Demonstration and immunolocalization of ATP diphosphohydrolase
in the pig digestive system
Jean
Sévigny,
Gilles
Grondin,
Fernand-Pierre
Gendron,
Julie
Roy, and
Adrien R.
Beaudoin
Département de Biologie, Faculté des Sciences,
Université de Sherbrooke, Sherbrooke, Québec, Canada
J1K 2R1
 |
ABSTRACT |
Two isoforms of
ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) have been previously
characterized, purified, and identified. This enzyme is an
ectonucleotidase that catalyzes the sequential release of
-
and
-phosphate groups of triphospho- and diphosphonucleosides. One of its putative roles is to modulate the extracellular
concentrations of purines in different physiological systems. The
purpose of this study was to define, identify, and localize these two
isoforms of ATPDase in the pig digestive system. ATPDase activity was
measured in pig stomach, duodenum, pancreas, and parotid gland. Enzyme assays, electrophoretograms, and Western blots with a
polyclonal antibody that recognizes both isoforms demonstrate the
presence of ATPDase in these organs. Immunolocalization
showed intense reactions with gastric glands (parietal and chief
cells), intestine (columnar epithelial cells), parotid gland, and
pancreas. Smooth muscle cells all along the digestive tract were also
highly reactive. Considering the variety of purinoceptors associated
with the digestive system, the ATPDase is strategically positioned to
modulate purine-mediated actions such as electrolyte secretion,
glandular secretion, smooth muscle contraction, and blood flow.
apyrase; ecto-ATPase; stomach; intestine; parotid; pancreas
 |
INTRODUCTION |
IN THE PAST DECADE, evidence has grown in support of
the concept that extracellular ATP and its dephosphorylated metabolites are involved in cell signaling in the different physiological systems
of vertebrates (20). ATP is released from cells by exocytotic and
nonexocytotic mechanisms; the latter are still undefined. Extracellular
concentrations of ATP and its metabolites appear to be modulated
primarily by ectonucleotidases, namely, an ATP diphosphohydrolase
(ATPDase), an ecto-ATPase, and the 5'-nucleotidase. The
5'-nucleotidase that converts 5'-nucleotides to nucleosides has been well described (38); however, this is not the case for the
ectoenzymes responsible for the hydrolysis of ATP to ADP and ADP to
AMP. Indeed, until recently, it was widely believed that distinct
enzymes were involved in the sequential hydrolysis of the
- and
-phosphate residues of ATP, i.e., an ecto-ATPase and an ecto-ADPase.
This notion was revised after the finding of an ecto-ATPDase in many
tissues. The latter enzyme, originally found in pig pancreas (19), has
been purified, characterized, and identified (8, 32-33). Its
encoding gene has been sequenced and corresponds to the gene of CD39, a
marker of activated lymphocytes (14). In addition, other
ectonucleotidases (7, 16, 17) that convert ATP to ADP have been
described (see Refs. 4, 18, and 28 for review). Several lines of
evidence have indicated that extracellular purines play primordial
roles in the gastrointestinal tract and some of its associated organs,
such as the salivary glands and the pancreas. These actions are
mediated by purinergic receptors and modulated by still undefined
nucleotidase activities. Extracellular nucleotides and their
dephosphorylated derivatives exert major physiological effects on the
digestive system, and ATPDase could potentially modulate the
concentrations of these nucleotides. It therefore appeared important to
determine the localization of ATPDase in the gastrointestinal tract. In
light of current literature describing the localization and properties of P1 and
P2 purinoceptors, our analysis
provides new insights into the physiological role of this enzyme. More
specifically, ecto-ATPDase reveals high levels of both immunoreactivity
and enzyme activity in the stomach, intestine, pancreas, and parotid gland.
 |
MATERIALS AND METHODS |
Materials
ATP, nitro blue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate
(BCIP), Tris, tetramisole, imidazole, ammonium molybdate, glycerin,
EDTA, sucrose, sodium deoxycholate, phenylmethylsulfonyl fluoride
(PMSF), malachite green, sodium azide
(NaN3), and mouse monoclonal
antibodies to rabbit IgG conjugated to alkaline phosphatase were
obtained from Sigma Chemical (St. Louis, MO). ADP, AMP,
and Triton X-100 were purchased from Boehringer-Mannheim (Laval, PQ). CaCl2,
MgCl2, and Tween 20 were obtained
from Fisher (Montréal, PQ). Transfer membrane Immobilon-P was
obtained from Millipore (Bedford, MA); Bradford reagent, BSA fraction
V, SDS, molecular mass standards, and polyacrylamide were obtained from
Bio-Rad (Mississauga, ON). All other reagents were of analytical grade.
