 |
INTRODUCTION |
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,
P2Y2 receptor activation by ATP or UTP-stimulated
Ca2+-dependent Cl
channels
(ICA) (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: P2X4 and P2X5 (8).
These ATP-gated cationic channels increased
Ca2+-dependent Cl
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 P2Y2 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 A2B 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 5'-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 been reported in numerous mammalian cells (19). Human airway
epithelial cells release ATP under basal conditions (20, 21) and by
mechanical stimulations such as membrane stretch (22, 23), shear stress
(24, 25), or hypotonicity-induced swelling (26-28). All adenine
nucleotides and nucleosides have been detected in the airway surface
liquid under basal conditions (29). Lazarowski et al. (30)
established that cell surface adenine nucleotide and nucleoside
concentrations are maintained by a balance between ATP release and cell
surface metabolism.
We recently demonstrated that exogenous ATP is dephosphorylated into
ADP, AMP, and adenosine at the surface of human nasal and bronchial
epithelial cells (31). Two ectoenzymes have been reported to
dephosphorylate AMP into adenosine on mammalian cells: ecto
5'-nucleotidase (ecto 5'-NT, CD73, eN; EC 3.1.3.5; Ref. 32) and
alkaline phosphatases (APs, EC 3.1.3.1; Ref. 33). Whereas ecto 5'-NT
specifically dephosphorylates nucleoside monophosphates (AMP
adenosine (32)), APs will metabolize a spectrum of substrates, including 5'-nucleotides (ATP
ADP
AMP
adenosine),
pyrophosphate, and p-nitrophenyl phosphate (33). The AP
family contains four ectoenzymes: intestinal AP (I AP), tissue
nonspecific AP detected in several organs including liver, bone, and
kidney (NS AP), placental AP (PLA AP), and germ-cell AP (G AP) reported
in testis and malignant tumors (34, 35). Two AP isoforms have been
localized in human airways: NS AP and PLA AP. NS AP activity was
detected by histochemistry on the epithelial surface lining the entire
respiratory system, except for Type I pneumocytes (36), which express
PLA AP (37). Bronchoalveolar fluid AP activity has been used for
decades to diagnose acute lung injury and chronic disorders like
idiopathic pulmonary fibrosis (38, 39). Because bronchoalveolar fluid and lung tissue AP activities are classically assayed with
p-nitrophenyl phosphate as substrate (38-40), the possible
role of human airway AP in the production of extracellular adenosine
has not been investigated. Furthermore, ecto 5'-NT activity has not
been investigated in human airways. Thus, the pharmacological
manipulation of endogenous adenosine levels by targeting these
ectoenzymes remains an unexplored area.
The purpose of this study was to identify the source(s) of
extracellular adenosine on the mucosal surface of human airway epithelia, distinguishing between permeation from the interstitium, synthesis and secretion from the epithelial cells, and/or cell surface
metabolism of released nucleotides. All experiments were performed on
primary epithelial cultures, freshly excised airway epithelia or
bronchial sections. Serosal to mucosal permeability was measured by
Ussing chamber experiments with [3H]adenosine, and the
transported species were identified by high-performance liquid
chromatography (HPLC). The expression of cytosolic CN-I was
investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). Based on our data suggesting that adenosine resulted from
cell surface conversion of ATP
ADP
AMP
adenosine (31), we
investigated in detail the biochemical properties and relative contribution of ecto 5'-NT and APs. Enzyme expression and tissue distribution were determined by RNase protection assays (RPA). Because
of the potential importance of adenosine in diseases associated with
airway inflammation (41), we investigated the impact of a prototypic
inflammatory cytokine, interleukin-1
(IL-1
), on ecto 5'-NT and
APs expression.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Well differentiated primary cultures of human
nasal and bronchial epithelial cells were grown as previously described
(42). In brief, cells were isolated from freshly excised nasal
turbinate and mainstem bronchi by protein digestion (43), and plated on porous Transwell Col filters (well diameter, 12 mm; pore size, 0.45 µM; Costar) in air-liquid interface medium (50:50 mixture of LHC basal and Dulbecco's modified Eagle's medium-H, 0.5 ng/ml epidermal growth factor, 50 nM retinoic acid, 0.5 mg/ml
bovine serum albumin, 0.8% bovine pituitary extract, 50 units/ml
penicillin, and 50 µg 47 µl streptomycin) (44). Once they reached
confluence, the cultures were maintained at an air-liquid interface
with air-liquid interface medium added only to the serosal compartment.
After 4 weeks, the cultures were composed of columnar ciliated cells (>90%) and secretory cells, covering a layer of basal-like cells (45). Enzyme assays were conducted on cultures of transepithelial electrical resistance
300
/cm2. Lactate dehydrogenase
activity was employed as a test of cellular integrity.
Enzyme Assays on Epithelial Cultures--
The epithelial
cultures were rinsed three times with Krebs buffer (KRB (in
mM)), 140 Na+, 120 Cl
, 5.2 K+, 25 HCO
, 2.4 HPO
, 1.6 Ca2+, 1.6 Mg2+, 5.2 glucose, and 25 HEPES
(pH 7.4), and preincubated in KRB (0.35 ml mucosal/serosal) for 30 min
at 37 °C (5% CO2, 95% O2). Reactions were
initiated with the substrate (AMP, UMP, CMP, GMP, or IMP) dissolved in
35 µl of KRB. Aliquots (10 µl) were collected over 10 to 60 min,
boiled 3 min, filtered, and analyzed by HPLC. For the determination of
pH sensitivity, HEPES was used to buffer solutions at pH 6.5 to 8.0, Tricine for solutions at pH 8.0 and 8.5, and CHES for solutions at pH
8.5 and 9.0. In these buffers, bicarbonate was omitted for better
control of pH.
Ussing Chamber Experiments--
Primary nasal epithelial
cultures grown on SnapwellsTM (Costar; Cambridge, MA) were
mounted in modified Ussing chambers (Physiologic Instruments, San
Diego, CA) with an aperture of 1.0 cm2. The epithelium was
bathed on each side with 5 ml of HEPES-free KBR (pH 7.4, 37 °C),
circulated by gas lift with 95% O2, 5%
CO2. The voltage was clamped to 0 mV, except for 3-s pulses
(±10 mV) every 60 s. Short-circuit current (Isc) and
transepithelial resistance were digitally recorded from the output of
voltage clamp. The permeability coefficients
(Pcoeff) for adenosine and mannitol were
measured. Mannitol Pcoeff was used as an index
of paracellular permeability. Following a 45-min preincubation period
with [3H]adenosine (0.01 µCi/µl; 1 µM)
and [14C]mannitol (0.2 µCi/µl) added to the serosal
bath, aliquots (500 µl) were collected from the mucosal side (sink)
every 30 min and replaced with 500 µl of HEPES-free KBR. Every 60-min
interval, 50 µl was collected from the serosal (source) side.
[14C]Mannitol and [3H]adenosine in the
source and sink buffer samples were measured using a liquid
scintillation counter (Beckman LS 6500 counter) and
Pcoeffs calculations were calculated with the
established equation: Pcoeff = (
Q/
t/S·A), where
Q/
t is the steady-state rate of appearance
of the tracer in the sink (cpm/s), S is the source
radioactivity (cpm/cm3), and A is the surface
area (cm2) (46). The units of Pcoeff
are cm/s. Mucosal and serosal buffer samples were also analyzed for
nucleoside and nucleobase composition by HPLC.
