Coexpression of ecto-5′-nucleotidase/CD73 with

specific NTPDases differentially regulates adenosine

formation in the rat liver
Michel Fausther, Joanna Lecka, Elwy Soliman, Gilles Kauffenstein, Julie Pelletier,
Nina Sheung, Jonathan A. Dranoff and Jean Sévigny
Am J Physiol Gastrointest Liver Physiol 302:G447-G459, 2012. First published 1 December 2011;
doi:10.1152/ajpgi.00165.2011

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Coexpression of ecto-5=-nucleotidase/CD73 with specific NTPDases
differentially regulates adenosine formation in the rat liver
Michel Fausther,1,2,3 Joanna Lecka,1 Elwy Soliman,2 Gilles Kauffenstein,1 Julie Pelletier,1 Nina Sheung,2
Jonathan A. Dranoff,2,3 and Jean Sévigny1
1
Centre de recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec (pavillon CHUL),
Québec and Département de microbiologie-infectiologie et d’immunologie, Faculté de médecine, Université Laval, Québec,
QC, Canada; 2Section of Digestive Diseases, Yale University School of Medicine, New Haven, Connecticut; and 3Division of
Gastroenterology and Hepatology, Department of Internal Medicine, College of Medicine, University of Arkansas for Medical
Sciences, Little Rock, Arkansas
Submitted 27 April 2011; accepted in final form 29 November 2011
Fausther M, Lecka J, Soliman E, Kauffenstein G, Pelletier J,
Sheung N, Dranoff JA, Sévigny J. Coexpression of ecto-5=-nucle-
otidase/CD73 with specific NTPDases differentially regulates adeno-
sine formation in the rat liver. Am J Physiol Gastrointest Liver Phys-
iol 302: G447–G459, 2012. First published December 1, 2011;
doi:10.1152/ajpgi.00165.2011.—Ectonucleotidases modulate puriner-
gic signaling by hydrolyzing ATP to adenosine. Here we characterized
the impact of the cellular distribution of hepatic ectonucleotidases,
namely nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39,
NTPDase2/CD39L1, NTPDase8, and ecto-5=-nucleotidase/CD73, and of
their specific biochemical properties, on the levels of P1 and P2 receptor
agonists, with an emphasis on adenosine-producing CD73. Immunostain-
ing and enzyme histochemistry showed that the distribution of CD73
(protein and AMPase activity) overlaps partially with those of
NTPDase1, -2, and -8 (protein levels and ATPase and ADPase activ-
ities) in normal rat liver. CD73 is expressed in fibroblastic cells
located underneath vascular endothelial cells and smooth muscle cells,
which both express NTPDase1, in portal spaces in a distinct fibroblast
population next to NTPDase2-positive portal fibroblasts, and in bile
canaliculi, together with NTPDase8. In fibrotic rat livers, CD73
protein expression and activity are redistributed but still overlap with
the NTPDases mentioned. The ability of the observed combinations of
ectonucleotidases to generate adenosine over time was evaluated by
reverse-phase HPLC with the recombinant rat enzymes at high “in-
flammatory” (500 ␮M) and low “physiological” (1 ␮M) ATP con-
centrations. Overall, ATP was rapidly converted to adenosine by the
NTPDase1⫹CD73 combination, but not by the NTPDase2⫹CD73
combination. In the presence of NTPDase8 and CD73, ATP was
sequentially dephosphorylated to the CD73 inhibitor ADP, and then to
AMP, thus resulting in a delayed formation of adenosine. In conclu-
sion, the specific cellular cocompartmentalization of CD73 with
hepatic NTPDases is not redundant and may lead to the differential
activation of P1 and P2 receptors, under normal and fibrotic condi-
tions.
P1 receptors; P2 receptors; ATP; fibrosis; carbon tetrachloride intox-
ication; nucleoside triphosphate diphosphohydrolases
VIA THE ACTIVATION OF SPECIFIC plasma membrane P1 and P2
receptors, extracellular nucleosides and nucleotides modulate
various physiological functions in virtually all tissues, includ-
ing blood vessels, heart, lungs, brain, smooth and skeletal
muscle, etc. (9). In the liver, these signaling agents regulate key
cellular pathways such as glucose metabolism, cell cycle pro-
gression, hepatic regeneration, cell volume regulation, ionic
Address for reprint requests and other correspondence: J. Sévigny, Centre de
Recherche en Rhumatologie et Immunologie, 2705 boul. Laurier, local T1-49,
G1V 4G2 Québec, QC, Canada (e-mail: Jean.Sevigny@crchul.ulaval.ca).
http://www.ajpgi.org 0193-1857/12 Copyright © 2012 the American Physiological Society
Am J Physiol Gastrointest Liver Physiol 302: G447–G459, 2012.
First published December 1, 2011; doi:10.1152/ajpgi.00165.2011.
secretion, and scar formation (5, 19, 43, 44). To regulate
extracellular nucleoside and nucleotide signaling and maintain
tissue homeostasis, liver cells express various ectonucleoti-
dases at their surface, including members of the ecto-nucleo-
side triphosphate diphosphohydrolase (NTPDase), ecto-nucle-
Downloaded from ajpgi.physiology.org on March 1, 2012
otide pyrophosphatase-phosphodiesterase, and alkaline phos-
phatase families, as well as ecto-5=-nucleotidase/CD73 (5, 19,
44). These ectoenzymes are thought to act in concert to
sequentially dephosphorylate extracellular nucleotides and
locally regulate their concentration in hepatic extracellular
fluids (55).
At physiological pH, plasma membrane-bound NTPDases,
namely NTPDase1/CD39, NTPDase2/CD39L1, and NTPDase8,
represent the major liver ectonucleotidase activities (18,
45). NTPDases hydrolyze various nucleoside tri- and di-
phosphates (e.g., ATP and ADP) with distinct affinities and
abilities. NTPDase1 hydrolyzes ATP and ADP equally well
whereas NTPDase2 preferentially breaks down ATP over
ADP, and NTPDase8 acts as a functional intermediate
between NTPDase1 and NTPDase2 (18, 26, 31). In the liver,
NTPDases are expressed in a cell-specific manner: NTPDase1 is
expressed by vascular endothelial cells and smooth muscle
cells (18, 45), NTPDase2 by cells in the vascular external
layer and by portal fibroblasts (14, 46), whereas NTPDase8
is restricted to the canalicular membrane domain of hepa-
tocytes (18).
The monophosphonucleosides (e.g., AMP) generated by
NTPDase activity are ultimately converted into the correspond-
ing nucleosides (e.g., adenosine) by ecto-5=-nucleotidase (12,
27, 49). Interestingly, the NTPDase substrates ATP and ADP
are natural inhibitors of the monophosphatase activity of ecto-
5=-nucleotidase (39). In the liver, ecto-5=-nucleotidase protein
expression has been associated with the canalicular domain of
hepatocyte plasma membrane and undefined cellular elements
of the portal space (2, 47).
Because of their complementary biochemical properties,
NTPDases and ecto-5=-nucleotidase represent central elements
of liver purinergic signaling. However, the potential influence
of the coordinated action of NTPDases and ecto-5=-nucleoti-
dase on local hepatic purinergic signaling has yet to be estab-
lished. In the present study, we have characterized the impact
of the cellular distribution of these hepatic ectonucleotidases
on the local levels of P1 and P2 receptor agonists. Here we
show that ecto-5=-nucleotidase is coexpressed with NTPDase1,
-2, and -8 in distinct liver compartments in normal and fibrotic
rat liver. These specific associations of ecto-5=-nucleotidase
G447
G448 FINE MODULATION OF ADENOSINE FORMATION BY LIVER ECTOENZYMES
with each hepatic NTPDase have the ability to differentially
regulate the signaling pathways modulated by extracellular
nucleotides and their metabolites in the liver.
EXPERIMENTAL PROCEDURES
Materials
Culture media were obtained from Invitrogen (Burlington, ON,
Canada). Adenosine 5=-(␣,␤-methylene)diphosphate (␣,␤-methylene-
ADP), 2-chloroacetaldehyde, 3,3=-diaminobenzidine (DAB), 1,4-
diaza-bicyclo[2.2.2]octane (DABCO), nucleotides, levamisole, tet-
rabutylammonium hydrogen sulfate were provided by Sigma-Aldrich
(Oakville, ON, Canada). The 4=,6-diamidino-2-phenylindole (DAPI)
dye was purchased from Molecular Probes (Eugene, OR) and aqueous
hematoxylin from Biomeda (Foster City, CA). Mouse monoclonal
antibody to rat CD31/PECAM-1 (clone TLD-3A12) was purchased
from BD Pharmingen (Mississauga, ON, Canada), to rat CD68 (clone
ED-1) from AbD Serotec (Raleigh, NC), and those to mouse/rat
␣-smooth muscle actin (SMA; clone 1A4) and rat vimentin (clone V9)
from Sigma-Aldrich. The antibodies to NTPDases and ecto-5=-nucle-
otidase can be obtained from ectonucleotidases-ab.com (Québec, QC,
Canada).
Plasmids
The plasmids encoding rat NTPDase1 (NM_022587) (26),
NTPDase2 (NM_172030) (30), NTPDase8 (AY536920) (18), and
ecto-5=-nucleotidase/CD73 (NM_021576) (8) were used for antiserum
generation and for cell transfection, as described below.
Animals and Antibody Production
Sprague-Dawley rats, Hartley guinea pigs and New Zealand rabbits
were obtained from Charles River Laboratories (St-Constant, QC,
Canada) or Harlan Sprague Dawley (Indianapolis, IN). All procedures
were approved by the Canadian Council on Animal Care and by the
Laval University Animal Ethics Advisory Committee and the Yale
Institutional Animal Care and Use Committee. Genetic immunization
was carried out by direct injection of a plasmid encoding rat ecto-5=-
nucleotidase (8), as previously described with other antigens (18), in
Hartley guinea pigs and New Zealand rabbits.
Chronic liver fibrosis was induced in rats by carbon tetrachloride
(CCl4) intoxication. For CCl4 treatment, male rats were provided with
drinking water containing 1% phenobarbital starting 2 wk before and
until the end of CCl4 treatment, to upregulate cytochrome P-450
enzyme levels (24). Rats were injected with CCl4 (0.1 ml sc, CCl4
diluted 1:6 in olive oil) three times per week for 6 – 8 wk. For control
experiments, rats were injected in identical fashion with olive oil
