From the Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599
Received for publication, August 7, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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Mechanically induced ATP release from
human airway epithelial cells regulates mucociliary clearance through
cell surface nucleotide receptors. Ectoenzymes detected on these cells
were recently shown to terminate ATP-mediated responses by sequential
dephosphorylation of extracellular ATP into ADP, AMP, and adenosine. We
now demonstrate that an ecto-adenylate kinase (ecto-AK) contributes to
the metabolism of adenine nucleotides on human airway epithelial
surfaces by the reversible reaction: ATP + AMP Mucociliary clearance
(MCC)1 represents the first
line of defense against lung infection. Inhaled bacteria are trapped
within a mucus blanket covering the epithelium and mechanically cleared by coordinated cilia beating activity (for a review, see Ref. 1).
Extracellular nucleotides are well recognized as regulators of the
epithelial functions supporting MCC, including mucus secretion, cilia
beat frequency (CBF), and ion channel activities involved in the
maintenance of epithelial surface liquid volume (for a review, see Ref.
2). Nucleotides are released by the epithelium under basal conditions
(3, 4) and in response to membrane stretch (5, 6), shear stress (7, 8),
and hypo-osmotic cell swelling (9-12). On the mucosal surface, they
interact with two members of the G-protein-coupled P2Y receptor (P2YR)
family: P2Y2 (13) and P2Y6 (14).
P2Y2 receptors are equally activated by ATP and UTP, but
not by ADP or UDP, whereas P2Y6 receptors are potently
activated by UDP and weakly activated by ADP. The serosal
epithelial surface expresses P2Y2 (15) and
P2Y1 (ADP > ATP > UTP) (6) receptors. Taylor et
al. (17) demonstrated that human airway epithelial cultures also
express members of the ionotropic P2X receptor (P2XR) family:
P2X4 and P2X5. Patch clamp and Ussing chamber
experiments indicated that ATP-induced Ca2+ entry through
these channels stimulates luminal Cl Over the last decade, several studies have demonstrated the role of
cell surface nucleotide metabolism in the regulation of MCC-related
epithelial functions (9, 10, 19, 22). ATP and UTP elicited large
transient increases in CBF on human nasal explants (19). However, ATP
also produced a post-peak sustained increase in CBF that was
prevented by the nonspecific adenosine receptor antagonist,
8-p-sulfophenyltheophylline. Hypotonicity-induced cell
swelling triggered ATP release and activation of volume-sensitive Cl We reported that human nasal and bronchial epithelial surfaces express
metabolic enzymes that sequentially dephosphorylate ATP into ADP, AMP,
and adenosine and UTP into UDP, UMP, and uridine (29-31). Lazarowski
et al. (32) proposed that extracellular nucleotide metabolism in human airways also involves transphosphorylating enzymes.
They identified a nucleoside diphosphate kinase (NDPK) activity on the
mucosal surface of human nasal epithelial cells. Classically described
as ubiquitous intracellular enzymes, NDPKs catalyze the transfer of
In the present study, we describe the biochemical and kinetic
properties, as well as the polarity, of ecto-AK expressed at the
surface of human bronchial epithelia. We measured the impact of ecto-AK
on the availability of adenine nucleotides for P2 (ATP and ADP) and P1
(adenosine) receptors regulating MCC. We tested the hypothesis that
ecto-AK could extend the duration of locally released ATP to promote
airway clearance of noxious agents. Biochemical assays were performed
on polarized primary cultures of bronchial epithelial cells from normal
donors and patients diagnosed with cystic fibrosis (CF), an inherited
obstructive lung disease characterized by impaired MCC, chronic
infection, and inflammation (for a review, see Ref. 41). This study
provides evidence that ecto-AK plays a major role in the prolongation
of P1 and P2 receptor-mediated MCC functions on human airway epithelial surfaces.
Cell Culture--
Well differentiated primary cultures of human
bronchial epithelial cells were grown as described previously (42). In
brief, the cells were isolated from freshly excised main stem bronchi by protein digestion (43) and plated on porous Transwell Col filters
(well diameter, 12 mm; pore size, 0.45 µM) in air-liquid interface medium (50:50 mixture of LHC basal and Dulbecco's
modified Eagle's medium-high glucose, 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/µl streptomycin) (44). Once they reached confluence, the
cultures were maintained in air-liquid interface with 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 with transepithelial electrical resistance Ectonucleotidase Assays--
Primary cultures of human
bronchial epithelial cells were rinsed three times with Krebs-Ringer
buffer (KRB): 140 mM Na+, 120 mM
Cl Synthesis of [3H]ADP and
[3H]UDP--
Tritiated ADP and UDP were obtained from
their respective nucleoside triphosphates by hexokinase reaction, as
described previously (46). In brief, 50 µCi of either
[3H]ATP or [3H]UTP (40-50 Ci/mmol) were
incubated with 10 units/ml hexokinase (30 min; 37 °C) in 0.2 ml of
KRB. The samples were boiled for 3 min to eliminate hexokinase
activity, and full conversion into [3H]ADP or
[3H]UDP was confirmed by HPLC.
Enzyme Release Assays--
Mucosal epithelial surfaces were
rinsed three times with KRB and incubated in KRB (350 µl of
mucosal/serosal) during 90 min at 37 °C (5% CO2/95%
O2). The conditioned buffer was collected and centrifuged
at 14,000 × g (4 °C; 20 min) to remove detached cells, debris, and large organelles. The supernatant was transferred to
tubes containing 1 mM [ Kinetic Properties of Human Airway Ecto-AK--
We established
the kinetic properties of the forward and reverse ecto-AK reactions for
ATP and ADP, respectively. For the forward reaction, the assays were
initiated with 1 mM AMP + 0.001-1 mM
[ HPLC Separation of Nucleotides--
The separation system
consisted of a Dinamax C-18 column and a mobile phase developed with
buffer A (10 mM KH2PO4 and 8 mM tetrabutyl ammonium hydrogen sulfate (TBASH), pH 5.3)
from 0 to 10 min, buffer B (100 mM
KH2PO4, 8 mM TBASH, and 10% 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 Scientific Instruments, Columbia, MD), and radioactivity was determined on-line with a Flo-One Radiomatic Materials--
All 5'-nucleotides and adenosine were purchased
from Roche Molecular Biochemicals. KH2PO4,
TBASH, and HEPES were obtained from Sigma. HPLC-grade water was
bought from Fisher Scientific. Cell culture media, bovine serum
albumin, bovine pituitary extract, epidermal growth factor, gentamicin,
penicillin, retinoic acid, and streptomycin were bought from
Invitrogen. [2,83H]ATP (40-50 Ci/mmol),
[2,83H]UTP (40-50 Ci/mmol), and
[ Data Analysis--
All enzyme assays were performed on primary
cultures of human bronchial epithelial cells obtained from at least
five different donors or CF patients. Rates of dephosphorylation were
calculated from the initial linear rate of substrate decay monitored by
HPLC and presented as nmol·min Detection of Ectonucleotidase and Ecto-AK Activities on
Airway Epithelia--
Polarized bronchial epithelial cultures were
assayed for nucleotide metabolism. Fig.