Identification of the AMP-hydrolyzing Enzymes--
Three enzymes
could be responsible for the conversion of extracellular AMP into
adenosine on human airway epithelial surfaces: cytosolic 5'-NT, ecto
5'-NT, and AP. The possible contribution of each enzyme was evaluated
with specific substrates and inhibitors. The non-hydrolyzable analog of
ADP,
,
-methylene ADP (
,
-met-ADP), inhibits ecto 5'-NT and
AP, but not cytosolic 5'-NT (32). Hence, complete inhibition of AMP
hydrolysis by
,
-met-ADP would rule out the contribution of
released cytosolic 5'-NT. Ecto 5'-NT activity was identified with
concanavalin A, a plant lectin that binds specifically to
-D-glucosyl and
-D-mannosyl residues of
glycoproteins (47). Concanavalin A has been reported to completely
inhibit the activity of ecto 5'-NT, without affecting AP (48). The AP activity was measured with a specific substrate (
-glycerophosphate) and inhibitor (levamisole). The substrate reports the biochemical activity of all AP isoforms (49), whereas levamisole sensitivity discriminates between NS AP, PLA AP, and I AP (50). The identity of the
AP isoform(s) was further addressed with L-phenylalanine (I
AP and PLA AP) and L-leucine (G AP) (34, 35). All
experiments were conducted as described above for enzyme assays.
Reactions were initiated with
-glycerophosphate mixed with AMP.
Alternatively, the cultures were preincubated 20 min with an inhibitor
(
,
-met-ADP, levamisole, amino acids, or concanavalin A) before
the onset of the reaction.
Kinetic Analysis of AMP Hydrolysis--
All assays were
conducted on the mucosal surface of human bronchial epithelial cultures
with [3H]AMP (0.1 mCi; 1-3000 µM), as
described above for enzyme assays. Samples were collected after
incubation periods that limited substrate hydrolysis to
10%, and
were analyzed by HPLC. Kinetic parameters for ecto 5'-NT and AP were
determined in the presence 10 mM levamisole and 5 mM concanavalin A, respectively. Michaelis constants
(Km) and maximal velocities
(Vmax) were obtained from the slope and the
ordinate of Woolf-Augustinson Hoftsee transformations, respectively. Catalytic efficiency (Cateff) was calculated from the
velocity (Vo) at Km divided
by Km (51).
Competition studies were conducted to determine whether ADP and/or ATP
inhibited the hydrolysis of nucleoside monophosphates. Because ecto
5'-NT (48) and AP (33) hydrolyze AMP and UMP at similar rates, UMP was
chosen over AMP as substrate for two reasons: 1) to rule out
interference from the adenylate kinase activity (ATP + AMP
2ADP) we
recently detected on these cells (52) and 2) to distinguish substrates
from inhibitors and their metabolites on the HPLC chromatograms.
Because we recently demonstrated that ATP and ADP are hydrolyzed into
AMP on these cells (31), the protocol was designed to prevent
interference of UMP hydrolysis by AMP produced from ATP and ADP
hydrolysis. Reactions were started with previously mixed UMP (10, 30, or 100 µM) and ATP or ADP (10, 30, 60, or 100 µM). Samples were collected after incubation periods that
limited AMP production from ATP and ADP to <0.1 µM.
These conditions also limited ATP conversion into ADP to
10%.
Inhibition patterns and constants (Ki) were derived
from Dixon plots.
Tissue Measurements of Enzyme Activities--
The epithelial
surface of human bronchial sections was assayed for AMP hydrolysis by
ecto 5'-NT and NS AP. From each tissue sample, three circular pieces
(diameter, 12 mm) were assayed in parallel with 5 mM AMP.
Each piece was inserted into a 12-mm porous Transwell and sealed along
the edges with silicone. Epithelial surfaces were rinsed three times
with KRB and preincubated in KRB (0.35 ml of mucosal/serosal) for 30 min at 37 °C (5% CO2, 95% O2). Reactions
were initiated with the substrate dissolved in 35 µl of KRB. Five
aliquots (10 µl) were collected over 10-30 min, boiled 3 min,
filtered, and analyzed by HPLC. All three pieces were submitted to a
second set of experiments. Surfaces were rinsed and tissues were
preincubated 30 min at 37 °C (5% CO2, 95%
O2) in KRB (0.35 ml of mucosal/serosal) containing 10 mM levamisole, 5 mM concanavalin A or vehicle.
Reactions were conducted as in the first set of experiments. Ecto 5'-NT
and NS AP activities were calculated from the differences in the rate
of AMP hydrolysis between control and treated experiments conducted on
the same piece of tissue. The piece assayed twice with 5 mM
AMP in the absence of enzyme inhibitor provided a control for tissue
integrity. This protocol was repeated on different sets of tissues with
0.01 mM AMP and 0.1 mM AMP. At the end of the
experiments, all sections were fixed and counterstained with
hematoxylin and eosin (H&E) for light microscopic examination of the
epithelium. A Nikon microphot SA microscope connected to a 3CC-Chilled
Camera (Sony, Marietta, GA) and interfaced to a powerMac 8100 were used
to capture the images via Adobe Photoshop.
HPLC Analysis--
All samples were analyzed by reversed-phase
paired-ion HPLC. The separation system consisted of a Dinamax C-18
column and a mobile phase developed with buffer A (10 mM
KH2PO4 and 8 mM TBASH, pH 5.3) from
0 to 10 min, buffer B (100 mM
KH2PO4, 8 mM TBASH, and 15% MeOH,
pH 5.3) from 10 to 20 min, and buffer A from 20 to 30 min. Absorbance
was monitored at 254 nm with an on-line model 490 multiwavelength
detector (Shimadzu Science Instruments Inc., Kyoto, Japan), and
radioactivity was determined on-line with a Flo-One Radiomatic
detector (Packard Instrument Co.).
Exposure of Human Airway Epithelia to IL-1
--
Primary
cultures of human bronchial epithelial cells were incubated during
24 h at 37 °C (5% CO2, 95% O2) with a
range of IL-1
concentrations reported to induce the cyclooxygenase
pathway (0.0-1.0 ng/ml) of human airway epithelial cells (53). Because polarized airway cultures respond to prolonged (>12 h) mucosal flooding by an increased acid production (54), the cytokine was added
to air-liquid interface medium in the serosal bath. At the end of the
challenges, the cultures were processed for total RNA extraction and
RPA analysis of enzyme expression.
