epithelia

“…Members of the three ecto-ATPase families (alkaline phosphatases, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), and nucleotide pyrophosphatase/phosphodiesterases (E-NPPs)) (22,23) have been identified on HBE cells (13-15, 24 -26), along with ecto-AK (2ADP 7 ATP ϩ AMP) (27,28). Also, Ado levels are regulated on HBEs by a balance between the production rate (AMP 3 Ado) by ecto-5Ј-NT and NSAP (13) and the elimination rate by Ado deaminase 1 (ADA1; Ado 3 Ino) and cellular uptake by concentrative nucleoside transporter 3 (CNT3) (29).…”

“…Nucleotides are released by the epithelium into the ASL layer under basal conditions (7,8), and their release rate increases in response to mechanical stress imparted by tidal breathing (8 -12). Nucleotide-mediated epithelial responses are modified by surface enzymes (ectonucleotidases) that convert a fraction of the ATP into Ado (13)(14)(15). This nucleoside activates airway epithelial signaling pathways through P1 receptors, typically the A 2B receptor, which stimulates ciliary beat activity (16) and Cl Ϫ /liquid secretion by CFTR (17).…”