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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 294:126–133, 2000 /2395/830130
Vol. 294, No. 1
Printed in U.S.A.
Chronic Oral Administration of ATP Modulates Nucleoside
Transport and Purine Metabolism in Rats1
KETTY KICHENIN and MICHEL SEMAN
Groupe d’Immunologie Denis Diderot, Université Paris 7, Hall des Biotechnologies, Paris, France
Accepted for publication March 8, 2000
This paper is available online at http://www.jpet.org
Extracellular purine nucleosides and nucleotides mediate
a wide range of pharmacological effects through two main
families of purine receptors, P1, which is sensitive to adenosine, and P2, which is sensitive to ATP, expressed on many
mammalian cell types, including enterocytes (Fredholm et
al., 1994). Extracellular nucleosides present in body fluids
also correspond to a pool of nucleotide precursors of the
salvage pathway. Nucleoside transporter (NT) proteins on
cell membrane are necessary for cellular uptake of physiological nucleosides (Cass et al., 1998). Two classes of NT
processes have been recognized. The first one, called equilibrative, exhibits the typical features associated with facilitated diffusion driven by the concentration gradient of
nucleosides. Two subtypes of equilibrative NT (es and ei)
have been identified according to their sensitivity to nitrobenzylmercaptopurine (NBMPR), and their genes (ENT1 and
ENT2) have been cloned in rat and humans (Griffith et al.,
1997a,b; Yao et al., 1997; Crawford et al., 1998). The second
class of NT process is concentrative and driven by transmembrane Na⫹ gradients. Concentrative NT thus are Na⫹-dependent but insensitive to NBMPR. Six concentrative NT proteins have been characterized (Cass et al., 1998), and two
Received for publication December 15, 2000.
1
This work was supported in part by Mayoly Spindler Laboratories (Chatou, France). K.K. was supported by a grant from the Ministere de la Recherche et de la Technologies (CIFRE).
ATP dose administered, whereas plasma adenosine concentration remained unchanged. This diminution likely resulted
from an increased ectonucleotidase activity, suggesting that
the chronic administration of ATP seems to induce a progressive adaptation of purine metabolism. This adaptive response
to free purine supplementation affects both intracellular metabolism and purine exchange between intracellular and extracellular compartments. This modification of free purine turnover
and delivery may affect physiological parameters under the
control of P1 and P2 purinoceptors described in different experimental models.
genes have recently been cloned (Che et al., 1995; Huang et
al., 1994; Ritzel et al., 1997, 1998). Equilibrative processes
can transport a wide variety of permeants from both sides of
the membrane, whereas concentrative ones display a relative
selectivity for the influx of purine or pyrimidine nucleosides
(Wang et al., 1997). The nature of NT proteins expressed on
plasma membrane varies among cell types, although both
equilibrative and concentrative transporters can coexist on
the same cell (Belt, 1983). Moreover, the transport rate of
nucleoside can change during cell cycle or after cell activation
(Smith et al., 1989; Kichenin et al., 2000a), in correlation
with the level of equilibrative or concentrative NT protein
expression.
The concentration of free nucleosides present in body fluids
can change with physiological (Chagoya de Sanchez et al.,
1983) or pathological (Burnstock, 1993; Driver et al., 1993)
situations or with the purine content of the diet (Clifford and
Story, 1976, LeLeiko et al., 1983; Porcelli et al., 1995). This
influences the transport rate of nucleosides via equilibrative
NT processes, which depends on the extracellular purine
concentration and accounts for pharmacological effects mediated by P1 and P2 purinoceptors.
In this study, we explored whether the oral administration
of natural purines over a long period of time could affect
purine metabolism and transport in rats. ATP was chosen
because it is susceptible to generate all of the purine metab-
ABBREVIATIONS: NT, nucleoside transporter; ENT, equilibrative nucleoside transporter; NBMPR, nitrobenzylmercaptopurine; HBSS, Hanks’
balanced salt solution.