Reverse Transcriptase-Polymerase Chain Reaction--
Total RNA
was extracted from primary cultures and freshly excised human airway
epithelial cells, as previously described (55). Oligonucleotide primers
were generated from the sequence of human ecto 5'-NT (CD73), cytosolic
AMP-specific 5'-nucleotidase (CN-I), and the four AP isoforms (Table
I). First strand cDNA was synthesized by RT reaction with 8 µg of total RNA and the outer antisense (AS2) primer, in a 20-µl reaction containing 10 mM dNTPs,
0.1 mM dithiothreitol, 40 units of RNase inhibitor, and 400 units of Superscript II RT in supplied buffer (Invitrogen). The
reaction was allowed to proceed for 1 h at 42 °C and stopped by
heating at 70 °C for 10 min. The cDNA was amplified by PCR with
the inner antisense (AS1) and the sense (S) primers by the "hot
start" technique: 36 cycles (1 min at 94 °C, 2 min at 57 °C, 3 min at 72 °C), followed by 10 min at 72 °C. In the case of CN-I,
the AS primer was poly(dT) (16) and the PCR product mixture contained
glyceraldehyde-3-phosphate dehydrogenase primers (BD Biosciences) as
internal control. The PCR was run in the presence of 2.5%
Me2SO in the following conditions: 35-55 cycles (1 min at
94 °C, 50 s at 45 °C, 25 s at 72 °C), followed by 10 min at 72 °C. The amplified PCR products were gel purified on 1%
agarose gel for ecto 5'-NT and APs, 1.5% agarose gel for CN-I and
glyceraldehyde-3-phosphate dehydrogenase (Qiagen gel purification kit).
The PCR products were ligated by TA cloning into the pCRII
vector (Invitrogen) and the ligations transformed in
One-Shot cells (Invitrogen). Plasmid DNA from individual
colonies was purified with a commercial kit (Qiagen) and screened by
automatic sequencing to confirm the identity of the product, verify the absence of mutations, and determine the orientation of the insert in
the vector. All PCR products were confirmed to be in 100% homology with the reported human cDNA sequences. RT-PCR reactions conducted with RNA in the absence of Superscript II RT yielded no signal on the
gels, ruling out the possibility of RNA contamination by genomic
DNA.
RNase Protection Assays--
Plasmids containing cDNA
fragments for human ecto 5'-NT and NS AP were linearized with
EcoRV. [32P]CTP-labeled antisense probes were
transcribed from the plasmid SP6 promoter site, according to the
manufacturer's recommendations (MAXIscript in vitro
transcription kit; Ambion, Austin, TX). The housekeeping gene probe
-actin was prepared at low specific activity by adding 2 µl of 1 mM cold CTP to the labeling mixture and reducing the
32P label to 10 µCi per reaction. The probes were
purified by electrophoresis on RNase-free 5% acrylamide, 8 M urea gels (250 volts, 1 h), eluted and hybridized
(8 × 104 disintegration/min) with 20 µg of total
RNA (RPA III kit; Ambion). The protected fragments were separated by
electrophoresis on 5% acrylamide, 8 M urea gels (250 volts, 1 h). The gels were dried on a vacuum dryer (60 °C,
4 h) and exposed to a storage phosphorscreen (Amersham
Biosciences) for 4-8 days. The screen was scanned with an
optical scanner (Storm; Amersham Biosciences) and the signals quantified using digital image analyzing software (ImageQuant; Amersham
Biosciences). Appropriate sense strand and yeast RNA controls yielded
no signal on the gels.
Statistical Analysis--
All experiments were performed on
cultures and tissues from at least three donors. Rates of hydrolysis
were calculated from the decrease in the amount of substrate monitored
by HPLC, and presented as
nanomoles·min
1·cm
2 of surface area. The
values were expressed as mean ± S.E. Unpaired Student's
t tests were used to assess the significance between means.
Paired Student's t tests were used when comparing
hydrolysis rates measured on the mucosal and serosal surfaces of the
same culture. Linear regressions, curve fits, and data transformations were performed with the computer programs Origin and Sigma plot.
Materials--
All 5'-nucleotides and adenosine were purchased
from Roche Molecular Biochemicals (Mannheim, Germany). Concanavalin A,
erythro-9-[2-hydroxyl-3-nonyl]adenine,
-glycerophosphate,
levamisole, L-phenylalanine, L-leucine,
,
-met-ADP, EDTA, KH2PO4, TBASH, Tricine,
CHES, and HEPES were obtained from Sigma. HPLC grade water was
purchased from Fisher Scientific (Pittsburgh, PA). Cell culture media,
bovine serum albumin, bovine pituitary extract, epidermal growth
factor, gentamicin, penicillin, retinoic acid, and streptomycin were
bought from Invitrogen and human cytokine IL-1
(rhIL-1
) from R&D
Systems. [3H]Adenosine (20 Ci/mmol) and
[3H]AMP (10 mCi/mmol) were obtained from Amersham
Biosciences, D-[1-14C]mannitol (0.1 mCi/mmol)
and [
-32P]CTP (10 mCi/mmol) were from PerkinElmer Life
Sciences. Salts and solvents were of analytical grade.
 |
RESULTS |
Adenosine Accumulation on the Apical Surface of Human Airway
Epithelia--
First, we explored the possibility that serosal
adenosine could reach the mucosal surface. Ussing chamber experiments
were performed on polarized cultures of human nasal epithelial cells. When [3H]adenosine (0.01 µCi/µl; 1 µM)
was added to the serosal bath, tritiated compounds accumulated in the
mucosal bath with a significantly higher Pcoeff
than for [14C]mannitol (Fig.
1A). However, HPLC analysis of
the tritiated species revealed that serosal [3H]adenosine
was converted into [3H]inosine and
[3H]hypoxanthine within 30 min in the serosal compartment
(Fig. 1B). The only radiolabeled molecule detected on the
mucosal surface over 30 min was [3H]hypoxanthine. These
results suggest that interstitial fluid does not constitute a source of
adenosine for the mucosal surface of airway epithelia.

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Fig. 1.
Origin of extracellular adenosine on the
mucosal surface of human airway epithelia. A and
B, Ussing chamber experiments on transepithelial and
paracellular adenosine transport. [3H]Adenosine (1 µM) and the paracellular marker
[14C]mannitol were added to the serosal bath of polarized
nasal epithelial cultures. Buffer samples were collected over 90 min to
calculate [3H]adenosine and [14C]mannitol
Pcoeffs. A, adenosine
Pcoeff was higher than mannitol
Pcoeff. B, HPLC analysis of serosal
and mucosal buffer samples collected over 30 min. Serosal
[3H]adenosine (ADO) was metabolized into
inosine (INO) and hypoxanthine (HP). Traces of HP
(but not ADO) were detected in the mucosal bath. C, RT-PCR
agarose gel indicating the absence of mRNA for cytosolic
5'-nucleotidase (CN-I). Lane 1, human placenta RNA
(negative control; 16); lane 2, human embryonic kidney
cells (HEK 293) overexpressing CN-I (positive control, 16); lane
3, human heart RNA (positive control) (16); lanes 4 and
5, freshly excised bronchial epithelium from 2 donors.
Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)-specific product of 983 bp was produced
by all reactions (internal control). D, production of
adenosine from ATP. On the mucosal surface of human bronchial
epithelial cells 0.1 mM ATP was dephosphorylated into ADP,
AMP, and ADO. No substrate or product was detected on the serosal
surface, and vice versa (data not shown). Values represent mean ± S.E. of five to seven independent experiments. *, p < 0.01.
|
|
We then tested whether adenosine could be generated within the cytosol
of columnar epithelial cells and released onto the mucosal surface. To
evaluate this possibility, we performed RT-PCR experiments for the
expression of CN-I (16, 17). Experiments performed with total RNA from
human heart or human embryonic kidney cells (HEK 293) produced a
positive band of expected size for CN-I (Fig. 1C), as
previously reported (16). In contrast, no reaction product was obtained
with total RNA from freshly excised human bronchial epithelial cells or
human placenta (negative control; Ref. 16), even after 55 PCR cycles
(not shown). These experiments indicated that extracellular adenosine,
detected on the mucosal surface of human airway epithelia does not
originate from intracellular compartments.