alone. Livers were harvested at 6 – 8 wk and processed for enzyme
histochemistry as described below.
Cell Transfection and Ectonucleotidase Activity Assays
Transfection of COS-7 cells using plasmids encoding either recom-
binant NTPDase1, -2, -8, or ecto-5=-nucleotidase and preparation of
crude protein extracts were carried out as previously described (31).
The COS-7 cell line was used because it exhibits very low basal levels
of nucleotide- and nucleoside-metabolizing enzyme activities (Refs.
29, 51 and data not shown).
For assays at high ATP concentration (500 ␮M), ectonucleotidase-
overexpressing crude protein extracts were added to obtain an enzy-
matic activity of 24 nmol Pi/min with ATP and AMP as substrate for
NTPDases and ecto-5=-nucleotidase, respectively, as determined by
the malachite green procedure according to Baykov et al. (3, 32). The
enzymatic reactions were performed at 37°C for 150 min in Ringer
buffer (120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 2.5 mM MgCl2, 1.2
mM MgSO4, 25 mM NaHCO3, 10 mM dextrose, 80 mM Tris·HCl, pH
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00165.2011 • www.ajpgi.org
7.4) and the products of ATP hydrolysis were determined by reverse
phase HPLC, as previously described (31).
For assays at low ATP concentration (1 ␮M), ectonucleotidase-
overexpressing crude protein extracts were prepared with an ATPase
(for NTPDase) and AMPase activity (for ecto-5=-nucleotidase) suffi-
cient to convert ⬃40% of the initial substrate within 5 min, as
determined by HPLC. Enzymatic reactions were performed at 37°C
for 30 min in a modified Krebs buffer (114 mM NaCl, 4.7 mM KCl,
1.2 mM KH2PO4, 1.16 mM MgSO4, 2.5 mM CaCl2, 2.5 MgCl2, 25.5
mM NaHCO3, 20 mM HEPES, pH 7.4). A modified Krebs buffer
was used here instead of Ringer buffer because of interference with
fluorescence detection observed with the latter. Briefly, 150-␮l
aliquots of reaction mixture were collected after various time
intervals and transferred to an equal volume of ice-cold 5%
(wt/vol) trichloroacetic acid (TCA) for deproteinization. The sam-
ples were centrifuged at 10,000 g for 3 min at 4°C and the
supernatants were subjected to lipid extraction with diethyl ether
(3:1, vol/vol; 5 cycles). The resulting samples were subsequently
transformed to etheno(ε)-derivatives, according to a modified der-
ivatization protocol by Levitt et al. (34, 36). This additional step
allowed the determination of 50-fold lower concentrations of
Downloaded from ajpgi.physiology.org on March 1, 2012
adenylated species by fluorescence detection.
In a representative derivatization assay, a 200-␮l aliquot of the
extracted sample was incubated for 60 min at 70°C in the presence
of 1 M chloroacetaldehyde and 25 mM Na2HPO4, pH 4.0, in a final
volume of 250 ␮l. The resulting fluorescent 1,N6-ethenoadenine
derivatives (ε-ATP, ε-ADP, ε-AMP, and ε-adenosine) were sepa-
rated by reverse-phase HPLC as previously described (31), where
the mobile phase used contained 25 mM tetrabutylammonium
hydrogen sulfate and 100 mM KH2PO4/K2HPO4, pH 7.0, in 10%
(vol/vol) MeOH. All experiments were performed in triplicate.
Samples to which crude protein extracts were added after reaction
had been stopped (i.e., after TCA addition) were used as controls
(baseline levels). Separation and detection of ethenylated deriva-
tives were performed with an automated Waters and Beckman
HPLC apparatus equipped with an RF-10AXL Shimadzu fluores-
cence detector (␭exc ⫽ 307 nm; ␭em ⫽ 410 nm). Quantification and
identification of the fluorescent adenylated derivatives were carried
out by comparison with the corresponding ε-purine standards.
Immunoblotting
Protein preparation, SDS-PAGE fractionation, and electroblotting
were performed as previously described (18). Following incubation
with primary antibodies, proteins of interest were visualized with the
appropriate horseradish peroxidase-conjugated secondary antibodies
(anti-rabbit IgG, Amersham Biosciences, Boston, MA; anti-guinea pig
IgG, Santa Cruz Biotechnology, Santa Cruz, CA) and the Chemilu-
minescent Reagent Plus (Perkin-Elmer, Boston, MA), according to the
manufacturer’s recommendations.
Immunofluorescence, Immunocytochemistry, and
Enzyme Histochemistry
COS-7 cells (105/coverslip) or 6-␮m sections of snap-frozen rat
liver specimens were fixed with cold acetone:phosphate-buffered
formalin (9:1). For immunofluorescence, fixed sections were incu-
bated with the following polyclonal antibodies against rat enzymes:
anti-NTPDase1 (rN1– 6L) (18), anti-NTPDase2 (BZ3– 4F) (14), anti-
NTPDase8 (rN8 – 8C) (18), and anti-CD73 (either r5=NT-4C or r5=NT-
9L), or corresponding preimmune serum samples. The appropriate
Alexa Fluor-conjugated secondary antibodies (Molecular Probes)
were then applied as previously described (18). In one set of double
immunostaining experiments, rabbit rN1– 6L and BZ3– 4F antibodies
were each used in combination with guinea pig r5=NT-4C serum, and
the guinea pig rN8 – 8C antibody was used with rabbit r5=NT-9L serum.
In the other set of experiments, mouse monoclonal antibodies against
rat CD31/PECAM-1 (clone TLD-3A12), rat ␣-SMA (clone 1A4) and
 FINE MODULATION OF ADENOSINE FORMATION BY LIVER ECTOENZYMES
rat vimentin (clone V9) were each used in combination with the rabbit
r5=NT-9L serum.
For immunocytochemistry, fixed cells were incubated with the
polyclonal CD73 antiserum (r5=NT-4C and r5=NT-9L) and the appro-
priate biotinylated secondary antibodies (Jackson ImmunoResearch
Laboratories, Westgrove, PA) before addition of Vectastain Elite
ABC reagent (Vector Laboratories, Burlington, ON, Canada). All
antibodies were used at a 1:1,000 working dilution, except for r5=NT-
9L, which was used at a final concentration of 1:2,000. Endogenous
peroxidase was quenched by incubating the fixed cells with 0.15%
(vol/vol) H2O2 (in PBS). Endogenous avidin and biotin were blocked
using the Avidin/Biotin Blocking Kit (Vector Laboratories). The
peroxidase reaction was performed in a solution containing the DAB
substrate (0.4 mg/ml) and 0.03% (vol/vol) H2O2 in PBS, and stopped
with extensive water rinsing.
For enzyme histochemistry, ectonucleotidase activities were local-
ized by using the Wachstein/Meisel lead phosphate method as mod-
ified by Braun et al. (see reference therein) (7). The enzymatic
reaction was carried out by incubating liver sections with various
nucleotides (200 ␮M) as substrates for 1 h. All experiments were
performed in the presence of levamisole (5 mM), an inhibitor of
tissue-nonspecific alkaline phosphatase (TNAP). In a separate set
of experiments, ␣,␤-methylene-ADP (1 mM) was used as an inhibitor
of ecto-5=-nucleotidase. Control assays (not shown) were performed
in the absence of nucleotide.
Nuclei were counterstained with either DAPI (immunofluores-
cence) or aqueous hematoxylin (immunocytochemistry and enzyme
histochemistry). Mowiol 4-88 medium (supplemented with DABCO
as antifade reagent for immunofluorescence only) was used as mount-
ing medium.
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00165.2011 • www.ajpgi.org
G449
Flow Cytometry and Isolation of Rat Liver Cell Populations
Hepatocytes, hepatic stellate cells, portal fibroblasts and nonparen-
chymal cell preparations were isolated from fresh rat liver tissue as
previously described (14, 16). Fixed primary isolated cells were
labeled by indirect immunofluorescence after incubation with a rabbit
polyclonal serum to rat CD73 (r5=NT-9L) followed by Alexa Fluor
488-conjugated polyclonal anti-rabbit IgG antibody (Molecular
Probes). Cell-surface expression of CD73 protein was assessed by
fluorescence-activated cell sorting, using a FACSCalibur (BD Biosci-
ences, Rockville, MD) sorter and Flowjo software Version 8.7 (Tree-
Star, Ashland, OR).
RESULTS
Although the hepatic expression of NTPDase1, -2, and -8
has previously been reported (14, 18, 45), the cellular local-
ization of ecto-5=-nucleotidase/CD73 had not been clearly
established. Therefore, we first looked at the distribution of
ecto-5=-nucleotidase in relation to NTPDases, using enzyme
histochemical and immunohistochemical methods.
Downloaded from ajpgi.physiology.org on March 1, 2012
Histochemical Detection of Ectonucleotidase Activities in
Normal and Fibrotic Rat Liver
All enzyme histochemistry assays were performed in the
presence of the TNAP inhibitor levamisole (5 mM) to exclude
any contribution of this enzyme to the apparent catalytic
activities measured with the nucleotide substrates. Neverthe-
less, no difference in ectonucleotidase activity could be de-
Fig. 1. Histochemical detection of liver ecto-
nucleotidase activities. Ectonucleotidase activ-
ities were located in serial sections of a normal
rat liver, using various nucleotides (200 ␮M)
as substrates. All 3 structural components of a
classical hepatic triad are depicted, including
branches of hepatic artery (A), portal vein (V),
and intrahepatic bile duct (BD). ATPase and
ADPase catalytic products are mainly detected
in all vasculature (arterial ⬎ venous), in the
connective tissue associated with the portal
space, and in the bile canaliculi (BC) (top and
middle). The AMP hydrolysis pattern is located
in structures similar to those where ATPase and
ADPase activities are found, although no signal
is observed either in endothelium or in vascular
smooth muscle (arrows, bottom). No ectonucle-
otidase activity is detected in cholangiocytes for
any of the substrates tested (asterisks, right). All
enzyme histochemical assays were performed in
the presence of levamisole (5 mM) to inhibit
tissue-nonspecific alkaline phosphatase (TNAP)
activity. Scale bar, 10 ␮m.
G450 FINE MODULATION OF ADENOSINE FORMATION BY LIVER ECTOENZYMES