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ABSTRACT
The effect of repeated oral administration of ATP on purine
transport and metabolism was investigated in rats. An increased ability of the gut to capture intraluminal purine nucleosides and to export ATP and nucleosides toward portal bloodstream was observed in rats after 30 days of treatment with 5
mg/kg/day ATP. This was accompanied in erythrocytes by an
increased transport of adenosine rapidly transformed into ATP,
which in turn was exported toward extracellular fluid. However,
these metabolic changes were associated with a paradoxical
and progressive diminution of plasma ATP level below that
found in control rats and that was not strictly dependent on the
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Biological Response to Chronic Oral Administration of ATP
olites of the salvage pathway and is the substrate of ectonucleotidases and ecto-protein kinases (Ziganshin et al.,
1994). Results reveal an adaptive metabolic response in
chronically supplemented rats characterized by an increased
nucleoside influx in gut and erythrocytes associated with an
increased ability to release ATP and purine nucleosides.
Whether this response is dependent on and mediates pharmacological signals through P1 or P2 receptors is discussed.
Materials and Methods
Adenosine Metabolism in Erythrocytes. Blood from control or
treated rats was collected on sodium citrate (3.8% w/v). After centrifugation for 10 min at 1200g, erythrocytes were washed and suspended in HBSS to reconstitute the initial hematocrit. A series of
microtubes containing 200 ␮l of erythrocyte suspension were incubated for 10 min at 37°C with 20 ␮l of a mixture of cold and
[14C]adenosine (666 kBq) at a final concentration of 1 ␮M. Individual
tubes were centrifuged (30 s, 1000g) at various time over a period of
10 min, and free purines were extracted from the washed pellet with
200 ␮l of 7% (w/v) trichloroacetic acid. Total radioactivity in the
supernatant and pellet was counted. Radioactivity corresponding to
the different purine metabolites was determined after HPLC separation as described above.
ATP Exportation. One milliliter of an erythrocyte suspension in
HBSS corresponding to the hematocrit was incubated with adenosine (1 ␮M) for 10 min at 37°C. Erythrocytes were rapidly washed
and resuspended in HBSS supplemented with dipyridamole (10 ␮M)
to inhibit nucleoside transport. Then 200-␮l aliquots were collected
at various times and centrifuged. Free purines were immediately
extracted from supernatant and pelleted for analysis.
Adenosine Transport. Adenosine transport was determined according to the method described by Harley et al. (1982). One hundred
microliters of erythrocyte suspension in transport medium (100 mM
NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.5)
was mixed at 25°C with 100 ␮l of [14C]adenosine (1 ␮M, 1.85 kBq).
The reaction was stopped by the addition of 200 ␮l of transport
medium supplemented with 20 ␮M NBMPR and dipyridamole. After
extraction, total radioactivity in the supernatant and pellet was
counted.
The volume of extracellular medium trapped in the cell pellet and
the total water space of the cell pellet were determined using [14C]sucrose and [3H]H2O, respectively, according to Plagemann and Aran
(1990). The intracellular volume of the erythrocyte was taken as the
difference between total water space and the sucrose space.
For the determination of kinetic constants, 100 ␮l of erythrocyte
suspension was mixed with different concentrations of [14C]adenosine. Nucleoside uptake was measured every 10 s during a period
of 2 min. The nucleoside uptake values were corrected for the amount
of [14C]nucleoside trapped in the extracellular space of the cell pellet,
and Vi was determined for each adenosine concentration. Vm and Km
values were determined from Lineweaver-Burke representations.
NBMPR Binding Measurement. Experiments were performed
according to Cass et al. (1981). Briefly, 200 ␮l of erythrocyte suspension was incubated with increasing [3H-benzyl]NBMPR concentrations in a total volume of 1 ml for 30 min at 37°C. They were then
centrifuged (30 s, 1000g) and washed, and the radioactivity in the
pellet and medium was counted. In parallel tubes, 10 ␮M excess cold
NBMPR was added to evaluate nonspecific binding.
Results
Intraluminal ATP Metabolism in Gut of Normal and
ATP-Treated Rats. The in vivo evolution of the purine
content in the intraluminal space of the gut was compared in
control and ATP-treated rats after the local injection of
[14C]ATP into an isolated jejunum section. HPLC analysis of
the intraluminal content 1.5 or 9 min after injection revealed
that ATP was rapidly transformed into ADP, AMP, adenine,
and uric acid (Fig. 1, A and B). No qualitative or quantitative
difference was observed between normal animals and rats
treated with 5 mg/kg/day ATP per os for 30 days.