Numerous cell types have the ability to release ATP (56) and to
transform the nucleotide into adenosine by cell surface enzymes (19).
Because human airway epithelial cells were reported to release ATP
(20-28) and to accumulate all adenine nucleotides on the mucosal
surface (ATP, ADP, AMP) (29), we tested whether extracellular adenosine
could be generated from mucosal cell surface nucleotide metabolism.
Fig. 1D shows that the mucosal surface of bronchial
epithelial cultures dephosphorylated 100 µM ATP into ADP,
AMP, and adenosine. No nucleotide or nucleoside was detected in the
opposite compartment, whether we administered ATP on the mucosal or
serosal surface. These results were consistent with the data showing
that adenosine does not permeate through tight junctions (see above).
Because these experiments indicated that adenosine accumulated in the
mucosal compartment primarily because of extracellular ATP hydrolysis,
we investigated the identity and polarity of the ectoenzyme(s)
responsible for the conversion of extracellular AMP into adenosine on
human airway epithelial surfaces. The mucosal surface of human
bronchial epithelial cultures metabolized 0.1 mM AMP with a
half-life of ~15 min (Fig.
2A). The nucleotide was converted into adenosine, inosine, and hypoxanthine (Fig.
2A). The adenosine deaminase inhibitor, 10 µM
erythro-9-[2-hydroxyl-3-nonyl]adenine (57), prevented the
accumulation of inosine and hypoxanthine (data not shown), supporting
cell surface generation from adenosine. The properties of AMP
hydrolysis on airway epithelial surfaces corresponded to those of
membrane-bound ectonucleotidases for the following reasons. First, the
substrate was hydrolyzed by intact cells and the product was released
into the buffer (Fig. 2B). Second, AMP hydrolysis followed a
linear relationship over time on both epithelial surfaces (Fig.
2B), making it unlikely that a significant amount of the
substrate had been hydrolyzed after entering the cells and products
released in the ectodomain. Third, KRB buffer conditioned by a 60-min
exposure to mucosal or serosal surfaces hydrolyzed 0.1 mM
AMP at rates that corresponded to <5% of total cell surface activity
(Fig. 2B). Collectively, these experiments demonstrate that
membrane-bound ectoenzymes are responsible for adenosine production at
the surface of human airway epithelia.

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Fig. 2.
Characterization of cell surface adenosine
production. A, HPLC analysis for the conversion of
extracellular AMP into adenosine. Mucosal 0.1 mM AMP was
converted into adenosine (ADO), inosine (INO),
and hypoxanthine (HP) on bronchial cultures. B,
AMP-hydrolyzing ectoenzymes are cell-associated. The mucosal surface
( ) hydrolyzed AMP at higher rates than the serosal ( ) surface.
KRB conditioned during 60 min on the mucosal ( ) or serosal ( )
surface hydrolyzed 0.1 mM AMP at <5% of total AMPase
activity. C, AMP hydrolysis on nasal and bronchial
epithelial cells. Mucosal activities (filled bars) were
3-fold higher than serosal activities (open bars). Reaction
rates were higher on bronchial cultures. Values represent mean ± S.E. of 4-12 independent experiments. *, p < 0.05;
**, p < 0.01.
|
|
The AMP hydrolytic activity measured on human bronchial epithelial
cells was also detected with epithelial cultures derived from human
nasal turbinate (Fig. 2C). Hydrolysis rates were 2-3-fold higher on bronchial than on nasal cultures. In addition, both cell
types exhibited enzyme activities that were 3-5-fold higher on the
mucosal surface than on the serosal surface. These results suggest that
the conversion of extracellular AMP into adenosine occurs throughout
airways, and that this activity predominates on the epithelia surface
facing the airway lumen.
Biochemical Properties of AMP Hydrolysis--
Human
bronchial epithelial cells were examined for their substrate
specificity, divalent cation, and pH sensitivity. The cultures displayed broad substrate specificity for nucleoside
monophosphates. On the mucosal surface, hydrolysis rates were in the
following order: AMP
UMP = CMP > GMP
IMP
(Fig. 3A). Mucosal AMPase
activity was not affected by Ca2+, but was significantly
increased by Mg2+ (Fig. 3B). Hydrolytic rates
were 30% higher in buffers containing 3 mM
Mg2+ or 3 mM Mg2+ and 3 mM Ca2+ than with 3 mM
Ca2+ alone (Table II). Assays
conducted on the serosal surface were not influenced by the buffer
composition in divalent cations (Table II). The pH dependence profile
of mucosal AMP hydrolysis followed a bimodal pattern, with optimal pH
around 7.5 and 9.0 (Fig. 3C). Serosal AMPase activity
produced a single activity peak at pH 7.5. The fact that mucosal and
serosal surfaces displayed different cation and pH sensitivities
suggested the presence of more than one AMP-hydrolyzing enzyme on human
airway epithelial surfaces.

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Fig. 3.
Biochemical properties of AMP hydrolysis by
human bronchial epithelial cells. A, substrate
specificity. The mucosal surface was assayed with 0.1 mM
nucleoside monophosphate in KRB, and buffer aliquots were analyzed by
HPLC. All substrates were hydrolyzed: AMP (A) UMP
(U) = CMP (C) > GMP
(G) > IMP (I). B, cation
sensitivity. Assays were performed on the mucosal surface with 0.1 mM AMP in KRB containing various concentrations of
Ca2+ ( ) or Mg2+ ( ). The reaction was
insensitive to Ca2+ and stimulated by Mg2+.
C, pH dependence. The profile for 0.1 mM AMP was
bimodal on the mucosal surface ( ), with a single peak on the serosal
surface ( ). Values represent mean ± S.E. of three to five
independent experiments. *, p < 0.05.
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Table II
Impact of divalent cations on AMP hydrolysis by mucosal and serosal
surfaces of human bronchial epithelial cells
Assays were conducted with 0.1 mM AMP in the
absence/presence of 3 mM Ca2+ and/or 3 mM Mg2+. Values represent means (±S.E.) of four
experiments.
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|
Identification of the Ectoenzymes--
We investigated the
identity of the enzymes responsible for AMP hydrolysis on human
bronchial epithelial surfaces under physiological conditions (pH 7.4).
The non-hydrolyzable analog of ADP,
,
-met-ADP, has been described
as a competitive inhibitor of ecto 5'-NT and AP that has no effect on
cytosolic 5'-NT (32). Fig. 4A
shows that
,
-met-ADP inhibited the hydrolysis of 0.1 mM AMP in a concentration-dependent manner on
both epithelial surfaces. AMP hydrolysis was completely abolished by
0.1 mM
,
-met-ADP, which ruled out the contribution of
released cytosolic 5'-NT. This finding is in agreement with the absence
of mRNA for CN-I (Fig. 1C).

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Fig. 4.