Isolation of Particulate Fractions
Organs from three young pigs, anesthetized by an intraperitoneal
injection of 33% chloral hydrate in saline (1 ml/kg), were frozen in
liquid nitrogen. Particulate fractions were prepared as previously
described (32). Briefly, tissues were homogenized with a Polytron in 10 vol of 95 mM NaCl, 0.1 mM PMSF, and 45 mM Tris, pH 7.6, at 4°C. The
homogenate was filtered through cheesecloth and centrifuged for 15 min
at 700 g. The supernatant was
centrifuged for 1 h at 100,000 g in an
SW41 Beckman rotor. The latter pellet was suspended in a solution of
0.1 mM PMSF and 1 mM NaHCO3, pH 10.0, with a potter Elvejehm homogenizer, loaded on a 40%
(wt/vol) sucrose cushion, and centrifuged for 90 min at 100,000 g in an SW41 Beckman rotor. The fluffy
layer was recovered on the cushion, washed twice with 1 mM
NaHCO3 and 0.1 mM PMSF, and
suspended in 7.5% glycerin and 5 mM Tris, pH 8.0, at a concentration
of 2-5 mg protein/ml. Enzyme activity was measured the same day.
ATPase and ADPase assays
Enzyme assays were carried out at 37°C in 1 ml of solution
containing (in mM) 8 CaCl2, 5 tetramisole, 50 Tris, and 50 imidazole, pH 7.5. The reaction was
started by adding 0.2 mM of the substrate (ATP or ADP) and stopped with
0.25 ml of the malachite green reagent. Pi was estimated according to the
method of Baykov et al. (2). Controls were run with the protein sample
added after the malachite green reagent. Enzyme activity was expressed
as nanomoles of Pi released per
minute per milligram of protein, which corresponds to milliunits (19).
Protein was estimated by the technique of Bradford (5), using BSA as a
standard of reference.
Electrophoresis and Immunoblotting Procedures
PAGE was carried out under nondenaturing conditions (32). Nucleotidase
activity was detected by incubating the gel for 3 h at 37°C in 10 mM CaCl2, 100 mM Tris-imidazole,
pH 7.5, and 4 mM ATP, ADP, or AMP. Released phosphate forms a white
Ca2+ precipitate at the reaction
site in the gel. Where indicated, proteins were separated by SDS-PAGE
in a polyacrylamide gradient (8-13.5%), as described by
Sévigny et al. (32). Immunoblotting procedures were carried out
as previously reported, using a rabbit antiserum raised against a
synthetic polypeptide corresponding to the
NH2-terminal 16-amino acid
sequence of the pig pancreas type I ATPDase, at a dilution of 1:10,000
(33). This antibody specifically recognizes both isoforms (type I and
II ATPDase) of the enzyme. Preparation and antibody specificity have
been previously described (32-33). The secondary antibody was a
mouse monoclonal anti-rabbit IgG (1:6,000) conjugated to alkaline
phosphatase, detected with NBT/BCIP.
Immunohistochemistry
Freshly dissected tissues were fixed overnight in 2% paraformaldehyde,
0.17% glutaraldehyde, and 4% sucrose, buffered at pH 7.4 with 0.1 M
sodium cacodylate buffer. Tissues were dehydrated in graded ethanol
solutions and embedded in paraffin. Paraffin sections were cut at
4-µm thickness and mounted on polyionic slides (Superfrost Plus;
Fisher). Paraffin was removed with xylene, and sections were rehydrated
through graded concentrations of ethanol to water and rinsed in
Tris-buffered saline (TBS; 150 mM NaCl, 0.1 M Tris, pH 7.5). Slides
were incubated for 10 min in TBS containing 0.1 M glycine and
subjected to a pressure cooker heat-induced epitope retrieval procedure
by incubating in 1 mM EDTA and 10 mM Tris, pH 8.0, for 9 min (23).