Adaptive Response of Gut in ATP-Treated Rats. To
evaluate the effect of oral treatment with ATP on intestinal
metabolism, we then analyzed the ATP concentration in the
draining portal blood stream immediately after intraluminal
deposition of a mixture of cold and 14C-labeled ATP into an
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Animals. Male Sprague-Dawley rats (250 g) were obtained from
the Center d’Elevage Depre (Doulcharel, France) and maintained in
our animal facilities under standard conditions. Treatment was performed with the daily administration of ATP or adenosine (Sigma,
Saint Quentin Fallavier, France) in 0.1 ml of sterile distilled water
with a gastric cannula. All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Chemicals. [14C]ATP (1.85–2.29 GBq/mmol) and [14C]adenosine
(1.85–2.29 GBq/mmol) were purchased from Amersham (Saclay,
France). [14C]Sucrose (2409 GBq/mmol), [3H]H2O (3.7 GBq/ml), and
Ecolume scintillation fluid were obtained from ICN (Orsay, France).
Hanks’ balanced salt solution (HBSS), NBMPR, dipyridamole, adenosine, ATP, and all other reagent-grade drugs were obtained from
Sigma.
Purine Extraction and Characterization. Immediately after
the collection of biological samples, purines were extracted with
trichloroacetic acid (7% w/v) for 30 min at 4°C. Samples were then
centrifuged, and supernatants were neutralized with a tri-N-octylamine/trichlorotrifluoroethane solution (0.5 M) before analysis,
according to Rapaport (1988). ATP dosage was determined by luminescence using a luciferin-luciferase kit assay (Roche Diagnostics,
Meylan, France) according to the manufacturer’s recommendations.
Luminescence was counted with a Top-Count luminometer (Packard
Instruments, Rungis, France). Radiolabeled purine metabolites were
analyzed after HPLC fractionation on a Licrosphere RP18 column
(125 ⫻ 4 mm; Merck, Nogent sur Marne, France) using a Waters
600E pump and a UV detector (Waters model 486). The mobile phase
consisted of a mixture of solution A (50 mM KH2PO4-KOH, pH 6) and
methanol (B). For the first 10 min, solution A was passed at a flow
rate of 0.5 ml/min. A linear gradient was then applied to achieve 80%
A/20% B after 5 min. Fractions were collected every 30 s. Then 2 ml
of scintillation fluid was added, and radioactivity was counted on a
Rack-beta counter (LKB, Saclay, France). For analysis of cold purine
metabolites, samples were first treated with chloroacetaldehyde to
obtain the N6 ethene-derived metabolites (Nithipatikom et al., 1994).
Derivatives were then separated by HPLC fractionation, and fluorescence was detected using a Fluostar fluorimeter (Bio-Tek Instruments, Winooski, VT).
Purine Absorption In Vivo. Fasted rats were anesthetized with
6 mg/kg sodium pentobarbital i.p. On dissection, a fraction of the
jejunum was isolated by introducing a proximal catheter (i.d., 0.86
mm; Biotrol no. 6; Biotrol, Chennevières-les-Louvres, France), 2 cm
under the stomach and a distal catheter (i.d., 1.014 mm; Biotrol no.
7) 10 cm below the stomach. The gut section was rinsed with 0.1 M
sterile NaCl equilibrated at 37°C. A heparin-treated catheter (i.d.,
0.3 mm; Biotrol no. 1) was introduced into one of the secondary
mesenteric venules up to the portal vein, which was clamped before
the liver. Then 4 ml of a solution providing 5 mg/kg cold purine mixed
with 74 kBq of the corresponding 14C-labeled molecule was injected
into the isolated section of the gut. Samples of portal blood and of
intraluminal content were collected every 90 s over a period of 10
min, after which animals were sacrificed by an overdose of sodium
pentobarbital. Samples were immediately extracted for analysis of
purine metabolites.