Identification of the AMP-hydrolyzing
ectoenzymes on human bronchial epithelial cells. A,
concentration-dependent inhibition of AMP hydrolysis by
, -met-ADP. Reactions were initiated with 0.1 mM AMP
and 0, 0.01, or 0.1 mM , -met-ADP in KRB on mucosal
(filled bars) and serosal (open bars) surfaces,
and buffer aliquots were analyzed by HPLC. B, detection of
mucosal ecto 5'-NT and AP activities. Reaction rates for 5 mM AMP decreased with increasing concentrations of
concanavalin A ( ), -glycerophosphate ( ), or levamisole
(···). C and D, polarity of ecto 5'-NT and
AP. On the mucosal surface, levamisole (L) and concanavalin
A (C) reduced reaction rates by 23 ± 3 and 78 ± 5% with 0.1 mM AMP (C) and by 83 ± 4 and
28 ± 3% with 5 mM AMP (D). Serosal
reactions (C and D) were insensitive to
levamisole and completely inhibited by concanavalin A. The two
inhibitors together (LC) abolished the reactions on both
surfaces. Values represent mean ± S.E. of four to seven
independent experiments. *, p < 0.05.
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Two types of ectonucleotidases could be involved in the metabolism of
extracellular AMP: ecto 5'-NT (32) and APs (33). Their activity at the
surface of human bronchial epithelial cells was detected with
concanavalin A (48) and
-glycerophosphate (49), respectively.
Dose-response curves were constructed with 5 mM AMP and
various inhibitor concentrations. On the mucosal surface, both
compounds reduced the rate of AMP hydrolysis in a saturable manner
(Fig. 4B). Concanavalin A inhibited 21 ± 4% of total
AMPase activity, with an IC50 of 9.0 µM. In
contrast,
-glycerophosphate reduced AMP hydrolysis by 84 ± 5%, with an IC50 of 26 µM. Similar results
were obtained when
-glycerophosphate was replaced by levamisole.
This non-competitive AP inhibitor reduced the rate of AMP hydrolysis by
81 ± 3%, with an IC50 of 17 µM (Fig.
4B). Based on IC50 values reported for
levamisole on NS AP (30 µM), PLA AP (1.7 mM),
and I AP (6.8 mM) (50), the AP isoform expressed on human
bronchial epithelial cells is likely to be NS AP. The identity of the
AP isoforms expressed in human airways was further addressed with
specific amino acids. The rate of AMP hydrolysis was not significantly
reduced by 10 mM L-phenylalanine (PLA AP and I
AP inhibitor) or by 10 mM L-leucine (G AP
inhibitor) (34, 35), respectively (data not shown). Collectively, these experiments indicate that ecto 5'-NT and NS AP are responsible for the
production of adenosine on human airway epithelial cells.
The polarity of ecto 5'-NT and NS AP on human bronchial epithelial
cultures was addressed with concentrations of concanavalin A (5 mM) and levamisole (10 mM) that produced
maximal inhibition (Fig. 4B). With 0.1 mM AMP,
levamisole and concanavalin A reduced mucosal rates of hydrolysis by
23 ± 3 and 78 ± 5%, respectively (Fig. 4C). The
two enzymes added together completely abolished AMP hydrolysis. On the
serosal surface, levamisole had no significant effect on AMP
hydrolysis, whereas concanavalin A completely inhibited the reaction.
These results indicate that ecto 5'-NT was expressed on both mucosal
and serosal surfaces, whereas NS AP activity was restricted to the
mucosal surface. The relative contribution of the two enzymes on
mucosal surfaces was strongly influenced by substrate concentration.
Assays conducted with 5 mM AMP generated total activities 5 times higher than with 0.1 mM substrate. Under these
conditions, levamisole and concanavalin A reduced mucosal rates of AMP
hydrolysis by 83 ± 4 and 28 ± 3%, respectively (Fig. 4D). Whereas ecto 5'-NT activity was already maximal with
0.1 mM AMP, NS AP activity was 5 times higher with 5 mM AMP. These results suggested distinct kinetic properties
for the two AMP-hydrolyzing enzymes.
Kinetic Properties of the AMP-hydrolyzing Enzymes--
The kinetic
properties of AMP hydrolysis were examined on the mucosal surface of
human bronchial epithelial cultures. Fig. 5A shows that the rate of AMP
hydrolysis decreased with increasing substrate concentration.
Hydrolytic rates did not saturate with millimolar AMP, suggesting the
presence of at least two catalytic sites with different substrate
affinities.

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Fig. 5.
Kinetic analysis of ecto 5'-NT and NS AP
activities on human bronchial epithelial cells. A,
properties of NS AP. Reactions initiated with 0.001-3 mM
[3H]AMP in the absence (- - -) or presence ( ) of 5 mM concanavalin A. Inset, Woolf-Augustinson
Hoftsee transformation fitted to two regressions (r = 0.96-0.99), with Km, Vmax,
and Cateff of 717 ± 49 µM, 2.8 ± 1.2 nmol·min 1·cm 2, and 0.006 ± 0.001 min 1, and 6 ± 8 µM, 1.2 ± 0.2 nmol·min 1·cm 2, and 0.021 ± 0.005 min 1, respectively. B, properties of
ecto 5'-NT. Reactions initiated with 0.001-3 mM
[3H]AMP and 10 mM levamisole.
Inset, Woolf-Augustinson Hoftsee transformation fitted to a
single regression (r = 0.99), with
Km, Vmax, and
Cateff of 14 ± 3 µM, 0.52 ± 0.05 nmol· min 1·cm 2, and 0.041 ± 0.006 min 1, respectively (n = 6; S.E. < 5% of the mean). C and D, impact of ADP and ATP
on UMP hydrolysis. Reactions initiated with UMP 10 µM
( ), 30 µM ( ), or 100 µM ( ) and ADP
(10, 30, 60, or 100 µM) or ATP (10, 30, 60, or 100 µM). Buffer aliquots were analyzed by HPLC. Dixon plots
showed (C) ADP and (D) ATP as competitive
inhibitors of UMP hydrolysis, with correlation coefficients of
0.96-0.99. Values represent mean ± S.E. of five independent
experiments.
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The profile was not significantly modified when ecto 5'-NT activity was
inhibited by 5 mM concanavalin A (Fig. 5A),
suggesting that AP activity dominated at high AMP concentrations, as
shown in Fig. 4D. Woolf-Augustinson Hoftsee transformation
revealed two kinetic components for AP: a high affinity and a low
affinity activity. The Km and
Vmax values of the high affinity activity were
36 ± 8 µM and 1.2 ± 0.2 nmol·
min
1·cm
2, respectively (Fig.
5A, inset). The Km and
Vmax values of the low affinity activity were
717 ± 49 µM and 2.8 ± 1.2 nmol·min
1·cm
2, respectively. The
Cateff of the high affinity activity was 4-fold higher than
the Cateff of the low affinity activity, with 0.021 ± 0.005 min
1 and 0.006 ± 0.001 min
1, respectively.
Experiments conducted in the presence of the AP inhibitor (10 mM levamisole) revealed a simple Michaelis-Menten equation
for the ecto 5'-NT reaction rate reaching saturation around 1 mM AMP (Fig. 5B). Woolf-Augustinson Hoftsee
transformation indicated a single high affinity activity, with
Km and Vmax values of 14 ± 3 µM and 0.52 ± 0.05 nmol·min
1·cm
2, respectively (Fig.