After a 10-min wash in TBS at room temperature, nonspecific binding
sites were blocked with 1% BSA and 1% fat-free skim milk in TBS for
30 min at room temperature. Sections were incubated overnight at
4°C with the ATPDase antiserum or the preimmune serum (1:100),
washed in TBS several times, and incubated with mouse monoclonal
anti-rabbit IgG conjugated to alkaline phosphatase at a dilution of
1:100 for 2 h at room temperature. After several washings in TBS,
visualization was obtained by the alkaline phosphatase reaction with
NBT/BCIP. Sections were mounted in 5% gelatin, 27% glycerin, and
0.1% sodium azide, preheated at 45°C. Photographs were taken under
bright-field illumination, with the use of a Zeiss photomicroscope on
Kodak T-Max 100 film.
 |
RESULTS |
Biochemical Data
As shown in Table 1, high levels of ATPase
and ADPase activities were found in the homogenates of stomach,
duodenum, pancreas, and parotid gland. ATPase and ADPase activities
were enriched by about five times in particulate fractions isolated
from these organs, with the highest levels in the stomach. Both ATPase
and ADPase activities from these organs were sensitive to sodium azide, a well-known inhibitor of ATPDase. From previous studies we know that
the only azide-sensitive ADPase activities are attributable to ATPDases
(4). The presence of the latter enzyme in these organs was also
assessed by looking at electrophoretograms of the particulate fractions
separated by PAGE under nondenaturing conditions. As shown in Fig.
1A,
electrophoretograms obtained with ATP and ADP as substrates correspond.
Migration distances in the polyacrylamide gel were comparable for
ATPDases from stomach, duodenum, and parotid gland but differed for the
liver enzyme, which was included for comparison. With the pancreas
particulate fraction, a barely detectable precipitate could be seen. In
contrast, a strong signal was observed with the enriched
ATPDase (isoform I) from the zymogen granule membrane
fraction, which had a specific activity of 3 U/mg protein. With all of
these preparations, there was no detectable reaction product with AMP
as the substrate (data not shown). ATPDase identity was confirmed in
these different organs by Western immunoblots, using a previously
described polyclonal antibody directed against a specific 16-amino acid
sequence of the pig enzyme. Good signals were found in stomach and
intestine, whereas immunoreactions were much weaker in the parotid
gland and pancreas (Fig. 1B).

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Fig. 1.
A: electrophoretograms of nucleotidase
activity after PAGE under nondenaturing conditions. A sample of 75 µg
of protein of particulate fractions from each tissue was loaded on a
4-7.5% polyacrylamide gradient [13 µg of protein in the
case of zymogen granule membrane (ZGM) of pancreas]. ATPase and
ADPase activities were localized by incubating the gel for 3 h at
37°C in 10 mM CaCl2, 100 mM
Tris-imidazole, pH 7.5, and 4 mM of either ATP or ADP. Liberated
Pi forms a white precipitate with
Ca2+ at reaction sites. Notice
similar migration patterns of ATPase and ADPase activities.
B: Western blots of particulate
fractions. Samples of 100 µg of protein of particulate fractions from
each tissue were separated by SDS-PAGE and immunoblotted as described
in MATERIALS AND METHODS. Positive
control consisted of a particulate fraction from bovine spleen (30 µg). The sample of zymogen granules, purified as described in Ref.
32, contained 15 µg of protein.
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Immunological Localization
Stomach.
Immunological localization shows that ATPDase is associated
with gastric glands. As shown in Fig.
2B,
at high magnification one finds staining of both parietal and chief
cell membranes all along the crypts in cardia and fundus regions.
Notice, however, that there is much less reactivity at the crypt
extremities (Fig. 2A). In chief
cells the reaction product was localized on the plasma membrane,
whereas in parietal cells the entire cell appeared to be stained (Fig.
2B). Since the latter cells possess
a network of canaliculi, which are invaginations of the plasma
membrane, the cytoplasmic staining probably reflects the labeling of
this network (Fig. 3,
A and
B). Strong reactions were observed
in stomach smooth muscle cells (muscularis externa) (Fig. 3,
C and
D). In these cells, ATPDase was
mainly found on the plasma membrane, with some variability in signal
intensity, suggesting a heterogeneity in the distribution of this
enzyme among these cells (Fig. 3). Similar reactivity was observed in
smooth muscles at the level of the intestine.