1A presents typical HPLC
traces for samples collected 0, 2, and 5 min after the addition of 0.1 mM [3H]ATP to the mucosal surface. The
nucleotide was converted into less phosphorylated compounds
([3H]ADP, [3H]AMP,
[3H]adenosine) and [3H]inosine. Time course
analysis showed that the disappearance of [3H]ATP
occurred during the accumulation of [3H]ADP and
[3H]AMP (Fig. 1B). [3H]Adenosine
concentration increased over the first 40 min and accounted for 50% of
all radioactive species by the end of the experiment. In contrast,
reactions initiated with 0.1 mM [3H]ADP
indicated that adenine nucleotide metabolism involves other activities
besides dephosphorylation. As observed with [3H]ATP,
[3H]ADP was converted into less phosphorylated
compounds ([3H]AMP, [3H]adenosine) and
[3H]inosine (Fig. 1C). The HPLC traces
also revealed the presence of [3H]ATP, suggesting that
[3H]ADP was phosphorylated. Time course analysis showed
transient accumulation of [3H]AMP and
[3H]ATP during the first 30 min, whereas adenosine and
inosine concentrations increased during the entire incubation period
(Fig. 1D). These results support a complex interplay between
dephosphorylating and phosphorylating activities on human airway
epithelial surfaces.
We explored the possibility that an ecto-AK was responsible for the
phosphorylation of ADP into ATP at the surface of human bronchial
epithelial cells. By definition, an AK supports the reversible
reaction: ATP + AMP Cell-associated Ecto-AK and ATPase Activities--
The mucosal
surface of human bronchial epithelial cultures and conditioned KRB were
assayed for ATPase and AK activities with 1 mM
[ Cation Dependence and Substrate Specificity of Ecto-AK--
Human
airway ecto-AK exhibited an absolute requirement for divalent cations
and adenine nucleotides, as reported for intracellular AKs (34, 49).
Enzyme assays were conducted on the mucosal surface of human bronchial
epithelial cultures with 0.1 mM [ Kinetic Properties of Human Airway Ecto-AK--
Experiments were
designed to compare the kinetic properties of the forward and reverse
ecto-AK reactions on the mucosal surface of human bronchial epithelial
cells. The forward reaction was measured with 1 mM AMP + 0.001-1 mM [ Polarity and Directionality of Ecto-AK on Airway Epithelial
Surfaces--
Nucleotide transphosphorylation was detected on
both mucosal and serosal surfaces of human bronchial epithelial
cultures (Table I). Enzyme assays,
performed with saturable substrate concentrations, indicated that
forward and reverse activities were 4-fold higher on the mucosal
surface (Table I, reactions 1-2; p < 0.01). These experiments also showed that ecto-AK favored AMP phosphorylation by
3-fold over [3H]ADP phosphorylation on both epithelial
surfaces (p < 0.01). These results were in agreement
with the higher affinity of the enzyme for ATP over ADP (Fig. 5,
A and B). The directionality of the reversible
ecto-AK reaction suggested that the enzyme could partially circumvent
the loss of high energy phosphate groups resulting from
dephosphorylation of AMP into adenosine.
Contribution of Ecto-AK to ADP Metabolism--
We evaluated the
contribution of ecto-AK to the metabolism of ADP on human bronchial
epithelial surfaces with the non-permeant AK inhibitor,
diadenosine pentaphosphate (Ap5A (37)). This
dinucleotide was shown to act as competitive inhibitor for the forward
AK reaction (ATP + AMP
The possibility remained that this inhibition reflected, at least in
part, interactions between Ap5A and ADP-dephosphorylating enzymes reported on these cells (4, 29-32). To address this question,
we took advantage of the substrate specificity and ion requirements of
ecto-AK (Fig. 4). The hydrolysis of 2 mM
[3H]UDP was reduced from 0.73 nmol·min Impact of Ecto-AK on the Metabolism of ATP--
We designed time
course experiments to investigate the impact of ecto-AK on ATP and
other receptor agonists (ADP and adenosine) generated during cell
surface metabolism. Since dephosphorylating ectoenzymes hydrolyze ATP
and UTP (for a review, see Ref. 51), whereas ecto-AK is specific
for adenine nucleotides (for a review, see Ref. 34) (Fig. 4,
C and D), the phosphotransferase activity would
be responsible for any discrepancy between the patterns of ATP and UTP
metabolism through time. These experiments were conducted with
substrate concentrations (10 µM) relevant to stimulated nucleotide release and P2 receptor activation (for a review, see Ref.
52). Fig. 7, A and
B, shows that initial hydrolytic rates for
[3H]UTP were lower than for [3H]ATP with
values of 7.9 ± 1.2 nmol·min
The reversible transphosphorylating activity of ecto-AK on adenine
nucleotides could be responsible for the differences in UTP and ATP
metabolism. The initial faster rates of [3H]ATP
degradation and [3H]ADP accumulation could reflect the
combination of [3H]ATP dephosphorylation into
[3H]ADP and phosphorylation of [3H]AMP into
[3H]ADP. During the following 15 min, the reversible
ecto-AK reaction competed with dephosphorylating enzymes to prolong the
pools of [3H]ATP, [3H]ADP, and
[3H]AMP. Adenine nucleotide concentrations eventually
decreased below the micromolar range, suggesting that phosphate groups
were escaping the nucleotide entrapment cycle, most likely through conversion of AMP into adenosine by the high affinity
ecto-5'-nucleotidase activity (Km = 14 µM) we described on these cells (30). Consequently,
ecto-AK would prolong P2 receptor agonist availability and delay
A2B receptor activation over a finite period of time following ATP release.
Lazarowski and Boucher (53) reported the presence of AMP on the mucosal
surface of human airway epithelial cultures. We hypothesized that
endogenous AMP concentrations would be sufficient to support
transphosphorylation events initiated by the addition of 10 µM [ Impact of Cystic Fibrosis on Human Airway Ecto-AK--
The airways
of CF patients are characterized by severe inflammatory responses to
chronic bacterial infection, resulting from defective MCC (for a
review, see Ref. 41). We investigated the long term effects of airway
obstruction, chronic infection, and inflammation on the activity of
human airway ecto-AK. Primary cultures of bronchial epithelial cells
from normal donors and CF patients were assayed for forward and reverse
ecto-AK activities with 2 mM [3H]ADP and 1 mM [ In the present study, we have demonstrated that extracellular
adenine nucleotide concentrations on human airway epithelial surfaces
are regulated by a complex interplay between dephosphorylating and
transphosphorylating ectoenzymes. Extracellular ATP was rapidly dephosphorylated by the stepwise reaction: ATP We first addressed the distribution of ecto-AK on human airway
epithelial surfaces. The primary bronchial cultures exhibited the
morphologic characteristics expressed in vivo (45) with a
layer of columnar ciliated and secretory cells covering 1-2 layers of
basal-like cells. Both forward and reverse ecto-AK reactions were
bilaterally distributed with 4-fold higher activities on the mucosal
than on the serosal surface. The mucosal surface is comprised of the
apical surface of the columnar cells and a thin liquid layer,
i.e. airway surface liquid. Time course experiments indicated that more than 90% of the mucosal ecto-AK activity remained associated to the epithelial surface. Similar results were reported for
human nasal epithelial cultures, whereby released AK represented ~20% of total ecto-AK activity measured on the mucosal surface (40).