5B, inset). Calculated Cateff was
0.041 ± 0.006 min
1, which is 2-fold higher than the
value obtained for the high affinity AP activity.
We investigated the impact of ATP and ADP on the hydrolysis of
nucleoside monophosphates by human bronchial epithelial cells. The
assays were conducted with UMP instead of AMP to distinguish the
substrate from the inhibitors and their metabolites on the HPLC
chromatograms (see "Experimental Procedures"). The rate of UMP
hydrolysis was inversely related to ATP and ADP concentrations. Dixon
plot analysis indicated a competitive pattern of inhibition for both
ADP (Fig. 5C) and ATP (Fig. 5D), with
Ki values of 7 and 10 µM,
respectively. These results suggest that extracellular ATP and ADP
participate in the control of adenosine concentrations on human airway
epithelial cells.
Tissue Measurements of Ecto 5'-NT and NS AP--
The relative
contributions of ecto 5'-NT and NS AP activities to AMP hydrolysis were
examined on the epithelial surface of freshly excised bronchial
sections. Fig. 6A shows that
the polarized cultures closely reproduced the in vivo
morphologic characteristics displayed by freshly excised bronchial
epithelia. Both preparations exhibited a layer of columnar ciliated and
secretory epithelial cells covering cuboidal basal-like cells. The
epithelial surface of the bronchial sections was assayed with 0.01-5.0
mM AMP, in the absence/presence of the ecto 5'-NT (5 mM concanavalin) or NS AP (10 mM levamisole)
inhibitor (see "Experimental Procedures"). Ecto 5'-NT activity was
5-fold higher than NS AP activity when assayed with 0.01 mM
AMP (Fig. 6B). In contrast, NS AP activity dominated with
0.1 and 5 mM AMP. These results are in agreement with the
cell culture model (Fig. 4, C and D), suggesting
that the two AMP-hydrolyzing enzymes address distinct pools of
nucleotides on human airway epithelial surfaces.

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Fig. 6.
Tissue measurements of ecto 5'-NT and NS AP
activities. A, typical brightfield (H&E)
sections of human airway epithelium from freshly excised main bronchus
and polarized bronchial cultures. B, AMP hydrolysis on the
epithelial surface of bronchus sections. Mucosal NS AP (filled
bars) and ecto 5'-NT (open bars) activities toward
0.01-5 mM AMP assayed with 5 mM concanavalin A
and 10 mM levamisole, respectively. Values represent
mean ± S.E. of four independent experiments. *, p < 0.05.
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Expression of Ecto 5'-NT and NS AP in Cultured and Excised
Epithelia--
The identity of the AP isoforms expressed on human
airway epithelial cells was addressed by RT-PCR with primers specific
for NS AP, PLA AP, I AP, and G AP (Table I). These experiments were performed with total RNA extracted from primary cultures of human nasal, bronchial, and bronchiolar epithelial cells. Control reactions were conducted with commercial human total RNA (BD Biosciences): liver
(NS AP), small intestine (I AP), placenta (PLA AP), and testis (G AP)
(35). Only two AP isoforms were detected in human airway epithelial
cells: NS AP and PLA AP. Agarose gels exhibited strong mRNA signals
for NS AP in all epithelial samples (Fig. 7A). In contrast, PLA AP was
not detected in nasal and bronchial epithelial cells and produced a
weak signal in bronchiolar epithelial cells. These results are
consistent with the inhibition data indicating that the AP activity,
detected on bronchial epithelial surfaces, corresponded to the NS AP
isoform (Fig. 4B). Similar results were obtained for RT-PCR
performed with total RNA from freshly excised human airway epithelia
(data not shown).

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Fig. 7.
Expression of ecto 5'-NT and NS AP in primary
cultures of human airway epithelial cells. A,
identification of the AP isoform by RT-PCR. Reactions conducted with
total RNA from cultured airway epithelial cells and primers for NS AP,
I AP, PLA AP, and G AP (Table I). Controls: commercial human RNA (BD
Biosciences) from liver (NS AP), small intestine (I AP), placenta (PLA
AP), and testis (G AP). B, co-expression of ecto 5'NT and NS
AP detected by RPA in nasal and bronchial cultures. The mRNA
levels, normalized with -actin, were 2-3 times lower in nasal
(N, filled bars) than bronchial
(B, open bars) epithelial cells. Values
corrected for the number of G-C binding sites represent mean ± S.E. of three independent experiments. *, p < 0.05.
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The co-expression of ecto 5'-NT and NS AP in primary cultures of human
nasal and bronchial epithelial cells was confirmed by RPA. Both enzymes
displayed mRNA levels that were 2-3 times higher in bronchial than
in nasal epithelial cells (Fig. 7B), findings consistent
with the biochemical measures of their relative cell surface AMPase
activities (Fig. 2C).
Impact of Environmental Conditions on Adenosine
Production--
Because the airways are continuously exposed to
inhaled bacteria, we investigated whether the aseptic and stable
environment provided by the cell culture conditions affected the
expression of ecto 5'-NT and NS AP. Fig.
8A shows that ecto 5'-NT
mRNA levels in epithelial cultures and freshly excised bronchial
epithelia were not significantly different. In contrast, NS AP mRNA
was 10-fold lower in culture (p < 0.01). These results
suggest that the contribution of NS AP to adenosine production in
human airways would be underestimated by experiments performed on
epithelial cultures. To further test this notion, we compared the
mRNA levels of ecto 5'-NT and NS AP in human epithelia freshly
excised from the nose, trachea, bronchi, and bronchioles. Fig.
8B shows that the two ectoenzymes were detected throughout
the respiratory tract. Ecto 5'-NT and NS AP were expressed at
comparable levels in nasal turbinate and tracheal epithelia. In lower
airways, ecto 5'-NT expression gradually decreased toward alveoli,
whereas NS AP mRNA increased with airway generation. These results,
combined with the ex vivo assays conducted on bronchial
sections (Fig. 6B), support a major role for NS AP in the
elimination of high (> 0.01 mM) nucleotide concentrations
below the tracheobronchial tree.

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Fig. 8.
Cell culture versus tissue
expression of ecto 5'-NT and NS AP. A, selective
down-regulation by the culture conditions. RPAs conducted with total
RNA from freshly excised (F, filled bars)
and cultured (C, open bars) human bronchial
epithelial cells. NS AP (but not ecto 5'-NT) mRNA was higher
in vivo than in culture. B, opposite
distributions for ecto 5'-NT (filled bars) and NS AP
(open bars) in human airways. RPAs conducted with total RNA
from freshly excised human nasal, tracheal, bronchial, and bronchiolar
epithelial cells. Values normalized with -actin and corrected for
the number of G-C binding sites represent mean ± S.E. of four
independent experiments. *, p < 0.05.