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Fig. 2.
Immunolocalization of ATP diphosphohydrolase (ATPDase) in stomach:
cardia region. A: very weak signal on
mucus-producing cells (arrowheads) and strong reaction on gastric
glands (body and fundus). B: higher
magnification shows reaction product concentrated on plasma membrane of
chief cells (arrowheads) with strong reactions in parietal cells
(arrows). C: control with preimmune
serum. Magnification: ×200 (A
and C), ×500
(B).
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Fig. 3.
Immunolocalization of ATPDase in stomach: fundus region and muscularis
externa. A: high immunoreactivity of
parietal cells. B: higher
magnification showing intense signal in parietal cells (arrows):
transverse section. C:
overview showing exceptionally high intensity of the immunoreaction
on smooth muscles (arrow). D: higher
magnification showing immunostaining on plasma membrane (arrowheads).
Notice variability in the signal. Some cells appear to be devoid of
reaction (arrows). Magnification: ×200
(A and
C), ×500
(B and
D).
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Intestine.
In the duodenum, ATPDase was found on columnar epithelial cells. Some
cells of the lamina propria were also highly reactive (Fig.
4, A,
B, and
D), including lymphocytes, which
appear to migrate into the epithelial layer (Fig.
4C). In the jejunum, ATPDase distribution was essentially similar, although the majority of reticular cells of the lamina propria were also highly reactive (Fig.
4, E and
F). In the ileum, the enzyme was
found in both columnar and reticular cells, and Peyer's patches showed
particularly intense reactions (Fig.
4G).

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Fig. 4.
Immunolocalization of ATPDase in the intestine.
A: duodenum columnar cells are
positive (arrowheads) together with some cells in the lamina propria
(arrow). B: immunoreactive lymphocytes
(arrowheads). C: small lymphocytes
described above show high immunoreactivity (arrowheads). Notice
different levels of migration through epithelial layer.
D: a dense immunoreaction is observed
on reticular cells of the duodenum lamina propria (arrow) (transverse
section). E: in jejunum, columnar
cells are positive (arrowheads), with some immunoreactivity in the
lamina propria (arrows). F: the
majority of reticular cells are highly reactive (arrow).
G: immunolocalization of ATPDase in
the ileum. Highly reactive columnar cells (arrowheads), reticular cells
(arrows), and Peyer's patch (large arrowhead). Magnification:
×200 (A,
E, and
G), ×500
(B-D and
F).
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Pancreas.
Pancreas ATPDase immunoreactivity was quite high considering the low
specific activity of the enzyme measured in the particulate fraction (see Table 1). Careful examination of the sections
revealed that the immunoreaction products were localized on basal and
lateral membranes as well as on the apical membrane of acinar cells,
whereas the signal over zymogen granules appeared less intense (Fig.
5, A-C).
ATPDase was also found on the ductal epithelium, with much less
reactivity associated with mucus-producing cells (Fig.
5D).

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Fig. 5.
Immunolocalization of ATPDase in the pancreas.
A: high level of immunoreactivity
associated with acinar cells (arrowheads).
B: at high magnification,
immunoreaction seems to be associated with basal (arrows) and lateral
membranes (arrowheads). C:
immunoreaction observed on apical membrane (arrowheads).
D: control with preimmune serum.
E: immunolocalization of ATPDase at
the level of ducts. The majority of ductal cells react strongly, but
some are devoid of reaction (arrow). Magnification: ×500
(A,
D, and
E), ×800
(B and
C).
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Parotid.
As shown in Fig. 6, the level of ATPDase
was very high in ductal epithelium and blood vessels, in contrast to
pancreas acinar cells. Parotid acinar cells were devoid of
immunoreactivity, except for a very light signal at the level of the
apical membrane, which suggests a shedding of the enzyme in the acinar
lumen. Myoepithelial cells, also identified as "basket cells,"
which are dispersed throughout the tissue, were also positive.

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Fig. 6.
Immunolocalization of ATPDase in the parotid gland.
A: all ducts (arrows) and blood
vessels show strong immunoreactivity (arrowheads).
B: strong immunoreactivity on duct
cells (arrow) with a light signal in acinar lumen (arrowheads).