Given the short half-life of extracellular nucleotides within the
airway surface liquid (i.e. 0.2 mM UTP;
t1/2 ~30 s (55)), these findings suggest that
human airway ecto-AK is likely to target extracellular nucleotides
released by the epithelium.
The kinetic properties of human airway ecto-AK at first glance may not
suggest a significant role in the modulation of physiological nucleotide concentrations on airway surfaces. Ecto-AK
transphosphorylated ATP and ADP with Km values of 23 and 43 µM, respectively. Although these values are lower
than for ecto-AK on rat brain synaptosomes (Km,
ATP = 460 µM (37)) and human endothelial cells
(Km, ATP = 135 µM (38)), ATP
concentrations on airway epithelial surfaces, measured by
luciferin-luciferase assays in bulk solutions, are >100-fold lower
under both basal (1-10 nM) and stimulated (5-200
nM) conditions (56, 57). It is important to consider,
however, that measurements of endogenously released ATP in large buffer
volumes may significantly underestimate near cell surface
concentrations. A biosensor technique, using
P2X2R-expressing PC12 cells as probes, detected ~13
µM ATP at the surface of osmotically challenged
intestinal epithelial cells, as compared with 5 nM by the
"bulk fluid" luciferin-luciferase assay (57). Single cell confocal
bioluminescence luciferase assays (58) and cell-attached luciferase
assays (59) also measured ATP concentrations as high as 10-80
µM in close vicinity to the site of stimulated release. These studies suggest that ecto-AK may influence physiological nucleotide concentrations on human airway epithelial surfaces, particularly following an osmotic or mechanical stimulation.
Most dephosphorylating and transphosphorylating ectoenzymes
characterized on human airway epithelial surfaces exhibit substrate affinities in the low micromolar range. Ecto-5'-nucleotidase hydrolyzed AMP into adenosine with a Km of 14 µM
(30), and ecto-nucleotide pyrophosphatase/phosphodiesterases (ATP A key finding of this work is the demonstration that
ecto-AK competes with dephosphorylating enzymes to maintain adenine
nucleotides on human airway epithelial surfaces. The higher affinity of
human bronchial ecto-AK for ATP over ADP translated into 3-fold higher rates for the forward (ATP + AMP Perhaps the most compelling evidence for the physiological relevance of
this nucleotide entrapment cycle was obtained from assays conducted
with endogenous nucleotides. Adenine nucleotide and nucleoside
concentrations measured on the mucosal surface of human nasal
epithelial cells by etheno derivatization assays in bulk fluid were:
ATP (10 nM), ADP (40 nM), AMP (70 nM), and adenosine (200 nM) (53). Because AMP
levels are significantly higher than ATP under basal conditions,
stimulated ATP release would drive the forward ecto-AK reaction (ATP + AMP Based on the properties of ecto-AK, the dephosphorylating ectoenzymes
(4, 29-32), P2XRs and P2YRs on human airway epithelial surfaces, we
propose the following model for adenine nucleotide-mediated MCC (Fig.
10). Immediately following locally
stimulated ATP release, the nucleotide concentration is at its highest,
in a range that activates P2XRs (EC50 = 10 µM; for a review, see Refs. 62 and 63). P2XRs may
desensitize rapidly, i.e. within ~30 s (16, 20),
suggesting that a prolonged ATP level may not be important for
signaling. On the other hand, P2YRs are activated by lower ATP
concentrations (EC50 = 0.1-1 µM; for a
review, see Ref. 21) and desensitize less rapidly. For instance,
Cl 2ADP. This
phosphotransferase exhibited a bilateral distribution on polarized
primary cultures of human bronchial epithelial cells with a 4-fold
higher activity on the mucosal surface. Ecto-AK presented an absolute
requirement for magnesium and adenine-based nucleotides. UMP, GMP, and
CMP could not substitute for AMP as
-phosphate acceptor, and UDP could not replace ADP. Apparent Km and
Vmax values were 23 ± 5 µM
and 1.1 ± 0.1 nmol·min
1·cm
2 for
ATP and 43 ± 6 µM and 0.5 ± 0.1 nmol·min
1·cm
2 for ADP. Ecto-AK
accounted for 20% of [
-32P]ATP
dephosphorylation, and the impermeant AK inhibitor, diadenosine pentaphosphate, reduced ADPase activity by more than 70% on both epithelial surfaces. Time course experiments on ATP metabolism demonstrated that ecto-AK significantly prolongs effective ATP and ADP
concentrations on airway epithelial surfaces for P2 receptor signaling
and reduces by 6-fold adenosine production. Our data suggest a role for
this nucleotide entrapment cycle in the propagation of purine-mediated
mucociliary clearance on human airway epithelial surfaces.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
secretion. In rabbit
trachea, epithelial P2XRs contribute to ATP-mediated increase in CBF
via Ca2+ influx (18).
channels by a mechanism that required cell surface
conversion of ATP into adenosine (10). In a human bronchial cell line
lacking P2Y2 receptors (Calu-3 (23)), the basal ion channel
activity of the cystic fibrosis transmembrane regulator (CFTR) was
inhibited by 8-p-sulfophenyltheophylline and by AMPCP (22),
an inhibitor of the cell surface conversion of AMP into adenosine (for
a review, see Ref. 24). Incidentally, adenosine has been shown to
trigger MCC-related epithelial responses, including CBF (19, 25) and ion transport (26-28).
-phosphate groups between nucleoside di- and triphosphates by the
reversible reaction: NTP + NDP
NDP + NTP (for a review, see Ref.
33). Transphosphorylation events taking place in most intracellular
compartments combine the activities of NDPKs and adenylate kinases
(AKs; ATP:AMP-phosphotransferases). In contrast to the broad substrate
specificity of NDPKs, AKs transfer phosphate groups between
adenine-based nucleotides by the reaction: MgATP + AMP
MgADP + ADP
(for a review, see Ref. 34). Cell surface AK (ecto-AK) activity was
recently detected on Sartorius frog muscles (35, 36), rat
brain synaptosomes (37), human umbilical vein endothelial cells (38,
39), and human nasal epithelial cells (40). However, the physiological
role of cell surface adenine nucleotide transphosphorylation has not
been explored.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
300 ohms·cm
2. Lactate dehydrogenase activity was employed
as a test of cellular integrity.
, 5.2 mM K+, 25 mM
HCO
-32P]ATP (0.1 µCi) as substrates. ADPase and UDPase activities were assayed with
[3H]ADP (0.5 µCi) and [3H]UDP (0.5 µCi), respectively. The forward ecto-AK reaction (ATP + AMP
2ADP)
was initiated with equal concentrations of [
-32P]ATP
(0.1 µCi) and AMP mixed previously and then quantified by the rate of
[
-32P]ADP production. The reverse ecto-AK reaction
(2ADP
ATP + AMP) was measured by the rate of [3H]ATP
production from [3H]ADP (0.5 µCi).
