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J. Biol. Chem., Vol. 278, Issue 15, 13468-13479, April 11, 2003
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From the 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 µM [3H]adenosine was not
transported to the mucosal compartment. Messenger RNA for the cytosolic
AMP-specific 5'-nucleotidase (CN-I) was not detected in human bronchial
epithelial cells, suggesting that mucosal adenosine did not originate
from intracellular pools. In contrast, extracellular 0.1 mM
ATP was rapidly dephosphorylated into adenosine on the mucosal
epithelial surface. We identified two ectonucleotidases that mediated
the conversion of AMP to adenosine: ecto 5'-nucleotidase
(ecto 5'-NT, CD73) and alkaline phosphatase (AP). Both mucosal and
serosal epithelial surfaces displayed ecto 5'-NT activity
(Km = 14 µM,
Vmax = 0.5 nmol·min
Ecto 5'-Nucleotidase and Nonspecific Alkaline
Phosphatase
TWO AMP-HYDROLYZING ECTOENZYMES WITH DISTINCT ROLES IN HUMAN
AIRWAYS*
§,,
, and
Cystic Fibrosis/Pulmonary Research and
Treatment Center, School of Medicine, University of North Carolina,
Chapel Hill, North Carolina 27599, the ¶ Department of Pulmonary
and Critical Care Medicine, Duke University Medical Center, Durham,
North Carolina 27710, and the Lineberger Comprehensive Cancer
Center, University of North Carolina,
Chapel Hill, North Carolina 27599
1·cm2), whereas AP activity was
restricted to the mucosal surface
(Km,high = 36 µM,
Vmax = 1.2 nmol·min1·cm2;
Km,low = 717 µM,
Vmax = 2.8 nmol·
min1·cm2). 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.
*
This work was supported by National Institutes of Health
Grants 34322, CFF R026, and CFF R001.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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