tected when omitting levamisole from the reaction solution
(data not shown), suggesting that TNAP activity was negligible
under the experimental conditions used. In the rat liver, en-
zyme histochemical assays using ATP as substrate revealed a
prominent staining activity in vascular endothelium and sub-
endothelium, in the connective tissue adjacent to the portal/
periportal space and surrounding central veins, in sinusoids,
and in bile canaliculi (Fig. 1; ATP). No nucleotide hydrolysis
was detected in ductular epithelium. Hepatic ADPase activity
displayed a distribution pattern similar to ATPase activity,
albeit with a slightly weaker staining intensity (Fig. 1; ADP).
When AMP was applied as substrate, catalytic products were
mainly detected in the subendothelium, in the connective tissue
associated with the perivascular and periductular areas, and in
the bile canaliculi. Note that ductular epithelium, vascular endo-
thelium, as well as smooth muscle were devoid of AMPase activity
(Fig. 1; AMP). Lack of endothelial AMPase activity was
observed in blood vessels of all types and calibers. When
␣,␤-methylene-ADP (1 mM) was added to specifically inhibit
ATPase, ADPase, and AMPase activities were observed in
fibrous septa surrounding hepatocyte nodules, in perivascular
connective tissue areas, as well as in cells localized within
hepatic sinusoids. Both ATPase and ADPase hydrolytic activ-
ities were still observed along the hepatocyte plasma mem-
brane, with a more prominent but disrupted canalicular dis-
tribution, whereas AMPase activity was increased at all
levels of the hepatocyte membrane. These observations,
although qualitative, indicate that levels and distribution of
ATPase, ADPase, and AMPase activities are regulated dur-
ing hepatic fibrosis and suggest their involvement in the
inflammatory liver response. This is in agreement with
previous studies showing that transcriptional regulation of
ectonucleotidase gene expression occurs during liver in-
flammation and fibrosis (15, 23, 40, 53).
Generation of Polyclonal Antibodies to Rat
Ecto-5=-Nucleotidase/CD73 and Specificity