-32P]ATP (0.1 µCi) or 1 mM [
-32P]ATP (0.1 µCi) + 1 mM AMP in 35 µl KRB for assessment of ATPase and ecto-AK
activities in a shaker bath (37 °C). These values were compared with
enzyme activities measured the following day on the same
cultures to ascertain the contribution of released enzymes to total
epithelial surface activity.
-32P]ATP (0.1 µCi), and reaction rates were
calculated from [
-32P]ADP production. For the reverse
reaction, the assays were initiated with 0.001-1 mM
[3H]ADP (0.5 µCi), and reaction rates were calculated
from [3H]ATP production. All experiments were conducted
on the mucosal surface of human bronchial epithelial cultures, as
described above for enzyme assays. Samples were collected after
incubation periods that limited substrate hydrolysis to
10%
and were analyzed by HPLC. Michaelis-Menten 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 (47).
detector (Packard, Canberra, Australia), as described previously (48).
-32P]ATP (3000 Ci/mmol) were obtained from Amersham
Biosciences. Salts and solvents were of analytical grade.
1·cm
2.
Forward and reverse ecto-AK activities were calculated from initial
linear rates of accumulation of the phosphorylated product. Values
were expressed as means ± S.E. Unpaired Student's
t tests were used to assess the significance between
measurements performed on different cultures. Paired T tests were used
for comparisons between mucosal and serosal surfaces of the same
culture or between conditioned buffer and the corresponding epithelial
surface. All linear regressions, curve, fits and data transformations
were performed with the PC computer programs Origin and Sigma plot.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ATP and ADP metabolism at
the surface of human bronchial epithelial cells. The epithelial
cultures were incubated with KRB (pH 7.4; 0.35 ml mucosal/serosal)
containing mucosal 0.1 mM [3H]ATP (0.5 µCi)
(A and B) or 0.1 mM
[3H]ADP (0.5 µCi) (C and D).
Buffer samples collected over 60 min and analyzed by HPLC revealed
[3H]ATP (A) and [3H]ADP
(C) dephosphorylation, as well as phosphorylation of
[3H]ADP into [3H]ATP (C and
D). Time course analyses indicated significant differences
in the patterns of [3H]ATP (B) and
[3H]ADP (D) metabolism on bronchial epithelial
cells: [3H]ATP ( ), [3H]ADP (
),
[3H]AMP (
), [3H]adenosine (
),
[3H]inosine (
). HPLC traces represent typical results
obtained from six independent experiments. Metabolic patterns represent
mean results from all experiments (S.E. < 10% of the mean).
2ADP (for a review, see Ref. 34). We already
demonstrated that these epithelial cultures support the reverse
reaction (2ADP
ATP + AMP; Fig. 1, C and D).
In the following experiments, we investigated the forward AK reaction
(ATP + AMP
2ADP). Fig. 2A
shows typical HPLC traces obtained from buffer samples collected 0, 2, and 5 min after the addition of 0.1 mM
[
-32P]ATP to the mucosal surface. The
dephosphorylation of [
-32P]ATP was detected by the
accumulation of inorganic phosphate ([32P]Pi). Reactions initiated with 0.1 mM [
-32P]ATP + 0.1 mM AMP
produced an additional radioactive compound that co-migrated with ADP
standards (Fig. 2B), suggesting that the
-phosphate group
of [
-32P]ATP was transferred to AMP to produce
[
-32P]ADP. These experiments demonstrated that human
airway epithelial surfaces exhibit both forward and reverse AK
activities.
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Fig. 2.
Detection of ecto-AK activity on human
bronchial epithelial cells. The epithelial cultures were incubated
with KRB (pH 7.4; 0.35 ml mucosal/serosal) containing mucosal 0.1 mM [ -32P]ATP (0.1 µCi) (A) or
0.1 mM [
-32P]ATP (0.1 µCi) + 0.1 mM AMP (B). Buffer samples (10 µl) collected
over 10 min were analyzed by HPLC. HPLC traces are presented for buffer
samples collected after 0, 2, and 5 min. As shown in
A, [
-32P]ATP dephosphorylation was detected
by [32P]inorganic phosphate
([32P]Pi) release. As shown in
B, addition of 0.1 mM
[
-32P]ATP + 0.1 mM AMP generated
[32P]Pi by dephosphorylation and
[
-32P]ADP by ecto-AK phosphorylation. HPLC traces
represent typical results from six independent experiments.
-32P]ATP and 1 mM
[
-32P]ATP + 1 mM AMP, respectively (see
"Experimental Procedures"). Similar levels of ATPase and AK
activities were detected in the conditioned KRB (Fig.
3A). On the epithelial
surface, the rate of [
-32P]ATP dephosphorylation by
ATPase activity was 5-fold higher than the rate of AMP phosphorylation
by AK activity (Fig. 3B). Importantly, released ATPase and
AK activities represented ~3 ± 1% and 11 ± 2% of total
surface activity, respectively. The cell surface enzymes, remaining on
the bronchial culture after the conditioned KRB was collected, were not
eluted by subsequent washes (five rapid changes in KRB). These
experiments indicate that the ectoenzymes responsible for the
interconversion of phosphate groups between extracellular nucleotides
were predominantly associated to the epithelial surface of human
bronchial epithelial cells.
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Fig. 3.
Contribution of released enzymes to cell
surface ecto-AK and ATPases. A, enzyme activities
released from the mucosal surface. KRB was conditioned by a 90-min
exposure to the mucosal surface. B, cell-associated
enzyme activities. The mucosal surface was assayed for total enzyme
activities. ATPase (filled bars) and ecto-AK (open
bars) activities were assayed in KRB (pH 7.4) with 0.1 mM [ -32P]ATP (0.1 µCi) and 0.1 mM [
-32P]ATP (0.1 µCi) + 0.1 mM AMP, respectively. Buffer samples (10 µl) collected
over 10 min were analyzed by HPLC. Released enzyme activities accounted
for <15% of total epithelial surface activity. Values represent
mean ± S.E. of five independent experiments (*, p < 0.01).
-32P]ATP + 0.1 mM AMP. The kinase activity was not detected in
Mg2+-free KRB containing millimolar Ca2+ (Fig.
4A). The rate of AMP
phosphorylation was proportional to the Mg2+ concentration.
Fig. 4B shows that UMP, GMP, and CMP could not substitute
for AMP as phosphate acceptor for the forward ecto-AK reaction. In
addition, UDP could not substitute for ADP as phosphate donor or
acceptor for the reverse ecto-AK reaction (Fig. 4, C and
D). Fig. 4C presents typical HPLC traces for
buffer samples collected 0, 5, and 10 min after the addition of 0.1 mM [3H]UDP to the mucosal surface. The
nucleotide was gradually dephosphorylated into [3H]UMP
and [3H]uridine but was not phosphorylated into
[3H]UTP (Fig. 4D). Taken together, these
results indicate the presence of a
Mg2+-dependent ecto-AK activity specific for
adenine nucleotides at the surface of human bronchial epithelial
cells.