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Vol. 294
isolated jejunum section. In normal rats, small amounts of
adenosine, hypoxanthine, and uric acid were detected in portal blood, in the absence of liver passage, 9 min after intraluminal injection of an amount of ATP corresponding to 5
mg/kg (Fig. 1C). On the contrary, all types of 14C-labeled ATP
metabolites (i.e., ADP, AMP, adenosine, inosine) were detected in the portal blood of rats treated with ATP for 30
days, indicating an increased absorption of purines resulting
from treatment (Fig. 1D). We then tested whether intestinal
cells could release ATP into blood stream by using the highly
sensitive luciferin-luciferase ATP assay. Unexpectedly, the
basal plasma ATP level in ATP-treated animals was much
lower than that in control animals (see below). Yet in these
animals, a rapid 1000-fold increase in portal plasma ATP
concentration was detected within 2 min after the introduction of ATP into a jejunum section (Fig. 2A). A lower (10⫻)
and slower (⬎5 min) increase was observed in control rats
under the same experimental conditions. This demonstrated
that gut can rapidly export ATP toward the bloodstream. It
also suggested an adaptive metabolic response of enterocytes
to chronic oral ATP treatment characterized by an increased
release of ATP and purine nucleosides toward blood. When 4
nmol of adenosine, corresponding to the amount of purines
present in a dose of 5 mg/kg ATP, was again introduced into
an isolated jejunum, a more important and rapid liberation of
ATP was observed in the blood of ATP-treated rats compared
with control animals (Fig. 2B). This indicated that the im-
proved ATP delivery was likely the consequence of an increased absorption of adenosine and purine nucleosides from
the lumen by enterocytes and an increased ATP synthesis
from these precursors.
Adenosine Transport in Red Cells from ATP-Treated
Rats. Changes in purine uptake and exportation observed in
the gut after chronic ATP treatment motivated an analysis of
NT processes in erythrocytes, which play an important role
in the regulation of extracellular purine level and are dependent on the purine salvage pathway for ATP synthesis. We
first compared the ability of erythrocytes from normal and
ATP-treated rats to capture exogenous adenosine by incubating 200 ␮l of washed red cells suspended in HBSS with 6.6
kBq of [14C]adenosine at 37°C. Samples of supernatant were
collected over a period of 5 min, and radioactivity was
counted. A faster decrease in extracellular radioactivity was
observed with erythrocytes from ATP-treated rats compared
with control animals as illustrated in Fig. 3A, suggesting an
accelerated uptake of adenosine. Direct adenosine transport
rate was thus evaluated. In red cells from ATP-treated rats,
a faster and higher level of adenosine transport was observed
than in red cells from control animals when 100 ␮l of erythrocyte suspension corresponding to the hematocrit was incubated with 1 ␮M adenosine in the transport medium (Fig.
3B). To explain this increase in adenosine transport, the
kinetic constants of ENT, which is the only process present
on red cells, were determined in erythrocytes from control
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Fig. 1. Intestinal metabolism
of ATP in vivo in control (bold
line) and ATP-treated (10 mg/
kg/day for 30 days) rats.
[14C]ATP (74 kBq) mixed with
cold purine to a final concentration of 5 mg/kg was introduced
into an isolated jejunum section. Metabolites were analyzed after HPLC fractionation
in the intraluminal (A and B)
and the plasma of portal blood
(C and D) at 1.5 min (A and C)
or 9 min (B and D) after administration. 1 indicates uric acid;
2, ATP plus ADP; 3, hypoxanthine; 4, AMP; 5, inosine; 6,
adenosine; and 7, adenine.
HPLC profiles are representative of independent determinations from four rats in each
group.
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Biological Response to Chronic Oral Administration of ATP
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Fig. 2. Plasma ATP levels in portal blood of control (E) and ATP-treated
(10 mg/kg/day for 30 days, F) rats. Blood was collected from the portal
vein at different times after the administration of [14C]ATP (A) or
[14C]Ado (B) (74 kBq mixed with cold purine to a final concentration of 5
mg/kg) in an isolated jejunum section in vivo. After extraction, ATP
concentration was measured by luciferin-luciferase luminescence assay.
Each point represents the mean of four animals, representative of two
independent experiments. Vertical bars represent the S.E. *P ⬍ .05
compared with control, calculated from paired Student’s t test.
and ATP-treated animals. The Vmax value for adenosine
transport increased from 2.53 ⫻ 10⫺3 in control animals to
6.53 ⫻ 10⫺3 pmol/␮l/s in erythrocytes from ATP-treated rats,
but the Km value remained unchanged.