Myoepithelial cells (basket cells) (wavy arrows) are also positive.
C: control with preimmune serum.
Notice acini (arrowheads) and secretory duct cells (arrow).
Magnification: ×125 (A),
×500 (B and
C).
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DISCUSSION |
This study reports the immunolocalization of ATPDase in the
different cells and tissues of the mammalian digestive system. Intense
reactions were found in stomach and intestine, in agreement with enzyme
assays. More specifically, ATPDase was found on epithelial cells,
smooth muscle cells, reticular cells, and lymphocytes in these organs.
Reactivity of the gastric glands was remarkably high in both chief
cells and parietal cells. In addition, ATPDase was localized on the
ductal systems of glands associated with the digestive tract. Similar
to the parenchymal cells of the gastric glands, pancreatic acini were
highly reactive, whereas parotid gland acini did not produce
significant levels of reaction. This wide distribution of ATPDase,
joined to the existence of a variety of purinoceptors, supports the
view that this enzyme plays a key role in the regulation of purinergic
actions and many vital processes throughout the digestive tract.
Obviously, there are still many missing links in this complex puzzle of
purine signaling. Perhaps the most crucial questions are the source and
the mechanisms of ATP release. There is a lack of knowledge regarding
these processes. Nevertheless, we explored the role of
ATPDase by looking at purinoceptor distribution and the
physiological effects on different cellular systems of the
gastrointestinal tract.
Stomach
High levels of ATPDase were measured in the stomach, and
immunolocalization indicated that the enzyme is mainly associated with
parietal cells, chief cells, and smooth muscle cells. In contrast,
ATPDase was barely detectable in mucus-producing cells. Several reports
have provided evidence that gastric acid secretion may be modulated by
adenosine receptors in rabbit and rat stomach (1, 25, 36) and that it
is mediated by an A2 receptor (1, 25). There is also some evidence for a
P2Y receptor in gastric gland
plasma membrane (35) and a P2U
receptor on mucus-producing cells (26). Indeed, ATP (but not
adenosine) induced a glycoprotein secretory response of isolated rabbit
gastric mucous cells in primary culture (26). In the rabbit antral
mucosa, blood flow is increased by adenosine, an effect mediated by an
A2 subtype purinoceptor (27). In
rat gastric fundus, nerve terminals presumably release ATP and thereby
influence smooth muscle activity. Indeed, in vitro studies on
longitudinal muscle strips showed that adenosine produced relaxation,
whereas ATP caused a phasic relaxation followed by a maintained spasm
(22). Ohno et al. (24) observed that on smooth muscle cells of the
guinea pig stomach fundus a transmural nerve stimulation evoked an
excitatory junction potential that could be blocked by suramin, which
is a putative P2 purinoceptor inhibitor. These observations, together with our immunolocalization of
ATPDase, indicate that this enzyme influences gastric acid and pepsin
secretions, mucus production, and contractility of the stomach.
Intestine
High levels of ATPDase activity were found in the pig small intestine,
in agreement with recent observations of ecto-ATPase on rat intestine
(30). Columnar epithelial cells and reticular cells of the mucosa were
highly reactive, and some significant immunoreactivity was found on
smooth muscle cells. ATPDase appears to be strategically located on the
luminal side of the intestine to modulate extracellular concentrations
of ATP and other triphospho- and diphosphonucleosides, which could
potentially come with chyme, including bile. ATPDase could also
modulate the response to the release of ATP from the nervous system at
the neuromuscular junction, thereby influencing intestinal motility.
Finally, the presence of the enzyme on blood vessels indicates that it
can influence the concentration of nucleotides in the circulatory
system. Hence important roles for the ATPDase can be postulated, since
purines exert major influences on peristalsis, electrolyte secretion, and blood flow (12, 15, 21, 27, 31). Concerning purinoceptor distribution and the physiological effects associated with these purinoceptors, there is evidence for an
A1-mediated inhibition of
peristalsis (12) in rat jejunum, whereas in the smooth muscles of the
same species, both adenosine and ATP enhanced spontaneous mechanical
activity (21). The effects of extracellular purines on intestinal
electrolyte secretion are supported by pharmacological studies and
Northern blot analysis (31) on the T84 cell line, which have indicated
that adenosine-stimulated
Cl
secretion of human
intestinal epithelia is mediated by an
A2b purinoceptor. Purines can also
alter intestinal blood flow. Indeed, sympathetic nerve stimulation
causes a constriction of submucosal arterioles of guinea pig ileum that
is mediated by ATP acting on P2X
receptors (11). Pennanen et al. (27) found a differential effect of
adenosine on blood flow to subregions of the upper gastrointestinal tract of the rabbit, an effect that was mediated by
A2 purinoceptors. After
ischemia-reperfusion of the rat intestine, adenosine arrests most of the inflammatory changes associated with reperfusion (15). These observations have potentially important implications for the
treatment of intestinal diseases, including diarrhea.