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We also tested the impact of the inflammatory mediator IL-
on the
expression of ecto 5'-NT and NS AP. Primary cultures of human bronchial
epithelial cells were exposed to serosal 0.1-1.0 ng/ml IL-1
in
air-liquid interface medium during 24 h and then processed for
RPA. Fig. 9 shows that NS AP mRNA
increased with IL-1
concentration, whereas ecto 5'-NT mRNA was
unaffected. Collectively, these two sets of experiments suggest that
the high affinity ecto 5'-NT modulates physiological nucleotide
concentrations, independently of the environmental conditions. In
contrast, the fact that NS AP was down-regulated in cell culture, but
up-regulated by IL-1
, suggests that the ectoenzyme could be
recruited by inflammatory mediators released on epithelial surfaces in
response to infection.

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Fig. 9.
Impact of IL-1 on
the expression of human airway ecto 5'-NT and NS AP. Primary
cultures of human bronchial epithelial cells were exposed to serosal
0.0-1.0 ng/ml IL-1 during 24 h. RPAs on total RNA showed that
NS AP (open bars), but not ecto 5'-NT (filled
bars), was up-regulated by IL-1 . Values normalized with
-actin and corrected for the number of G-C-binding sites represent
mean ± S.E. of three independent experiments. *,
p < 0.05.
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DISCUSSION |
The current understanding of airway defense mechanisms against
bacterial infection acknowledges a complex interplay between ATP and
adenosine receptor-mediated epithelial functions (2-15). However,
despite numerous studies demonstrating that ATP is released from the
mucosal surface of human airway epithelia (20-28), the origin of
extracellular adenosine has not been established. In the present work,
we considered three possible sources of adenosine for the mucosal
surface: 1) paracellular or transepithelial transport from the
interstitium; 2) cytosolic formation and release from epithelial cells
facing the lumen; and 3) cell surface metabolism of locally released
ATP. First, Ussing chamber experiments demonstrated that serosal
[3H]adenosine did not permeate into the mucosal
compartment, but was locally converted into [3H]inosine
and [3H]hypoxanthine (Fig. 1, A and
B). This observation was confirmed by applying ATP to the
serosal compartment and searching by HPLC for the appearance of purine
nucleotides/nucleosides on the mucosal surface (Fig. 1D).
Second, we provided evidence that adenosine was not generated
intracellularly for transport to the mucosal surface. Specifically, we
sought evidence for the mRNA encoding the cytosolic enzyme for
adenosine production, CN-I (16, 17), and no evidence for expression of
this transcript was found by RT-PCR (Fig. 1C). Moreover, we
recently reported that extracellular adenosine was actively removed
from the mucosal surface of human nasal epithelial cultures by a
concentrative Na+-dependent nucleoside
transporter (58). These properties are consistent with the vectorial
transport system described for adenosine across intestinal epithelia
(59, 60). Taken together, these findings indicate that interstitial and
cytosolic pools of nucleosides do not contribute to the endogenous
adenosine concentrations detected on the mucosal surface of human
airway epithelia (29).
The abundant literature describing basal and stimulated ATP release
from the mucosal surface of airway epithelia (20-28) raised the
possibility that released ATP could represent the major source of
adenosine through cell surface metabolism. Indeed, cell surface conversion of extracellular ATP into adenosine has been reported in
most mammalian tissues (19). In the present study, we have shown that
ATP is sequentially dephosphorylated into ADP, AMP, and adenosine on
airway epithelial surfaces. The physiological importance of
constitutive ATP release and conversion to adenosine was emphasized by
the substantial inhibitory effect (>70%) of 0.3 mM
,
-met-ADP on the basal cystic fibrosis transmembrane regulator
activity of Calu-3 cells (11). This compound has been described as a
competitive inhibitor of ecto 5'-NT and AP, preventing cell surface
conversion of AMP to adenosine (32). The fact that ATP, ADP, AMP, and
adenosine were confined to the epithelial surface on which ATP was
administered suggested that mucosal and serosal surfaces represent
distinct compartments with respect to nucleotide pools and
purine-mediated signaling pathways. Patch clamp studies on Calu-3 cells
demonstrated the close proximity between the site of ATP release, the
enzymes responsible for adenosine production, A2B
receptors, G proteins, and cystic fibrosis transmembrane regulator (11). Taken together, these findings support local ATP release and
metabolism as the major source of adenosine for P1 receptor activation
on the mucosal surface of human airway epithelia.
The present work identifies the ectoenzymes directly responsible for
the production of adenosine from AMP on the mucosal surface of human
airway epithelia. The conversion of extracellular AMP into adenosine
was detected on nasal and bronchial epithelial cells, suggesting
widespread expression throughout human airways. Several ectoenzymes
have been reported to dephosphorylate AMP in the ectodomain: ecto 5'-NT
and APs. The biochemical properties of AMP hydrolysis on primary
cultures of human bronchial epithelial cells supported the functional
expression of more than one ectoenzyme. Mucosal surfaces hydrolyzed
nucleoside monophosphates with a substrate specificity (AMP
UMP = CMP > GMP
IMP) intermediate between that of
purified ecto 5'-NT (AMP = UMP > CMP > GMP > IMP
(48)) and purified AP (AMP = UMP = CMP = GMP = IMP
(33)). Mammalian ecto 5'-NT and AP have been reported to display
different cation and pH sensitivities. Purified ecto 5'-NT from rat
glioblastoma was insensitive to Ca2+ and Mg2+
(47). In contrast, human liver AP (61) and kidney AP (62) activities
were enhanced 2-3-fold by Mg2+, suggesting that an AP
could be responsible for the Mg2+-sensitive AMPase activity
we detected on the mucosal surface of human bronchial epithelial cells
(Fig. 3B). In the presence of Mg2+, the optimum
pH of purified ecto 5'-NT and AP were reported in the range 7.5-8.0
(47, 63) and 9-10 (33, 61, 62), respectively. We showed that assays
conducted in the presence of Ca2+ and Mg2+
generated two peaks of activity (pH 7.5 and 9.0) on the mucosal surface
and a single peak (pH 7.5) on the serosal surface (Fig. 3C).
Bimodal pH dependence profiles (pH 8.0 and 10.0) were also reported for
AMPase activities measured on intact cells or plasma membrane
preparations, the alkaline peak of activity exhibiting an absolute
requirement for Mg2+ (64, 65). Collectively, these results
suggest that ecto 5'-NT was responsible for the activity peak detected
on both surfaces around pH 7.5, whereas the alkaline activity
restricted to the mucosal surface corresponded to APs.
The identity of the AMP-hydrolyzing enzymes expressed on human
bronchial epithelial cells at pH 7.4 was further investigated with
specific inhibitors. The activity of ecto 5'-NT was revealed with
concanavalin A, a lectin reported to have no effect on APs (48). When
reactions were initiated with 0.1 mM AMP, this compound inhibited 80 and 100% of total activity measured on mucosal and serosal surfaces, respectively. The remaining mucosal AMPase activity was completely inhibited by
-glycerophosphate and levamisole, non-competitive (49) and competitive (50) inhibitors of APs, respectively. Conversely, serosal AMP hydrolysis was insensitive to
levamisole. These results were in agreement with the polarity of ecto
5'-NT and AP activities suggested by the pH and cation sensitivity experiments.