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Fig. 4.
Biochemical properties of ecto-AK on human
bronchial epithelial cells. A, absolute
requirement for Mg2+. Epithelial cultures were incubated in
KRB (pH 7.4) containing 0.1 mM [ -32P]ATP
(0.1 µCi) + 0.1 mM AMP in the mucosal bath. Buffer
samples (10 µl) collected over 10 min were analyzed by HPLC. KRB
contained 1.5 mM Ca2+ + 0, 0.5, or 1.5 mM Mg2+. B-D, ecto-AK specificity
for adenine-based nucleotides. As shown in B, mucosal
0.1 mM [
-32P]ATP (0.1 µCi)
phosphorylated 0.1 mM AMP but not UMP, CMP, or GMP.
C, HPLC traces for the metabolism of mucosal 0.1 mM [3H]UDP (0.1 µCi). As shown in
D, time course assays showed that [3H]UDP
(
) was dephosphorylated into [3H]UMP (
) and
[3H]uridine (
) but not phosphorylated into
[3H]UTP. Bar diagrams show means ± S.E. of 6-8
independent experiments (*, p < 0.05). HPLC traces
represent typical results and metabolic patterns mean results (S.E. < 10% of the mean) from all experiments.
-32P]ATP. Fig.
5A shows that the rate of
[
-32P]ADP production increased with substrate
concentration and saturated with 0.6 mM
[
-32P]ATP. The reverse reaction was measured with
0.001-3 mM [3H]ADP. The production of
[3H]ATP saturated at a higher substrate concentration
than the forward reaction (Fig. 5B; p < 0.05). Woolf-Augustinson Hoftsee analysis indicated that the
Km for [
-32P]ATP was 2-fold lower
than for [3H]ADP with corresponding values of 23 ± 5 µM and 43 ± 6 µM, respectively (Fig. 5, A and B, insets;
p < 0.05). The Vmax of the
forward reaction was 2-fold higher than for the reverse reaction with
values of 1.1 ± 0.1 nmol·min
1·cm
2
and 0.5 ± 0.1 nmol·min
1·cm
2,
respectively (Fig. 5, A and B, insets;
p < 0.05). Calculated Cateff
were 0.025 ± 0.005 min
1 and 0.014 ± 0.002 min
1 for [
-32P]ADP and
[3H]ATP production, respectively (p < 0.05).
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Fig. 5.
Kinetic properties of human bronchial
ecto-AK. A, catalytic properties of the forward
reaction, initiated with 1 mM AMP + 0.001-1 mM
[ -32P]ATP (0.1 µCi). [
-32P]ADP
production saturated with 0.6 mM
[
-32P]ATP. Inset, Woolf-Augustinson Hoftsee
transformation fitted to one regression (r = 0.96-0.99) with Km, Vmax,
and catalytic efficiencies of 23 ± 5 µM, 1.1 ± 0.1 nmol·min
1·cm
2, and 0.025 ± 0.005 min
1, respectively. B, catalytic
properties of the reverse reaction, initiated with 0.001-3
mM [3H]ADP (0.5 µCi). Inset,
Woolf-Augustinson Hoftsee transformation fitted to one regression
(r = 0.97-0.99) with Km,
Vmax, and catalytic efficiencies of 43 ± 6 µM, 0.5 ± 0.1 nmol·min
1·cm
2, and 0.014 ± 0.002 min
1, respectively. Samples were collected after
incubation periods that limited substrate hydrolysis to
10% and then
analyzed by HPLC. Values represent mean ± S.E. of five
independent experiments.
Measurements of ecto-adenylate kinase activity at the surface of human
bronchial epithelial cells
2ADP) and non-competitive inhibitor for the
reverse AK reaction [2ADP
ATP + AMP] (50). We tested the impact
of Ap5A on the phosphorylation of ADP by human bronchial
ecto-AK. Dose-response curves were constructed on mucosal and serosal
surfaces with 2 mM [3H]ADP and a range
(0-0.5 mM) of Ap5A concentrations. Fig.
6A shows that
[3H]ATP production was inhibited in a
dose-dependent manner and completely abolished by 0.5 mM Ap5A. In the absence of ecto-AK activity,
the pattern of [3H]ADP metabolism (Fig. 6B)
closely resembled the pattern of [3H]UDP metabolism (Fig.
4D). This Ap5A concentration reduced
[3H]ADP metabolism by 60-70% on mucosal and serosal
surfaces (Fig. 6C).
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Fig. 6.
Contribution of ecto-AK to ADP metabolism on
human bronchial epithelial cells. Epithelial cultures were
incubated with KRB (pH 7.4) containing bilateral 2 mM
[3H]ADP (0.5 µCi), and buffer samples collected over 60 min were analyzed by HPLC. A,
concentration-dependent inhibition of [3H]ATP
production on mucosal (filled bars) and serosal (open
bars) surfaces by Ap5A. B, in the
presence of 0.5 mM Ap5A, the metabolic pattern
of 0.1 mM ADP was similar to that of 0.1 mM UDP
(Fig. 4D). , ADP;
, AMP;
, adenosine;
, inosine.
C, ADPase and ecto-AK activities detected on mucosal
and serosal surfaces. Enzyme activities measured without (filled
bars, total ADPase) or with (open bars, ADPase without
ecto-AK) 0.5 mM Ap5A were 2-3-fold higher on
the mucosal surface (p < 0.01). Complete inhibition of
ecto-AK with 0.5 mM Ap5A reduced the rate of
ADP hydrolysis by 70% on both surfaces (*, p < 0.01).
Values represent ± S.E. of 5-7 independent experiments.
1·cm
2 to 0.59 nmol·min
1·cm
2 by 0.5 mM
Ap5A on the mucosal surface (Table I). Since
[3H]UDP is not a substrate of ecto-AK (Fig.
4C), the difference between these two reaction rates would
represent inhibition of dephosphorylating enzymes by Ap5A
(Table I, reaction 8). Similar results were obtained when
[3H]ADP metabolism was assayed in Mg2+-free
KRB (data not shown), which does not support ecto-AK activities (Fig.
4A). Reverse AK activity, calculated from
Ap5A-sensitive ADPase activity (Table I, reaction 5) and
corrected for nonspecific Ap5A interactions (Table I,
reaction 8), was not significantly different (Table I, reaction 9) from
values calculated by ATP production (Table I, reaction 2). Altogether,
these experiments clearly demonstrate that ADP metabolism occurs mostly
(~60%) by transphosphorylation on human airway epithelial surfaces.
1·cm
2
and 12.1 ± 2.3 nmol·min
1·cm
2,
respectively (p < 0.01). However, whereas
[3H]UTP and [3H]UDP were undetectable after
30 min (Fig. 7A), [3H]ATP and
[3H]ADP concentrations remained above 1 µM
throughout the experiment (Fig. 7B). Uridine concentration
rapidly plateaued at 5-6 µM within 20 min, whereas
adenosine gradually reached 4 µM by the end of the
experiment.
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Fig. 7.