Adenosine transport in red cells from control animals was
completely inhibited by NBMPR according to a biphasic
mode (Jarvis and Young, 1986) (Fig. 4A). About 30% of this
transport was inhibited by NBMPR at a concentration of 10
nM, whereas the remaining transport activity was inhibited
by NBMPR with an IC50 value close to 1 ␮M. In red cells from
treated rats, the adenosine transport sensitive to low
NBMPR concentration was not affected, but the remaining
activity was NBMPR-resistant, even at a high concentration,
leading to only partial inhibition (Fig. 4A). This demonstrated that the oral administration of ATP over a period of
30 days was able to improve and to modify the NT process in
red cells. The number of NBMPR-binding sites was not, however, significantly different on erythrocytes from control and
ATP-treated rats (Fig. 4B), yet the Kd value for NBMPR,
Fig. 3. Adenosine metabolism in erythrocytes from control (E) and ATPtreated (5 mg/kg/day for 30 days) (F) rats. A, capture of extracellular
adenosine. Microtubes containing 200 ␮l of erythrocytes (in reconstituted
hematocrit were incubated in HBSS for 10 min at 37°C. Then, [14C]Ado
(0.66 kBq), mixed with cold purine to a final concentration of 1 ␮M, was
added. Reaction was stopped at various times, and extracellular radioactivity was counted after centrifugation (30 s, 1200g). B, adenosine transport. Erythrocyte suspension (100 ␮l) in transport medium corresponding
to the hematocrit was incubated with [14C]Ado (1.85 kBq, 1 ␮M) at 25°C.
The reaction was stopped by the addition of NBMPR and dipyridamole
(10 ␮M) and then centrifuged (30 s, 1200g). Radioactivity in the pellet
was counted. C, determination of transport kinetic constants. Vi was
determined in transport medium with various concentrations of [14C]Ado.
Kinetic constants were deduced from Lineweaver-Burke representations.
Each point represents the mean of four animals, representative of two
independent experiments. Vertical bars represent S.E. *P ⬍ .05, **P ⬍
.01 compared with control, calculated from paired Student’s t test.
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Vol. 294
Fig. 4. A, inhibition of adenosine transport by NBMPR in erythrocytes
from control (E) and ATP-treated (10 mg/kg/day for 30 days) (F) rats.
Erythrocytes suspended in transport medium were preincubated with
NBMPR for 5 min before the addition of [14C]Ado (1.85 kBq, 1 ␮M).
Adenosine uptake was then determined in triplicate after 5 min by
measuring the radioactivity in cell pellet. Adenosine uptake in erythrocytes preincubated without NBMPR was arbitrarily taken as 100%. B,
determination of NBMPR binding sites in erythrocytes from control (E)
and ATP-treated (10 mg/kg/day for 30 days) (F) rats. Erythrocytes were
incubated with [3H-benzyl]NBMPR for 30 min at 37°C. Parallel experiments were performed in the presence of 10 ␮M cold NBMPR to determine the nonspecific binding. Binding sites were determined after Scatchard representation of radioactivity bound to cells. Each point
represents the mean of four animals.
which is in the range of 50 pM, was generally two to three
times higher for red cells from ATP-treated rats. This modification could account for the resistance to NBMPR inhibition
of adenosine transport. Together, this demonstrated that the
oral administration of ATP over a period of 30 days was able
to improve and to modify the NT process in red cells.
Purine Metabolism in Red Cells from ATP-Treated
Rats. To further explore the consequence of increased nucleoside transport in red cells from ATP-treated rats, intracellular purine metabolites were analyzed after extraction from
erythrocytes as soon as 30 s after contact with extracellular
[14C]adenosine. Adenosine, adenine, AMP, uric acid, and a
small amount of ATP were detected in red cells from control
rats, but most extracellular adenosine captured by erythrocytes from ATP-treated animals was rapidly converted into
ATP (Fig. 5). Thus, although red cells synthesize ATP
through the glycolysis pathway, adaptation to ATP treatment improved their ability to produce ATP from extracellular purine precursors. Accordingly, a 2-fold increase in total
ATP content was observed 30 s after incubation with adenosine in erythrocytes from treated animals (Table 1). Conversely, when resuspended into fresh HBSS, red cells from
ATP-treated animals exported about 50 times more ATP
than those from control animals. This was assessed by incubating cells with 1 ␮M adenosine for 10 min. After washing,
the time course of ATP concentration in supernatants was
followed by luminescence ATP assay.