Downloaded from ajpgi.physiology.org on March 1, 2012

ecto-5=-nucleotidase activity, overall AMP hydrolysis was re-
duced in all regions, suggesting that this enzyme contributes to
most, if not all, AMPase activity present in the rat liver at
physiological pH (Fig. 2). In support of these findings, enzyme
histochemical assays performed with other ecto-5=-nucleoti-
dase substrates, namely CMP, GMP, and IMP, showed similar
patterns of activity (not shown).
When enzyme histochemistry assays were performed in
livers from rats subjected to CCl4 intoxication (Fig. 3), hepatic
fibrosis induction was associated with obvious alterations of
ectonucleotidase activity distribution. Strong signals for
To determine the cellular distribution of ecto-5=-nucleoti-
dase and the various NTPDases in the liver, we used a panel of
polyclonal antibodies developed in our laboratory. The speci-
ficity of polyclonal sera to NTPDase1 (rN1– 6L), NTPDase2
(BZ3– 4F), and NTPDase8 (rN8 – 8C) has previously been val-
idated (14, 18). Guinea pig and rabbit polyclonal antibodies
directed against rat ecto-5=-nucleotidase have been generated
and their specificity tested by the following immunological tech-
niques. By immunocytochemistry, positive dark brown staining
could be seen only with ecto-5=-nucleotidase-transfected COS-7
cells incubated in presence of either r5=NT-4C guinea pig serum or

Fig. 2. Histochemical detection of liver ecto-5=-nucleotidase activity. Ecto-5=-nucleotidase activity was located in normal rat liver sections with AMP (200 ␮M)
as substrate. Top: prominent signals for hepatic AMPase activity in the connective tissue associated with perivascular areas (arrows), and in bile canaliculi
(asterisks). Bottom: ecto-5=-nucleotidase inhibitor ␣,␤-methylene-ADP (␣␤-MeADP; 1 mM) reduces AMPase activity in all structures (see arrows and asterisks).
All enzyme histochemical assays were performed in the presence of the TNAP inhibitor levamisole (5 mM). Scale bar, 20 ␮m.

AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00165.2011 • www.ajpgi.org

 FINE MODULATION OF ADENOSINE FORMATION BY LIVER ECTOENZYMES
r5=NT-9L rabbit serum, respectively (Fig. 4, A and B; serum). The
same antisera detected a strong protein band with a molecular
weight of ⬃66 kDa by immunoblotting in the lane corresponding
to the crude protein extracts from ecto-5=-nucleotidase-transfected
cells. No protein was detected in the lanes with crude protein
extracts from nontransfected cells, confirming the specificity of
both types of polyclonal antibodies (Fig. 4, A and B; immunoblot).
Furthermore, both r5=NT-4C and r5=NT-9L antibodies displayed
similar distribution patterns for hepatic ecto-5=-nucleotidase, as
determined by immunohistochemistry and immunofluorescence
(see next section; data not shown).
Immunolocalization of NTPDases and Ecto-5=-Nucleotidase
in Normal and Fibrotic Rat Liver
Immunofluorescence labeling in the rat liver with the polyclonal
antibody rN1–6L showed prominent staining for NTPDase1 in vas-
cular endothelium and smooth muscle cells as well as in liver
resident macrophages, namely Kupffer cells (detected as
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00165.2011 • www.ajpgi.org
G451
Fig. 3. Histochemical detection of liver ectonucleotidase activ-
ities in CCl4-induced hepatic fibrosis model. Ectonucleotidase
activities were located in liver sections from vehicle (control)
and CCl4-treated rats, using ATP, ADP, or AMP (200 ␮M) as
substrate. All 3 structural components of a classical hepatic
triad are depicted: branches of hepatic artery, portal vein, and
the intrahepatic bile duct. ATPase, ADPase, and AMPase
activities in control animals followed a distribution pattern
Downloaded from ajpgi.physiology.org on March 1, 2012
similar to that described in Fig. 1. In contrast, in fibrotic
animals, overlapping signals for ATP, ADP, and AMP hydro-
lytic products were detected in fibrous bands surrounding
hepatic nodules. Moreover, staining for ATPase and ADPase
activities was observed in hepatic sinusoids and hepatocyte
basolateral membrane, and more prominently, although in a
diffuse manner, in bile canaliculi. All enzyme histochemical
assays were performed in the presence of the TNAP activity
inhibitor levamisole (5 mM). CV, central vein; FS, fibrous
septum. Scale bar, 10 ␮m.
CD68-positive; Fig. 5A and data not shown). As seen with
BZ3– 4F serum, NTPDase2 was detected in the subendothelial
basement membrane cells, in portal fibroblasts, and in peri-
ductular “bundle-shaped” structures, in the connective tissue of
portal space area, likely representing intrahepatic nerves (Fig.
5B). Staining with the rN8 – 8C polyclonal antibody showed
that NTPDase8 expression was restricted to bile canaliculi
(Fig. 4C). Both r5=NT-4C and r5=NT-9L polyclonal sera re-
vealed strong ecto-5=-nucleotidase expression in the connective
tissue associated with the perivascular and periductular areas,
as well as in bile canaliculi, with a faint immunostaining in the
basal membrane of hepatocytes (Fig. 4; ECTO-5=NT, all pan-
els). Interestingly, ecto-5=-nucleotidase expression was absent
from the vascular endothelium (Fig. 5; ECTO-5=NT, all pan-
els). Double immunofluorescence staining showed that al-
though ecto-5=-nucleotidase did not strictly colocalize with
NTPDase1, both proteins were located in the same structures in
adjacent cells, such as NTPDase1-positive Kupffer cells pres-
G452 FINE MODULATION OF ADENOSINE FORMATION BY LIVER ECTOENZYMES

Fig. 4. Specificity of the polyclonal antibodies to

rat ecto-5=-nucleotidase. Immunocytochemistry
was performed with intact nontransfected (insets)
or transfected COS-7 cells with an expression
vector encoding rat ecto-5=-nucleotidase. Cells
were incubated with either preimmune serum or
guinea pig (r5=NT-4C; top) or rabbit (r5=NT-9L;
bottom) polyclonal antibody to ecto-5=-nucleoti-
dase. Immunoreactivity is present only in trans-
fected cells incubated with either antibody. Scale
bar, 20 ␮m. Immunoblot analysis of lysates from
nontransfected COS-7 cells (ctrl) next to lysates
from COS-7 cells transfected with the ecto-5=-
nucleotidase expression vector (CD73) shows a
single band of ⬃66 kDa found exclusively in
lysates from transfected cells, again confirming
the specificity of both guinea pig r5=NT-4C and
rabbit r5=NT-9L sera. A: guinea pig antibody

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(r5=NT-4C; right) or its preimmune serum (left).
B: rabbit antibody (r5=NT-9L; right) or its preim-
mune serum (left).

ent in perivascular and/or periductular connective tissue, and
on the basolateral surface of the endothelial cell layer in close
contact with the perivascular extracellular matrix (Fig. 5A,
merged). Likewise, although ecto-5=-nucleotidase did not co-
localize with NTPDase2, both enzymes were expressed in the
same vicinity in the perivascular and periductular connective
tissue areas (Fig. 5B, merged). By contrast, NTPDase8 and
ecto-5=-nucleotidase colocalized in bile canaliculi (Fig. 5C,
merged).
We further investigated ecto-5=-nucleotidase expression in
the rat liver using the following cell markers: cell adhesion
molecule PECAM-1 for vascular endothelium, ␣-SMA for
smooth muscle cells and myofibroblasts, and vimentin for
fibroblasts and other liver mesenchymal cells (42). Ecto-5=-
nucleotidase labeling was clearly distinct from that observed
for PECAM-1 and ␣-SMA, but partly overlapped with vimen-
tin staining in the perivascular and periductular areas (Fig. 6;
merged, all panels). We further confirmed the expression of
ecto-5=-nucleotidase in primary liver cell preparations by flu-
orescence-activated cell sorting analysis. These data demon-
strate that in normal rat liver, ecto-5=-nucleotidase is expressed
by NTPDase8-positive hepatocytes and a nonparenchymal cell
type that is distinct from hepatic stellate cells and NTPDase2-
positive portal fibroblasts (Fig. 7).
We also investigated the distribution of ectonucleotidases
NTPDase1, -2, -8, and ecto-5=-nucleotidase in specimens from
CCl4-induced fibrosis by immunohistochemistry (Fig. 8). Pos-
itive staining for NTPDase1 expression could be observed in
several proliferating vascular structures and (likely) inflamma-
tory cells in liver fibrotic areas and intersinusoidal spaces.
NTPDase2 distribution was noticeably reorganized and present
in fibrous septa surrounding hepatic nodules, as previously
described (15). NTPDase8 expression, while still restricted to
hepatocyte bile canaliculi, appeared rather reduced and diffuse
in these structures compared with control liver. Finally, in-
creased ecto-5=-nucleotidase expression was observed in fi-
brotic septa and sinusoidal spaces between hepatocytes. Also,
its canalicular distribution at the hepatocyte membrane level
was diffuse and less prominent relatively to control liver.
Again, as noted in normal rat liver, the distribution profiles of
NTPDase1, -2, -8, and ecto-5=-nucleotidase enzymes paral-
leled those described for ATPase, ADPase (NTPDases), and
AMPase (ecto-5=-nucleotidase) activities in fibrotic livers.
Taken together, these data show that the histochemical local-
ization of ATPase and ADPase activities correlates with the
combined distribution of NTPDase1, -2, and -8, as detected by
immunohistochemistry, whereas the AMPase activity corresponds
to the immunohistological expression pattern of ecto-5=-nucleoti-
dase in normal and fibrotic rat livers. In addition, our results
suggest that ecto-5=-nucleotidase is expressed by a fibroblastic
population that is devoid of NTPDase activity in normal rat liver.
HPLC Analysis of ATP Hydrolysis by Different Hepatic
NTPDases in Combination with Ecto-5=-Nucleotidase
Based on the localization determined as above for NTPDase
and ecto-5=-nucleotidase activities and protein expression, we
evaluated how the net biochemical activity resulting from the pairing
of ecto-5=-nucleotidase with either NTPDase1, NTPDase2, or
NTPDase8 might affect the generation of nucleotide and nu-
cleoside species in the liver. As described in EXPERIMENTAL
PROCEDURES, ATP hydrolysis assays were performed at a high,
enzyme-saturating substrate concentration (500 ␮M) such as
found under pathological conditions such as inflammation or
hepatic failure (21, 25, 55), as well as at low more physiolog-
ical substrate concentration (1 ␮M) (10, 33). Aliquots of the