Contribution of ecto-AK to UTP and ATP
metabolism on human bronchial epithelial cells. The mucosal
surface was assayed in KRB (pH 7.4) containing mucosal 0.01 mM [3H]UTP (0.5 µCi) (A) or 0.01 mM [3H]ATP (0.5 µCi) (B).
Mucosal buffer samples collected over 20 min were analyzed by HPLC.
, [3H]UTP and [3H]ATP;
,
[3H]UDP and [3H]ADP;
,
[3H]UMP and [3H]AMP;
,
[3H]uridine and [3H]adenosine;
,
inosine. The patterns represent mean results from seven independent
experiments (S.E. < 10% of the mean).
-32P]ATP. Fig.
8A shows HPLC traces for KRB
samples collected 0, 1, and 2 min after the addition of
[
-32P]ATP to the mucosal surface. The reaction
generated [32P]inorganic phosphate and two peaks that
co-migrated with ADP and UTP standards. The rapid conversion of
endogenous AMP into [
-32P]ADP (Fig. 8B)
suggests that ecto-AK plays a significant role in the availability of
extracellular adenine nucleotides supporting MCC in human airways. The
production of [
-32P]UTP corroborated earlier reports
of ecto-NDPK activity on these cultures (32), whereby
-phosphate was
transferred from [
-32P]ATP to endogenous UDP.
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Fig. 8.
Detection of ecto-AK activity with endogenous
nucleotides on human bronchial epithelial surfaces. Assays were
conducted in KRB (pH 7.4) containing mucosal 0.01 mM
[ -32P]ATP (0.1 µCi). Buffer samples (10 µl)
collected over 10 min were analyzed by HPLC. A, HPLC
traces showing that ecto-AK converted endogenous AMP into
[
-32P]ADP. Small amounts of [
-32P]UTP
were also identified by nucleotide standards, indicating ecto-NDPK
activity. As shown in B, [
-32P]ADP
accumulation reached a maximum within 2 min. HPLC traces represent
typical results (S.E. < 10% of the mean), and values (B)
are mean ± S.E. of four independent experiments.
-32P]ATP + 1 mM AMP,
respectively. Fig. 9A
indicates that CF was associated with ~2-fold higher forward and
reverse ecto-AK activities on the mucosal surface (p < 0.05). Interestingly, the disease had no significant effect on serosal
ecto-AK. Fig. 9B shows that total ADPase activity was
enhanced 2-fold by CF on the mucosal epithelial surface. Assays
repeated in Mg2+-free KRB indicated that ecto-AK was mostly
responsible for the impact of CF on ADP metabolism (Fig.
9B). These experiments demonstrated that ecto-AK activity is
enhanced by CF on the mucosal surface of human airway epithelia.
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Fig. 9.
Impact of CF on human bronchial ecto-AK.
As shown in A, CF enhanced ecto-AK activity on the
mucosal surface but not on the serosal surface. Epithelial cultures
from normal donors or CF patients were incubated with KRB (pH 7.4)
containing 1 mM [ -32P]ATP (0.1 µCi) + 1 mM AMP (Forward Reaction) or 2 mM
[3H]ADP (0.5 µCi; Reverse Reaction). Buffer
samples (10 µl) collected over 60 min were analyzed by HPLC for
[
-32P]ADP or [3H]ATP production. As
shown in B, the impact of CF on ADP metabolism was
limited to ecto-AK. Assays were conducted in KRB (pH 7.4) containing 2 mM [3H]ADP, without (0.5 µCi; total ADPase)
or with (ADPase
AK) 0.5 mM Ap5A. Values
represent ± S.E. of 5-7 independent experiments with epithelial
cultures from normal donors (filled bars) or CF patients
(empty bars) (*, p < 0.05).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ADP
AMP
adenosine, as generally reported for mammalian cells (for a review, see
Ref. 54). Experiments conducted with ADP as the initial substrate produced unexpected results that could not be revealed by standard phosphate assays. The reaction exhibited dephosphorylating activity by
the production of AMP and adenosine, as well as phosphorylating activity by the accumulation of ATP. The observation that
Ap5A prevented ATP formation suggested that an ecto-AK was
responsible for the phosphorylation of ADP (37). Indeed, experiments
performed herein clearly showed that human bronchial epithelial
surfaces display both forward (ATP + AMP
ADP + ADP) and reverse (ADP + ADP
ATP + AMP) reactions characteristic of an AK activity (for a
review, see Ref. 34).
AMP + PPi) dephosphorylated ATP with a
Km of 13 µM (60). Nonspecific alkaline
phosphatase displayed high affinity (Km = 36 µM) and low affinity (Km = 717 µM) activities toward AMP (30). Lazarowski et
al. (32) demonstrated that an ecto-NDPK influences endogenous concentrations of purine and pyrimidine nucleotides (ATP + UDP
ADP + UTP) on human nasal epithelial surfaces. When expressed in the human
1231N1 astrocytoma cell line, this enzyme presented a higher substrate
affinity for ADP (Km = 17 µM) than for
ATP (Km = 93 µM) (46). Collectively,
these data suggest substantial interactions between dephosphorylating
and transphosphorylating reactions on human airway epithelial surfaces.
ADP + ADP) than the reverse (ADP + ADP
ATP + AMP) reaction. This directionality could reduce the loss
of phosphate groups through AMP dephosphorylation. We explored this
possibility by taking advantage of the substrate specificity of
AKs for adenine nucleotides (Fig. 4) (for a review, see Ref.
34). Time course experiments on the metabolism of ATP and UTP indicated
that ecto-AK significantly extends the duration of micromolar adenine
nucleotides on epithelial surfaces while reducing adenosine production
(Fig. 7). The kinetic constant of ecto-AK for the forward reaction is
comparable with the affinity of ecto-5'-nucleotidase for AMP on these
cultures (KmAMP = 14 µM (30)). Consequently,
ecto-AK could significantly interfere with the conversion of AMP into
adenosine by ecto-5'-nucleotidase, delaying the transition between P2
and P1 receptor-mediated signaling pathways on airway epithelial surfaces.
ADP + ADP), thus minimizing nucleotide loss through AMP
dephosphorylation. The fact that [
-32P]ADP
accumulated from endogenous AMP and an
[
-32P]ATP concentration (10 µM; Fig. 8),
mimicking stimulated release (57-59), supports such a role for ecto-AK
on human airway epithelial surfaces. Furthermore, since ADP metabolism
occurs mostly through ecto-AK on these cells, this ADP pool could
constitute a significant source of extracellular ATP through the
reverse reaction (ADP + ADP
ATP + AMP). Consequently, during a
limited period following ATP release, ecto-AK could efficiently compete
with dephosphorylating ectoenzymes to prolong the availability of ATP
and ADP for P2 receptor activation.
currents (61) and CBF (19) induced by 100 µM ATP on airway epithelial surfaces remained above 60%
of maximal response for at least 15 min. Therefore, ecto-AK may have a
more pronounced effect on P2Y2R activation and
P2Y2R-linked effectors because ecto-AK may significantly
prolong the duration of ATP in the relevant concentration range (1-2
µM; Fig. 8B). As nucleotide concentrations decrease with time, the high affinity dephosphorylating activities (Kms = 5-17 µM (31)) will eventually
dominate over ecto-AK (Kms = 23 µM and
43 µM), and basal MCC functions will be maintained
through adenosine receptor activation (19, 25-28).