Levamisole has been used to discriminate between NS AP
(IC50 = 30 µM), PLA AP (IC50 = 1.7 mM), and I AP (IC50 = 6.8 mM)
(50). The high sensitivity of the mucosal AMPase activity to levamisole (IC50 = 17 µM; Fig. 4B) suggests
that NS AP would be the major AP isoform expressed on human airway
epithelial cells. In addition, the reaction was resistant to 10 mM L-phenylalanine (PLA and I AP inhibitor) and
10 mM L-leucine (G AP inhibitor) (34, 35). This
identification of NS AP as the dominant AP isoform expressed in
proximal airways was consistent with mRNA expression studies. Two
AP isoforms were detected by RT-PCR in total RNA extracted from
cultured or freshly excised human nasal, bronchial, and bronchiolar epithelial cells: NS AP and PLA AP (Fig. 7A). Whereas strong
signals were obtained for NS AP in all RNA fractions, PLA AP mRNA
was limited to bronchiolar epithelial cells. The localization of NS AP
and PLA AP in human lungs has been investigated by histochemical and
immunocytochemical procedures (36, 37).
L-p-Bromotetramisole-sensitive L-phenylalanine-resistant NS AP activity was detected on
the epithelial surface lining the entire respiratory system (36). In
contrast, PLA AP distribution was restricted to peripheral lung
parenchyma: respiratory bronchioli, alveolar ducts, alveolar sacs, and
alveoli (37). Therefore, the identity of the human airway AP isoforms provided in this work by functional assays and RT-PCR was supported by
studies of their tissue distribution.
The polarized primary cultures of human nasal and bronchial epithelial
cells exhibited the in vivo morphologic characteristics of
proximal airway epithelia (45), with columnar ciliated and secretory
cells covering basal-like cells. Based on the biochemical properties of
AMP hydrolysis on these cells, ecto 5'-NT would be expressed on mucosal
and serosal surfaces of airway epithelia, whereas NS AP would be
restricted to the mucosal surface. In rat nasal respiratory epithelium,
ecto 5'-NT displayed a polarity consistent with these results (66).
Ecto 5'-NT was localized by histochemistry to the mucosal surface of
columnar epithelial cells and the underlining basal cells. Human airway
NS AP was detected predominantly on the apical plasma membrane of
columnar epithelial cells (36). These in vivo studies
support the polarity we obtained for ecto 5'-NT and NS AP activities on
the culture model.
Novel information on the tissue distribution of the AMP-hydrolyzing
ectoenzymes was provided by quantitative analysis of mRNA levels
throughout human airways with cultured and freshly excised epithelial
preparations. First, ecto 5'-NT and NS AP expression were 2-3 times
higher in bronchial than in nasal epithelial cultures. Second, we
demonstrated with freshly excised epithelial cells that the two
ectoenzymes exhibit opposite expression patterns throughout airways.
Ecto 5'-NT mRNA levels gradually declined from nasal to bronchiolar
epithelia, whereas the expression of NS AP increased with airway
generation. This constitutes the first report of ecto 5'-NT in
mammalian airway epithelia below the nasal cavity. Interestingly,
opposite gradient distributions for the two ectoenzymes were also
reported for mammalian intestinal mucosa, with decreasing ecto 5'-NT
immunostaining (67) and increasing AP activity (68) from the small
intestine to the colon.
The relative contribution of the two AMP-hydrolyzing ectoenzymes on the
mucosal surface of human bronchial epithelial cells was strongly
influenced by substrate concentration. Experiments conducted on
epithelial cultures (Fig. 4, C and D) and freshly excised bronchial tissues (Fig. 6B) indicated that 5 mM AMP was eliminated 5-6 times more rapidly by NS AP than
by ecto 5'-NT. In contrast, ecto 5'-NT activity dominated 5-fold over
NS AP when assayed with 10 µM AMP (Fig. 6B).
Further analysis of the substrate concentration-enzyme activity
relationship revealed different kinetic properties for the two
ectoenzymes (Fig. 5, A and B). Ecto 5'-NT
presented a single high affinity activity for AMP, with a
Km of 14 µM, a value that falls within
the range reported for purified ecto 5'-NT (5-20 µM)
(47, 63). In contrast, NS AP exhibited two kinetic components: a high
affinity (Km = 36 µM) and a low
affinity (Km = 717 µM) activity. These findings are consistent with the kinetic properties reported for NS AP
purified from rat osseous plates at pH 7.5, with Km values of 82 µM and 1.3 mM for ATP (69).
Human osteosarcoma NS AP assayed at pH 7.4 also displayed high and low
affinity activities, with Km values of 25 and 780 µM, respectively (51). The fact that ecto 5'-NT and NS AP
possess high affinity activities suggests that they both participate in
the metabolism of nucleotide concentrations (
10 µM)
detected locally following ATP release from human airway epithelial
cells (70-72). However, the 2-fold higher Cateff of ecto
5'-NT predicts that this enzyme would be more efficient than NS AP at
producing adenosine from physiological AMP concentrations, as
demonstrated by their respective contribution to the hydrolysis of 10 µM AMP on bronchial cultures and tissues. Finally, AMP
hydrolysis was competitively inhibited by ATP (Ki = 7 µM) and ADP (Ki = 10 µM) on human bronchial epithelial cells (Fig. 5,
C and D), as previously reported for purified rat renal ecto 5'-NT (Ki = 0.03-30 µM)
(63, 73, 74). Consequently, the conversion of AMP into adenosine
would represent a rate-limiting step in the production of P1 receptor
agonists from ATP release on airway surfaces.
The purinergic modulation of MCC on human airway epithelial surfaces
involves a complex interplay between nucleotide and nucleoside concentrations, ectonucleotidases, P2 and P1 receptors. Mechanical stimulation, such as coughing-induced shear stress, raises local cell
surface ATP to concentrations (1-10 µM) (70-72) that
initiate P2 receptor-mediated MCC functions (2-9). Desensitization of the P2Y2 receptors by prolonged exposure to micromolar
nucleotides (4, 75) might be avoided by the rapid dephosphorylation of ATP and ADP into AMP we reported on human airway epithelial surfaces (21, 31). Through the conversion of extracellular AMP into adenosine,
ecto 5'-NT and NS AP would provide the agonist (adenosine) required for
the smaller but more sustained MCC responses, mediated by
A2B receptor regulation of CBF (4, 5) and ion transport (12-15).
In summary, we have demonstrated that ecto 5'-NT and NS AP are
responsible for the production of adenosine on the mucosal surface of
human airway epithelial cells. The relatively high efficiency of ecto
5'-NT suggests that this enzyme would play a major role in the
regulation of adenosine-mediated epithelial functions. On the other
hand, all APs dephosphorylate not only AMP but also ADP and ATP (33).
Under pathological conditions, trauma generates quantities of
extracellular nucleotides that may cause damage to the epithelium. For
example, airway epithelial cultures have been reported to express
P2X7 receptors (8), an ATP-gated channel known to induce
apoptosis (76). The high-capacity NS AP could protect these airways
against the deleterious effects of high ATP concentrations. The fact
that IL-1
enhanced 5-fold NS AP activity and expression supports a
role for this enzyme in airway defenses during periods of inflammation.
We would note, however, that the broad substrate specificity of APs
suggests that NS AP could be involved in other airway functions,
including bacterial endotoxin neutralization (77) and sphingosine
1-phosphate receptor signaling (78, 79).