Paradoxical Effect of ATP Treatment on ATP Plasma
Level. The influence of ATP treatment on plasma ATP level
was investigated in rats treated with 5 mg/kg ATP via the
oral route over a period of 30 days. Blood samples were
collected every fourth day. Plasma and red cell ATP concentrations were measured by luminescence ATP assay. Adenosine concentration was measured by fluorometric detection
after derivation and HPLC fractionation. Surprisingly, a dramatic reduction in plasma ATP level was gradually observed
down, after 21 days of treatment, to about 1% of the initial
value or of the concentration found in water-treated counterparts (Fig. 6A). No significant variation of intraerythrocyte
ATP concentration was detected (Fig. 6B). Extracellular ATP
breakdown is accomplished by various ectoenzymes generating ADP and AMP (Ziganshin et al., 1994), which are further
catabolized by successive dephosphorylation into adenosine
by ecto-5⬘-nucleotidases (Zimmermann, 1996). We thus
looked for variation in the adenosine level in both plasma and
red blood cells from control and ATP-treated rats. No modification of the adenosine plasma concentration was seen (Fig.
6C), but a small increase was observed in erythrocytes conversely related to the decrease of plasma ATP concentration.
Hence, long-term oral treatment with ATP led to a paradoxical and dramatic decrease in the plasma ATP level correlated with a moderate accumulation of adenosine into erythrocytes.
Influence of ATP Dose on Plasma ATP Level. The
influence of the dose of ATP used for treatment on plasma
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Fig. 5. Adenosine metabolism in erythrocytes from control (bold line) and
ATP-treated (5 mg/kg/day for 30 days) rats. Erythrocyte suspension (200
␮l) corresponding to the hematocrit was incubated in HBSS for 10 min at
37°C. Then, [14C]Ado (0.66 kBq) mixed with cold adenosine to a final
concentration of 1 ␮M was added. After 30 s, cells were centrifuged
(1000g), and the intracellular purine content was analyzed after HPLC
fractionation. 1 indicates uric acid; 2, ATP plus ADP; 3, hypoxanthine; 4,
AMP; 5, inosine; 6, adenosine; and 7, adenine. Values are representative
of four independent experiments.
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Biological Response to Chronic Oral Administration of ATP
131
TABLE 1
ATP concentration and release in erythrocytes from control or ATPtreated (5 mg/kg/day for 30 days) rats preincubated with adenosine
Intracellular ATP: microtubes containing 200 ␮l of erythrocytes (in reconstituted
hematocrit) were incubated in HBSS for 10 min at 37°C. Then, 1 ␮M adenosine was
added. Samples were collected every 30 s, and intracellular ATP concentration was
measured after centrifugation (30 s, 1200g) and extraction. Extracellular ATP:
erythrocytes were incubated for 10 min in HBSS with adenosine (1 ␮M). After rapid
washing, they were resuspended in fresh HBSS with dipyridamole (10 ␮M). Cells
and supernatants were separated by centrifugation every 30 s, and ATP concentration was determined. ATP concentration was determined by luciferin-luciferase
luminescence assay. Each value represents the mean of triplicate determinations
from four independent experiments ⫾ S.E.
Erythrocyte ATP
Supernatant ATP
Time
Control
min
0.5
1
1.5
ATP-Treated
⫻ 10
7.1 ⫾ 2.5
5 ⫾ 0.7
5.7 ⫾ 1.3
⫺4
Control
⫻ 10
M
14 ⫾ 2.1
9.3 ⫾ 0.2
11 ⫾ 1.3
ATP-Treated
1.4 ⫾ 0.4
0.6 ⫾ 0.34
0.2 ⫾ 0.1
⫺9
M
75 ⫾ 37
19 ⫾ 8
16 ⫾ 4.4
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ATP level was evaluated by treating animals with 1, 5, and
10 mg/kg ATP over a period of 20 days. A strong decrease in
ATP plasma level was observed (Fig. 7A) for the three doses.
This suggested that the effect of chronic ATP supplementation was not strictly dependent on the dose of free purines
delivered during treatment. Adenosine is a pharmacologically active ATP metabolite that, contrary to ATP, can be
uptake by nucleoside transporters in the gut and used for
nucleoside salvage. We thus tested whether adenosine could
reproduce the effect of ATP on plasma ATP level in chronically treated animals. As seen in Fig. 7B, an important decrease in plasma ATP level was observed in rats after 20 days
of oral treatment with 1, 5, or 10 mg/kg adenosine. This effect
could be compared with that obtained in ATP-treated rats
and was not clearly dose-dependent.