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Fig. 5. Comparative distribution of liver nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5=-nucleotidase (ECTO-5=NT). Double immuno-
fluorescence labeling of NTPDases and ecto-5=-nucleotidase was performed in serial sections of rat liver. A: NTPDase1 staining is associated with the liver
vascular network and Kupffer cells. B: NTPDase2 labeling is detected in the connective tissue of portal and septal spaces and perivascular areas. C: NTPDase8
signal is present exclusively in bile canaliculi. A–C: ecto-5=-nucleotidase immunostaining is observed in the connective tissue found in portal space or
perivascular areas as well as in bile canaliculi. Double staining of NTPDase1 and ecto-5=-nucleotidase reveals that both ectoenzymes are expressed by different
but adjacent cell populations in the vasculature, hepatic sinusoids, and hepatic extracellular matrix (A, MERGED). Similarly, NTPDase2 and ecto-5=-nucleotidase
proteins are both present at the surface of distinct cell types of hepatic connective tissue (B, MERGED). Both NTPDase8 and ecto-5=-nucleotidase colocalize
in bile canaliculi (C, MERGED). Scale bar, 10 ␮m except for A, bottom, where scale bar is 20 ␮m. DAPI, 4’,6-diamidino-2-phenylindole.

reaction mixtures were collected at different time points, and
nucleotide and nucleoside contents were evaluated by HPLC.
The time course of ATP hydrolysis and time-dependent gen-
eration of adenosine were analyzed for the different pairs of rat
ectonucleotidases observed above for each liver structure.
High ATP concentration (500 ␮M). In the presence of
NTPDase1 and ecto-5=-nucleotidase, ATP was hydrolyzed rap-
idly to adenosine, with transient increases of ADP and AMP
concentrations (Fig. 9, NTPDase1 and CD73, high). Although
AMP was rapidly produced, the formation of adenosine by
ecto-5=-nucleotidase appeared to be slow during the first hour,
likely because of the inhibitory effect of ATP and ADP
transiently present in the medium at that early stage. When
both ATP and ADP concentrations decreased afterward, aden-
osine formation increased until full conversion of AMP to
adenosine. When ATP was incubated with NTPDase2 and
ecto-5=-nucleotidase, it was readily converted into ADP, but
very poorly into AMP, as predicted by the fact that ADP is a

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G454 FINE MODULATION OF ADENOSINE FORMATION BY LIVER ECTOENZYMES

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Fig. 6. Comparative distribution of liver ecto-5=-nucleotidase with cell markers. Double immunofluorescence labeling of ecto-5=-nucleotidase and cell markers
for vascular endothelial cells [platelet/endothelial cell adhesion molecule-1 (PECAM-1)], myofibroblastic cells (␣-smooth muscle actin), and nonmyofibroblastic
cells (vimentin) was performed in rat liver sections. Although ecto-5=-nucleotidase immunostaining is clearly distinct from that of PECAM-1 and ␣-SMA, it
partially overlaps with vimentin staining. Scale bar, 10 ␮m.

poor substrate of NTPDase2 (Fig. 9, NTPDase2 and CD73,
high). Because of the protracted accumulation of ADP in the
assay medium and the low abundance of AMP substrate for
ecto-5=-nucleotidase, adenosine formation was accordingly
limited. Of note is the observation that under the conditions
described, rat NTPDase2 was active only for 50 – 60 min. The
ATP hydrolysis profile obtained with the pairing of NTPDase8
with ecto-5=-nucleotidase showed a rapid ADP accumulation,
peaking after 20 min, which was followed by the delayed
generation of AMP, which reached a plateau between 50 and
120 min (Fig. 9, NTPDase8 and CD73, high). These observa-
tions concur with the fact that NTPDase8 activity is higher
with ATP than with ADP. As a result, ecto-5=-nucleotidase
activity was transiently inhibited by the presence of ADP,
which delayed adenosine formation compared with the hydro-
lysis profile obtained with the NTPDase1 plus ecto-5=-nucle-
otidase pair.
Low ATP concentration (1 ␮M). In the presence of the
NTPDase1 and ecto-5=-nucleotidase pair, ATP was directly
converted to AMP, ADP being rapidly hydrolyzed after it was
formed (Fig. 9, NTPDase1 and CD73, low). Therefore, aden-
osine formation by ecto-5=-nucleotidase almost paralleled
AMP appearance in the reaction mixture. After about 20 min,
⬃80% of ATP added was hydrolyzed to adenosine. The ADP
generated upon ATP hydrolysis by the NTPDase2 plus ecto-
5=-nucleotidase pair accumulated in the reaction medium, with
little AMP production, and as a result, adenosine was produced
only in very small amounts (Fig. 9, NTPDase2 and CD73,
low). The pattern of ATP hydrolysis by NTPDase8 plus ecto-
5=-nucleotidase was very similar to the latter combination and
resulted in the continuous accumulation of ADP (Fig. 9,
NTPDase8 and CD73, low) with limited production of AMP
and adenosine. In the presence of 1 ␮M ATP, the level of ATP
and/or ADP produced was apparently too low to inhibit ecto-
5=-nucleotidase. In this condition, the rate of adenosine forma-
tion was solely dependent on the substrate (AMP) generated.
This contrasts with the data obtained at a high ATP concen-
tration (Fig. 9 NTPDase8 and CD73, high) where the “higher”
concentration of ADP generated was hampering the ability of
ecto-5=-nucleotidase to hydrolyze AMP to adenosine, in agree-
ment with our previous observations (35).
Note that in these assays we did not detect any degradation
products of adenosine, indicating the lack of expression of
adenosine-metabolizing enzymes in COS-7 cells, in keeping
with the report of Tkacz et al. (51).