View larger version (30K):
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Fig. 10.
Ecto-AK and adenine
nucleotide-mediated MCC on human airway epithelial surfaces.
Mechanical stimulation induces bilateral ATP release, which is
prolonged by the nucleotide entrapment cycle of ecto-AK or
dephosphorylated into adenosine. Activation of bilateral
P2Y2 (ATP), P2X4 (ATP ADP), and
P2X5 (ATP
ADP) receptors, and serosal P2Y1
receptors (ADP > ATP), leads to cytosolic Ca2+
mobilization and stimulation of MCC-related cellular functions: CBF,
mucin secretion, and Ca2+-dependent
Cl
channels (ICA). Adenosine maintains basal
CBF and cystic fibrosis transmembrane regulator (CFTR)
conductance by bilateral activation of A2B receptors.
In conclusion, we have demonstrated the co-existence of ATP-consuming
and ATP-generating pathways on human airway epithelial surfaces. The
biochemical and kinetic properties of ecto-AK suggest that the
transphosphorylase activity could participate in the propagation of MCC
along airway epithelial surfaces. Following stimulated ATP release,
ecto-AK would compete with dephosphorylating enzymes to maintain
effective nucleotide concentrations for P2Y and possibly P2X receptor
activation, resulting in enhanced
Ca2+-dependent Cl secretion, CBF,
and mucus secretion and, ultimately, acceleration of MCC.
Interestingly, we have shown that ecto-AK is up-regulated in chronic
obstructive airway diseases like CF. As extracellular nucleotides are
key regulators of MCC in CF (for a review, see Ref. 1), up-regulation
of AK activity may be an adaptive response in these diseases.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Eduardo Lazarowski and Scott Donaldson for critical comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants CFF R026 and CFF Picher 00G0 from the Cystic Fibrosis Foundation.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.
To whom correspondence should be addressed: School of Medicine,
University of North Carolina, 7010 Thurston-Bowles Bldg., Chapel Hill,
NC, 27510. Tel.: 919-966-7047; Fax: 919-966-7248; E-mail:
pichm@med.unc.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M208071200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MCC, mucociliary clearance;
Ecto-AK, ecto-adenylate kinase;
CF, cystic
fibrosis;
CBF, ecto-adenylate kinase;
AMPCP, ,
-methyleneADP;
NDPK, nucleoside diphosphate kinase;
KRB, Krebs-Ringer buffer;
TBASH, tetrabutyl ammonium hydrogen sulfate;
P2YR, P2Y receptor;
P2XR, P2X
receptor;
HPLC, high pressure liquid chromatography.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Boucher, R. C.
(1999)
J. Physiol.
516,
631-638 |
2. | Donaldson, S. H., and Boucher, R. C. (1998) in The P2 Nucleotide Receptors (Turner, J. T. , Weisman, G. A. , and Fedan, J. S., eds) , pp. 413-424, Humana Press, Totowa, NJ |
3. | Taylor, A. L., Kudlow, B. A., Marrs, K. L., Gruenert, D. C., Guggino, W. B., and Schwiebert, E. M. (1998) Am. J. Physiol. 275, C1391-C1406[Medline] [Order article via Infotrieve] |
4. | Donaldson, S. H., Lazarowski, E. R., Picher, M., Knowles, M. R., Stutts, M. J., and Boucher, R. C. (2000) Mol. Med. 6, 969-982[Medline] [Order article via Infotrieve] |
5. | Felix, J. A., Woodruff, M. L., and Dirksen, E. R. (1996) Am. J. Respir. Cell Mol. Biol. 14, 296-301[Abstract] |
6. |
Homolya, L.,
Steinberg, T. H.,
and Boucher, R. C.
(2000)
J. Cell Biol.
150,
1349-1359 |
7. |
Grygorczyk, R.,
and Hanraham, J. W.
(1997)
Am. J. Physiol.
272,
C1058-C1066 |
8. |
Watt, W. C.,
Lazarowski, E. R.,
and Boucher, R. C.
(1998)
J. Biol. Chem.
273,
14053-14058 |
9. |
Wang, Y.,
Roman, R.,
Lidofsky, S. D.,
and Fitz, G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12020-12025 |
10. |
Musante, L.,
Zegarra-Moran, O.,
Montaldo, P. G.,
Ponzoni, M.,
and Galietta, L. J. V.
(1999)
J. Biol. Chem.
274,
11701-11707 |
11. | Lange, K. (2000) J. Cell. Physiol. 185, 21-35[CrossRef][Medline] [Order article via Infotrieve] |
12. | Braunstein, G. M., Roman, R. M., Clancy, J. P., Kudlow, B. A., Taylor, A. L., Shylonsky, V. G., Jovov, B., Peter, K., Jilling, T., Ismailov, I. I., Benos, D. J., Schwiebert, L. M., Fitz, J. G., and Schwiebert, E. M. (2001) J. Biol. Chem. 279, 6621-6630[CrossRef] |
13. | Mason, S. J., Paradiso, A. M., and Boucher, R. C. (1991) Br. J. Pharmacol. 103, 1649-1656[Abstract] |
14. |
Lazarowski, E. R.,
Paradiso, A. M.,
Watt, W. C.,
Harden, T. K.,
and Boucher, R. C.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2599-2603 |
15. | Paradiso, A. M., Mason, S. J., Lazarowski, E. R., and Boucher, R. C. (1995) Nature 377, 643-646[CrossRef][Medline] [Order article via Infotrieve] |
16. | Lê, K.-T., Babinski, K., and Séguéla, P. (1998) J. Neurosci. 18, 7152-7159[Abstract] |
17. |
Taylor, A. L.,
Schwiebert, L. M.,
Smith, L. M.,
King, C.,
Jones, J. R.,
Sorscher, E. J.,
and Schwiebert, E. M.
(1999)
J. Clin. Invest.
104,
875-884 |
18. |
Korngreen, A.,
Ma, M.,
Priel, Z.,
and Silberberg, S. D.
(1998)
J. Physiol.
508,
703-720 |
19. |
Morse, D. M.,
Smullen, J. L.,
and Davis, C. W.
(2001)
Am. J. Physiol.
280,
C1485-C1497 |
20. | Ramirez, A. N., and Kunze, D. L. (2002) Am. J. Physiol. 282, H2106-H2116[Medline] [Order article via Infotrieve] |
21. | Kennedy, C., and Leff, P. (1995) Trends Pharmacol. Sci. 16, 168-174[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Huang, P.,
Lazarowski, E. R.,
Tarran, R.,
Milgram, S. L.,
Boucher, R. C.,
and Stutts, M. J.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
14120-14125 |
23. |
Communi, D.,
Paindavoine, P.,
Place, G.,
Parmentier, M.,
and Boeynaems, J. M.
(1999)
Br. J. Pharmacol.
127,
562-568 |
24. |
Le Hir, M.,
and Kaissling, B.
(1993)
Am. J. Physiol.