Pancreas
ATPDase was originally localized in the pig pancreas by a rather
nonspecific cytochemical approach (3). In the present study, the
presence of ATPDase was confirmed in basolateral and apical membranes
of acinar cells and zymogen granules, in agreement with our original
observations. Intriguingly, a strong signal was observed on the ductal
epithelium. Very little is known about the role of purines at this
level of the gland. Activation of Cl
and
K+ conductances was observed after
exposure of CFPAC-1 cells to ATP and UTP. These cells respond to
nucleotide receptor activation with a transient increase in
intracellular Ca2+ that stimulates
these ionic currents (10). The role of ATPDase in zymogen granules
remains a matter of speculation. Why the 78-kDa protein is truncated to
a 54-kDa form is another intriguing question that remains unanswered
(32). Moreover, the sectioned fragment remains associated with the
zymogen granule membrane (unpublished observations).
Parotid
In the pig parotid gland, ATPDase was mainly associated with duct
epithelium, with very little, if any, immunoreactivity on acinar cells.
Myoepithelial cells dispersed among acini produced a significant
signal. There have been reports of
Ca2+-dependent ATPases associated
with isolated parotid acinar cells, but as judged by their biochemical
properties, these ATPases are different from ATPDases. However, a
nucleoside triphosphatase described by Sato et al. (29) in bovine
parotid gland and an apyrase described by Valenzuela et al. (34) in a
microsomal fraction of rat salivary gland appear to correspond to the
ATPDase described in this study and hence could well be associated with ductal epithelial and myoepithelial cells. In agreement with these findings, some studies on rat parotid acini show very little
ecto-ATPase activity (9). Some P2Z
purinoceptors have been described on rat parotid acinar cells, which
respond to ATP and mediate a Ca2+
increase caused by both an influx and a mobilization from intracellular stores (13). Another receptor,
P2X4, an ATP-gated ion channel, is
expressed in serosal cells of salivary glands (6). Xu et al. (37)
characterized and localized Ca2+
signaling receptors in rat submandibular salivary gland ducts (37).
They showed that the ATP receptors were localized in the luminal
membrane of the epithelial cell. If a similar localization for these
ATP receptors exists in the pig parotid gland, the ATPDase could play a
key role in the transport of electrolytes by modulating the
extracellular ATP concentration in the salivary gland ducts.
Our study shows that ATPDase is present in large amounts in secretory
epithelia from stomach, intestine, pancreas, and parotid gland. These
results, in conjunction with the presence of purine-gated channels,
lead us to believe that the enzyme modulates electrolyte secretion all
along the digestive tract. The presence of the enzyme and
P2 purinoceptors on smooth muscle
cells also suggests that the enzyme is involved in digestive tract
motility and blood flow. Localization of the ATPDase supports the view
that this ectonucleotidase is a prominent modulator of the action of
extracellular purines and provides new clues for the interpretation of
the role of ectonucleotidases in the digestive system.
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ACKNOWLEDGEMENTS |
We thank Johanne Proulx for technical assistance.
 |
FOOTNOTES |
This work was supported by grants from Fonds pour la Formation de
Chercheurs et l'Aide à la Recherche du Québec (FCAR) and from the Natural Sciences and Engineering Research Council of Canada
(NSERC). J. Sévigny received studentships from FCAR and from the
Heart and Stroke Foundation of Canada.
Address for reprint requests: A. R. Beaudoin, Département de
Biologie, Faculté des Sciences, Université de Sherbrooke,
Sherbrooke, Québec J1K 2R1, Canada.
Received 1 December 1997; accepted in final form 5 May 1998.
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