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 FINE MODULATION OF ADENOSINE FORMATION BY LIVER ECTOENZYMES
Fig. 7. Flow cytometry analysis of ecto-5=-nucleotidase expression in liver
primary cell populations. Cell populations were prepared as described in
EXPERIMENTAL PROCEDURES. Ecto-5=-nucleotidase (CD73) was labeled using
rabbit polyclonal r5=NT-9L serum. Isolated hepatocytes and nonparenchymal
cells (NPCs) are positive for ecto-5=-nucleotidase staining whereas portal
fibroblasts (PFs) and hepatic stellate cells (HSCs) are mostly negative.
DISCUSSION
In the present study, we demonstrate a correlation between
ecto-5=-nucleotidase/CD73 protein expression and AMPase
activity in both normal and fibrotic rat livers. In normal rat
liver, both attributes of ecto-5=-nucleotidase were detected in
the canalicular and sinusoidal membrane domains of hepato-
cytes as well as in the connective tissue surrounding central
veins or associated with the portal and larger septal space
areas. Quite notably, ecto-5=-nucleotidase protein expression
and AMPase activity were absent from the cell surface of
hepatic vascular endothelium and bile duct epithelium in rat, as
shown here, and in mouse (data not shown). The absence of
ecto-5=-nucleotidase from murine endothelial cells contrasts
with the vascular localization reported for its human ortholog
(Ref. 1 and data not shown). Our examination of ecto-5=-
nucleotidase distribution by double fluorescence immunostain-
ing with specific cell markers as well as flow cytometry
analysis of primary liver cells further indicates that ecto-5=-
nucleotidase is mainly expressed in hepatocytes and nonmyo-
fibroblastic (nonparenchymal) liver cells that are likely distinct
from portal fibroblasts and hepatic stellate cells. Importantly,
the facts that 1) levamisole, an inhibitor of alkaline phospha-
tase activity, had no effect on the AMPase activity observed
(data not shown), 2) hydrolysis of AMP, CMP, GMP, and IMP
(Figs. 1 and 2 and data not shown) all matched ecto-5=-
nucleotidase immunolocalization using two different antibod-
ies (Figs. 4 and 5 and data not shown), and 3) this activity was
decreased by ␣,␤-methylene-ADP, an ecto-5=-nucleotidase in-
hibitor (Fig. 2) suggest that ecto-5=-nucleotidase is responsible
for most if not all nucleoside monophosphatase activity present
in the liver. Ecto-5=-nucleotidase therefore appears to be the
enzyme responsible for the formation of extracellular adeno-
sine at physiological pH in the rat liver. This enzyme would
therefore be expected to play a key role in the regulation of P1
receptor activation.
When we compared the distribution of ecto-5=-nucleotidase
to that of hepatic NTPDase1, -2, and -8 with respect to the
same parameters, i.e., protein expression and ectonucleotidase
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G455
activity (14, 18) in normal rat liver, different combinations of
NTPDase and ecto-5=-nucleotidase expression patterns were
found: 1) NTPDase1 (vascular endothelium, smooth muscle
cells, and Kupffer cells) and ecto-5=-nucleotidase, although not
actually expressed in the same cells, were collectively present
at the junction between the vascular tunica media and the
perivascular connective tissue or at the sinusoidal face of
hepatocyte membranes; 2) NTPDase2 (portal fibroblasts) and
ecto-5=-nucleotidase were also expressed by neighboring cells
located within the portal/periportal connective tissue; and fi-
nally 3) NTPDase8 and ecto-5=-nucleotidase were coexpressed
by hepatocytes in bile canaliculi. It is noteworthy to mention that
ecto-nucleotide pyrophosphatases/phosphodiesterases, which have
the ability to hydrolyze tri- and diphosphate nucleosides in
principle, are also expressed in the liver (48). However, their
biochemical properties, namely a lower substrate affinity than
NTPDases and optimal hydrolytic activity at alkaline rather
than physiological pH, did not allow us to evaluate their
potential contribution to liver purinergic signaling in our con-
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dition settings.
Induction of experimental liver fibrosis in CCl4-treated rats
was associated with changes in the distribution of all hepatic
ectonucleotidase activities. Overall, ATPase, ADPase, and
AMPase activities were increased in liver fibrotic areas, in
sinusoidal spaces, and in fibrous septa in the periphery of
hepatic nodules, a hallmark of CCl4-induced fibrosis. In the
same fibrotic livers, NTPDase1 protein was associated with
proliferating blood vessels, NTPDase2 protein with the fibrous
septa surrounding hepatic nodules, and NTPDase8 protein with
structurally altered bile canaliculi of hepatocytes. Ecto-5=-
nucleotidase protein expression was detected in both apical and
sinusoidal domains of hepatocyte plasma membrane as well as
in fibrous septa. Again, as observed in normal rat liver, hepatic
AMPase activity and expression of ecto-5=-nucleotidase over-
lapped with the localization of hepatic ATPase and ADPase
activities as well as the expression of NTPDase1, -2, and -8.
We addressed the potential impact of this specific distribu-
tion of NTPDases and ecto-5=-nucleotidase on extracellular
nucleotide levels by performing a biochemical ATP hydrolysis
assay in the presence of each pair formed by ecto-5=-
nucleotidase with either NTPDase1, -2, or -8. It is notewor-
thy that the hydrolytic patterns observed in Fig. 9 are in
agreement with the reported kinetic properties of each
NTPDase (31) and with the fact that ATP and ADP are
physiological inhibitors of ecto-5=-nucleotidase (35). Indeed, at
500 ␮M ATP, a significant decrease in ATP and ADP concen-
tration was required to observe the effective production of
adenosine from ecto-5=-nucleotidase. As expected, adenosine
generation was fastest in the presence of NTPDase1, which
rapidly hydrolyzed both ATP and ADP to the ecto-5=-nu-
cleotidase substrate, AMP. In contrast, in the presence of
NTPDase2, adenosine production was very low because of the
very limited hydrolysis of ADP by this enzyme. The result
obtained with NTPDase8 was somewhat intermediate between
those observed for NTPDase1 and -2 at 500 ␮M ATP, which
correlates with a transient production of ADP by NTPDase8
(18, 31), with adenosine production being inhibited when ATP
and ADP levels exceed 50 –100 ␮M, even at high AMP levels
(200 ␮M). Interestingly, the situation was different for hydro-
lysis of low ATP concentration (1 ␮M), where ATP and ADP
levels were insufficient to inhibit ecto-5=-nucleotidase, and as
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Fig. 8. Comparative distribution of liver NTPDases and ecto-
5=-nucleotidase in CCl4-induced hepatic fibrosis model. En-
zyme localization of the ectonucleotidases NTPDase1, -2, -8
and ecto-5=-nucleotidase was determined by immunohisto-
chemistry in liver sections from vehicle (control) and CCl4-
treated rats. In control animals, the expression patterns of
NTPDase1, -2, -8 and ecto-5=-nucleotidase are similar to those
described in Fig. 5. In contrast, in fibrotic animals, NTPDase1
expression is mainly associated with proliferating vascular
structures. NTPDase2 expression is almost exclusively de-
tected in perinodular fibrous areas. NTPDase8 protein is only
found in bile canaliculi, although with a diffuse distribution.
Immunoreactivity to ecto-5=-nucleotidase is localized at the
level of both canalicular and basolateral membrane domains in
hepatocytes, and prominently in fibrous septa surrounding
hepatic fibrotic nodules. Scale bar, 10 ␮m.