Discussion
Results reported herein demonstrate that the daily oral
administration of 5 mg/kg ATP during 1 month leads to a
modulation of purine transport and metabolism in rats. An
adaptive response is observed characterized by 1) an increased uptake of nucleosides in the intestine accompanied
by an increased exportation of ATP and nucleosides toward
portal blood, 2) an increased purine metabolism in red cells,
and 3) a dramatic reduction in plasma ATP level.
The rapid degradation of ATP observed in the gut is consistent with the existence of many enzymatic activities on the
brush barrier of the small intestine that are responsible for
the breakdown of nucleotides of dietary origin (Mohamedali
et al., 1993). It is also well known that most of the radioactivity found in animals fed radiolabeled nucleotides is found
in the gut (Salati et al., 1984), which is in good agreement
with the important salvage activity of this organ (LeLeiko et
al., 1983). However, the increased exportation of nucleosides
and ATP from gut toward blood observed in ATP-treated rats
compared with controls after the introduction of a same
amount of radioactive ATP in the lumen was not anticipated.
It suggests that in the small intestine, chronic ATP treatment regulates nucleoside transporters and stimulates the
nucleoside salvage pathway. The mechanisms that could account for this regulation are still unknown. However, they
might involve purine receptors on enterocytes (Burnstock,
1993). ATP-sensitive P2 and adenosine sensitive P1 receptors
have both been described on these cells (Fredholm et al.,
Fig. 6. ATP and adenosine levels in plasma and erythrocytes from rats
(n ⫽ 6) treated with oral ATP (5 mg/kg/day). A, plasma ATP level. B,
erythrocyte ATP level. C, plasma and erythrocyte adenosine level. ATP
concentration was measured using luciferin-luciferase luminescence assay. Adenosine concentration was measured by fluorescence after derivation with chloroacetaldehyde and HPLC fractionation. Each point represents the mean ⫾ S.E. *P ⬍ .05 compared with day 0, calculated from
paired Student’s t test.
1994), which can deliver signals responsible for the response
observed.
A second consequence of repeated oral administration of
ATP is a change in erythrocyte purine metabolism characterized by improved adenosine uptake, ATP synthesis, and
ATP exportation by red cells. This might result from an
increased availability of extracellular nucleotide precursors
in plasma originating from the gut. Adenosine has been reported to activate glycolysis in rat erythrocytes (Gutierrez-
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Kichenin et al.
Juarez et al., 1992) and to stimulate ATP synthesis (MarinezValdez et al., 1982) within 30 min after intraperitoneal
injection in vivo. In our experiments, red cells were collected
from treated rats 4 to 6 h after the last oral administration.
Moreover, plasma adenosine level remained constant
throughout the treatment in ATP supplemented rats. It thus
seems unlikely that the improved uptake of adenosine and
ATP synthesis are simply the consequence of a recent administration of ATP, which would be rapidly degraded into adenosine and other purine nucleosides by ectonucleotidases
present on the plasma membrane of many cell types. They
could possibly reflect a regulation of ATP metabolism in red
cells resulting from the induction of enzymes by a pharmacological effect of extracellular purines on erythrocyte precursors.
Interestingly, like the adenosine plasma level, the concentration of adenosine in red cells was stable throughout the
treatment. The adenosine gradient driving the equilibrative
transport rate of nucleosides was thus theoretically equivalent in normal and ATP-treated rats. However, adenosine
influx was higher in red cells from treated animals. Two
equilibrative transporters (es and ei) are expressed on erythrocytes and allow nucleoside capture. The es transporter,
which is the predominant and major adenosine transporter
on rat erythrocytes, is sensitive to NBMPR inhibition,
whereas the ei transporter is not (Jarvis and Young, 1986).
The es transporter density on red cells and the Km value were
not significantly affected by treatment, but the Vmax value for
adenosine transport was strongly increased. This improved
transport rate was correlated with the increased ATP synthesis and exportation observed in red cells from ATPtreated rats. The affinity for NBMPR was reduced. A conformational change of the es transporter might account for
these effects. This change might be induced by ATP itself.
Indeed, Delicado et al. (1994) recently showed on chromaffin
cells that ATP can modulate nucleoside transporter function
at both intracellular and extracellular levels. The consequence of this regulation is an increased nucleoside transport
rate with an increased Vmax value and no modification of the
Km value for the permeant. These effects resemble those
reported here in ex vivo experiments with red cells from
ATP-treated rats. Experiments to be published on human
cells confirm that extracellular ATP or analogs can modulate
nucleoside transport in vitro. Hence, oral ATP administration may progressively stimulate an autocrine regulation of
adenosine uptake and ATP synthesis in red cells and possibly
in enterocytes and other cell types.