264,
F377-F387 |
25. | Lieb, T., Wijkstrom Frei, C., Frohock, J. I., Bookman, R. J., and Salathe, M. (2001) J. Physiol. 538, 633-646 |
26. | Lazarowski, E. R., Mason, S. J., Clarke, L., Harden, T. K., and Boucher, R. C. (1992) Br. J. Pharmacol. 106, 774-782[Abstract] |
27. |
Stutts, M. J.,
Fitz, J. G.,
Paradiso, A. M.,
and Boucher, R. C.
(1994)
Am. J. Physiol.
267,
C1442-C1451 |
28. |
Clancy, J. P.,
Ruiz, F. E.,
and Sorscher, E.
(1999)
Am. J. Physiol.
276,
C361-C369 |
29. | Picher, M., and Boucher, R. C. (1999) Pediatr. Pulmonology Suppl. 19, 311 (abstr.) |
30. | Picher, M., Burch, L. H., Hirsh, A. J., Spychala, J., and Boucher, R. C. (January 30, 2003) J. Biol. Chem. 10.1074/jbc.M300569200 |
31. | Picher, M., and Boucher, R. C. (2001) Drug Dev. Res. 52, 66-75[CrossRef] |
32. |
Lazarowski, E. R.,
Boucher, R. C.,
and Harden, T. K.
(2000)
J. Biol. Chem.
275,
31061-31068 |
33. | Lacombe, M.-L., Milon, L., Munier, A., Mehus, J. G., and Lambeth, D. O. (2000) J. Bioenerg. Biomembr. 32, 247-258[CrossRef][Medline] [Order article via Infotrieve] |
34. | Van Rompay, A. R., Johansson, M., and Karlsson, A. (2000) Pharmacol. Ther. 87, 189-198[CrossRef][Medline] [Order article via Infotrieve] |
35. | Cascalheira, J. F., and Sebastiao, A. M. (1992) Eur. J. Pharmacol. 222, 49-59[Medline] [Order article via Infotrieve] |
36. | Dunkley, C. R., Manery, J. F., and Dryden, E. E. (1966) J. Cell. Physiol. 68, 241-248 |
37. | Nagy, A. K., Shuster, T. A., and Delgado, V. (1989) J. Neurochem. 53, 1166-1172[Medline] [Order article via Infotrieve] |
38. |
Yegutkin, G. G.,
Henttinen, T.,
and Jalkanen, S.
(2001)
FASEB J.
15,
251-260 |
39. | Yegutkin, G. G., Henttinen, T., Samburski, S. S., Spychala, J., and Jalkanen, S. (2002) Biochem. J. 367, 121-128[CrossRef][Medline] [Order article via Infotrieve] |
40. | Donaldson, S. H., Picher, M., and Boucher, R. C. (2001) Am. J. Respir. Cell Mol. Biol. 26, 209-215 |
41. |
Wine, J. J.
(1999)
J. Clin. Invest.
103,
309-312 |
42. | Gray, T. E., Guzman, K., Davis, C. W., Abdullah, L. H., and Nettesheim, P. (1996) Am. J. Respir. Cell Mol. Biol. 14, 104-112[Abstract] |
43. | Wu, R., Yankaskas, J., Cheng, E., Knowles, M. R., and Boucher, R. C. (1985) Am. Rev. Respir. Dis. 132, 311-320[Medline] [Order article via Infotrieve] |
44. | Lechner, J. F., and LaVeck, M. A. (1985) J. Tissue Cult. Methods 9, 43-48 |
45. |
Matsui, H.,
Davis, C. W.,
Tarran, R.,
and Boucher, R. C.
(2000)
J. Clin. Invest.
105,
1419-1427 |
46. |
Lazarowski, E. R.,
Homolya, L.,
Boucher, R. C.,
and Harden, T. K.
(1997)
J. Biol. Chem.
272,
20402-20407 |
47. | Fedde, K. N., Lane, C. C., and Whyte, M. P. (1988) Arch. Biochem. Biophys. 264, 400-409[Medline] [Order article via Infotrieve] |
48. | Lazarowski, E. R., Watt, W. C., Stutts, M. J., Boucher, R. C., and Harden, T. K. (1995) Br. J. Pharmacol. 116, 1619-1627[Abstract] |
49. |
Hamada, M.,
Sumida, M.,
Okuda, H.,
Watanabe, T.,
Nojima, M.,
and Kuby, S. A.
(1982)
J. Biol. Chem.
257,
13120-13128 |
50. | Kuby, S. A., Hamada, M., Gerber, D., Tsai, W., Jacobs, H. K., Cress, M. C., Chua, G. K., Fleming, G., Wu, L. L., Fischer, A. H., Frischat, A., and Maland, L. (1978) Arch. Biochem. Biophys. 187, 34-52[Medline] [Order article via Infotrieve] |
51. | Zimmermann, H. (2000) Naunyn-Schmiedeberg's Arch. Pharmacol. 362, 299-309[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Dubyak, G. R.,
and El-Moatassim, C.
(1993)
Am. J. Physiol.
265,
C577-C606 |
53. | Lazarowski, E. R., and Boucher, R. C. (2001) Pediatr. Pulmonology Suppl. 22, 193 |
54. | Beaudoin, A. R., Sévigny, J., and Picher, M. (1996) in ATPases Biomembranes (Lee, A. G., ed), Vol. 5 , pp. 369-401, Greenwich, CT |
55. | Tarran, R., Grubb, B. R., Parsons, D., Picher, M., Hirsh, A. J., Davis, C. W., and Boucher, R. C. (2001) Mol. Cell 8, 149-158[Medline] [Order article via Infotrieve] |
56. | Lazarowski, E. R., and Harden, T. K. (1997) Br. J. Pharmacol. 127, 1272-1278 |
57. |
Hazama, A.,
Shimizu, T.,
Ando-Akatsura, Y.,
Hayashi, S.,
Tanaka, S.,
Maeno, E.,
and Okada, Y.
(1999)
J. Gen. Physiol.
114,
525-533 |
58. |
Newman, E. A.
(2001)
J. Neurosci.
21,
2215-2223 |
59. | Beigi, R., Kobatake, E., Aizawa, M., and Dubyak, G. R. (1999) Am. J. Physiol. 276, C267-C278[Medline] [Order article via Infotrieve] |
60. |
Picher, M.,
and Boucher, R. C.
(2000)
Am. J. Respir. Cell Mol. Biol.
23,
255-261 |
61. |
Clarke, L. L.,
Harline, M. C.,
Otero, M. A.,
Glover, G. G.,
Garrard, R. C.,
Krugh, B.,
Walker, N. M.,
Gonzalez, F. A.,
Turner, J. T.,
and Weissman, G. A.
(1999)
Am. J. Physiol.
276,
C777-C787 |
62. | Soto, F., Garcia-Guzman, M., and Stühmer, W. (1997) J. Membr. Biol. 160, 91-100[CrossRef][Medline] [Order article via Infotrieve] |
63. | Williams, M., and Jarvis, M. F. (2000) Biochem. Pharmacol. 59, 1173-1185[CrossRef][Medline] [Order article via Infotrieve] |