adenosine generation was limited only by the rate of AMP
production by NTPDases. Of note, under the latter conditions,
NTPDase8 behaved somewhat like NTPDase2 since it hydro-
lyzed ATP to ADP, with only minimal production of AMP,
which greatly limited adenosine generation by ecto-5=-nucleoti-
dase. This observation is in agreement with the Km value of
NTPDase8 for ADP, which is much higher than for NTPDase1
(18, 31).
What functional importance could such a distribution of
NTPDases and ecto-5=-nucleotidase have in the liver? At

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Fig. 9. ATP hydrolysis profile by the different combinations of ecto-5=-nucleotidase with hepatic NTPDases. Hydrolysis products of 500 ␮M (high) and 1 ␮M
(low) ATP were analyzed over time, as indicated in EXPERIMENTAL PROCEDURES. At high substrate concentration, ATP is rapidly and completely converted into
adenosine in the presence of both NTPDase1 and ecto-5=-nucleotidase (CD73), whereas adenosine is hardly produced by the NTPDase2 ⫹ ecto-5=-nucleotidase
combination. ATP hydrolysis in the presence of NTPDase8 ⫹ ecto-5=-nucleotidase combination generated a transient accumulation of ADP and AMP that
resulted in a delayed (compared with the NTPDase1 ⫹ ecto-5=-nucleotidase combination) but substantial adenosine production (when compared with the
NTPDase2 ⫹ ecto-5=-nucleotidase combination). At low substrate level, the incubation of ATP with NTPDase1 ⫹ ecto-5=-nucleotidase leads to its rapid
dephosphorylation to adenosine without an increase in ADP level. In the presence of either NTPDase2 or NTPDase8 together with ecto-5=-nucleotidase, the
accumulation of generated ADP was accompanied by poor AMP production because of limited ADP hydrolysis and thus resulted in a modest adenosine
generation by ecto-5=-nucleotidase. ADO, adenosine.

physiological pH, NTPDase1, -2, and -8 enzymatic activities
account for the major bulk of hepatic ectonucleotidase activity,
degrading nucleoside tri- and diphosphates into their mono-
phosphate counterparts, which are essential for the activity of
ecto-5=-nucleotidase, the rate-limiting ectoenzyme for adeno-
sine formation (5). Thus the biochemical data presented here
demonstrate that the precise expression pattern of the different
NTPDases in the liver is of physiological relevance because
they can distinctly modulate P1 and P2 receptor activation.
Indeed, in a given cellular microenvironment, the coupling of
each hepatic NTPDase species with its ecto-5=-nucleotidase
complementary activity would feed extracellular purinergic
mediators for different signal transduction pathways, according
to the identity of the purinoceptors expressed at the cell
membrane. In our assay, we used two different ATP concen-
trations, namely 1 ␮M, which is within the range of nucleotide
levels measured in the conditioned media of liver cell lines
(33), and reported for circulating nucleo(s/t)ides (50 nM to 1
␮M) in both sinusoidal blood and bile flows (10), and 500 ␮M
ATP, a value close to those potentially reached in the
extracellular medium during inflammatory liver diseases
(21, 25, 55). Our results show that the substrate concentra-
tion used can influence the net biochemical activity of the
different NTPDase/ecto-5=-nucleotidase pairs, suggesting that
the cellular context is an important parameter for the role of
these ectonucleotidases in extracellular nucleotide signaling.
Consequently, purinergic receptors expressed in the vicinity of
a particular NTPDase/ecto-5=-nucleotidase combination might
be differentially regulated depending on the prevailing biolog-
ical conditions. Moreover, this hypothesis supports the notion
that NTPDase1, -2, and -8 likely play nonredundant roles in the
modulation of purinergic signaling, since ATP hydrolysis and
adenosine generation profiles noticeably change as a function
of the NTPDase species (see Fig. 9). Several liver functions
could be affected by this specific distribution of NTPDases and
ecto-5=-nucleotidase: 1) ion transport by cholangiocytes, an
extracellular nucleotide-dependent mechanism that is essential
to bile formation (20, 37–38, 57); 2) purine salvaging pathways
in hepatocytes, as the liver represents the main source of
nucleo(s/t)ides for extrahepatic tissues deficient in de novo
purine biosynthesis (11); 3) cell proliferation, an important
feature of liver tissue, which involves P2 receptor signaling in
hepatocytes, sinusoidal endothelial cells, and portal fibroblasts (6,
22, 28, 50); 4) cell death, since stimulation of either P1 or P2
receptors in hepatocytes has been shown to induce apoptosis (52,
56); and finally 5) inflammation, since hepatocytes, liver immune
cells such as Kupffer cells and T lymphocytes, portal fibroblasts,
as well as hepatic stellate cells express different P1 and P2
receptors that might modulate their activation and/or deactivation
states in liver diseases (4, 13, 17, 41, 54).
In summary, the specific distribution of NTPDase1, -2, and -8
in conjunction with ecto-5=-nucleotidase in the liver differentially
regulates extracellular nucleo(s/t)ide levels and, therefore, may
also differentially regulate the associated signaling pathways.
ACKNOWLEDGMENTS
The authors thank Dr. H. Zimmermann (J. W. Goethe University, Frankfurt,
Germany) for providing the plasmid encoding for rat ecto-5=-nucleotidase/

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G458 FINE MODULATION OF ADENOSINE FORMATION BY LIVER ECTOENZYMES
CD73 and Dr. M. J. Fernandes and V. Gagné (Rheumatology and Immunology
Research Centre, CHUL Research Center) for technical assistance with con-
focal microscopy. We also thank Dr. R. Poulin (Scientific Proofreading and
Writing Service, CHUQ Research Center) for editing this paper.
GRANTS
This work was supported by the Canadian Institutes of Health Research
(CIHR; MOP-102472 to J. Sévigny) and by the National Institutes of Health
(NIH/NIDDK R01 DK076735, R01 DK070849 and the Yale Liver Center DK
45710 awards to J. A. Dranoff). M. Fausther was the recipient of a Doctoral
Scholarship from the Government of Gabon and is currently receiving the 2011
Roger L. Jenkins Post doctoral Fellowship from the American Liver Founda-
tion, E. M. Soliman of a Student Research Grant from the Egyptian Govern-
ment Ministry of Higher Education, G. Kauffenstein of fellowships from the
Institut National de la Santé et de la Recherche Médicale in partnership with
the Fonds de Recherche en Santé du Québec (FRSQ) and the Heart and Stroke
Foundation of Canada in partnership with the CIHR and the Canadian Stroke
Network, and J. Sévigny of a New Investigator award from the CIHR and a
Junior 2 Scholarship from the FRSQ.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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