Repeated oral administration of ATP also led to a progressive diminution of plasma ATP level. This seems quite paradoxical because the administration of doses corresponding
to few micromoles reduces normal plasma ATP level, which is
in the range of 1 to 3 ␮M, below 100 nM despite an increased
ATP delivery from the gut. In preliminary experiments, we
observed a similar phenomenon in cynomolgus monkeys
treated with ATP. Moreover, this diminution was clearly not
dependent on the dose administered and was rather similar
in rats given 1 or 10 mg/kg/day ATP. It may be accounted for
by the induction in ectonucleotidases on plasma cell membranes. Our attempts to demonstrate this induction on blood
vessels and liver led to inconclusive results due to the high
ecto-ATPase activity already present in these tissues, which
degrades ATP within a few milliseconds (Ziganshin et al.,
1994). Conversely, this activity is low on erythrocytes from
mammals (Bencic et al., 1997) and was not stimulated by the
treatment (data not shown). The diminution of plasma ATP
level seems a progressive physiological reaction to control the
potential pharmacological effects via P2 receptors that would
result from an increased delivery of ATP to the blood by
enterocytes and red cells. A similar diminution was observed
in adenosine-treated rats, demonstrating that the biological
response observed results from metabolic changes and not
simply from a pharmacological effect of ATP on the intestine.
This progressive change in ATP concentration did not affect
the uric acid level in biological fluids at the end of the treatment (data not shown).
It is questionable whether purine nucleosides and nucleotides present in the food can have a similar effect. It is
noteworthy, however, that in food and meat, consistent with
the very short half-life of ATP, most of the free purine nucleotides and nucleosides are present as IMP and inosine (LeLeiko et al., 1983), which are not effective ligands of purinoceptors but can be transported into cells via various NT
systems (Huang et al., 1993). These metabolites may not
have regulatory effects on the purine metabolism of enterocytes and represent, in addition, nucleoside precursors that
can be used by intestinal microorganisms that have no ectonucleotidases on their surface. A direct coupling of these
enzymes with nucleoside transporters on the surface of enterocytes might explain why the direct administration of ATP
could specifically induce a pharmacological effect that would
not normally occur in the gut with food intake.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 7. Influence of the dose of ATP (A) or adenosine (B) administered on
plasma ATP level after 30 days. Plasma ATP level was determined using
luciferin-luciferase luminescence assay. Each point represents the
mean ⫾ S.E. of six animals. *P ⬍ .05 versus control calculated from
paired Student’s t test.
Vol. 294
2000
Biological Response to Chronic Oral Administration of ATP
Acknowledgments
We thank Dr. A. Geloso for valuable advice on the surgical preparation of animals and C. Joberty, C. Joulin, P. Aumond, and A.
Tomas for expert technical assistance.
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Send reprint requests to: Dr. Ketty Kichenin, Groupe d’Immunologie Denis
Diderot, Université Paris 7, Hall des Biotechnologies, Tour 54, CP7124, 2 place
Jussieu, 75251 Paris Cedex 05, France. E-mail: [email protected]
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Several experimental or clinical attempts have been made
to use ATP or adenosine for the treatment of pain (Sawynok,
1998), cardiovascular diseases (Daval et al., 1991), cancer
(Rapaport, 1993), and brain disorders (Williams, 1993). Most
of these experiments have been done using high doses of
purines by the i.v. route. Our present results demonstrate
that it is possible to obtain a biological response to purines by
chronic oral treatment. Experiments from our laboratory
support this conclusion. Indeed, an important peripheral vascular response characterized by peripheral vasodilatation
and increased paO2, is observed in rabbits after 14 days of
treatment with ATP, which, again, is not seen with a single
oral administration (Kichenin et al., 2000b). This treatment
also profoundly affects the metabolism of skeletal muscles
(K. Kichenin and M. Seman, unpublished findings). The repeated oral administration of pharmacologically active purines, such as ATP or adenosine, seems thus able to modify
purine metabolism in vivo and to regulate physiological parameters that are known to be under the control of purinoceptors.
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