ADP Acting on P2Y13 Receptors Is a Negative Feedback
Pathway for ATP Release From Human Red Blood Cells
Lingwei Wang, Göran Olivecrona, Matthias Götberg, Martin L. Olsson,
Maria Sörhede Winzell, David Erlinge
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Abstract—Red blood cells may regulate tissue circulation and O2 delivery by releasing the vasodilator ATP in response to
hypoxia. When released extracellularly, ATP is rapidly degraded to ADP in the circulation by ectonucleotidases. In this
study, we show that ADP acting on P2Y13 receptors on red blood cells serves as a negative feedback pathway for the
inhibition of ATP release. mRNA of the ADP receptor P2Y13 was highly expressed in human red blood cells and
reticulocytes. The stable ADP analogue 2-MeSADP decreased ATP release from red blood cells by inhibition of cAMP.
The P2Y12 and P2Y13 receptor antagonist AR-C67085 (30 ␮mol/L), but not the P2Y1 blocker MRS2179, inhibited the
effects of 2-MeSADP. At doses where AR-C67085 only blocks P2Y12 (100 nmol/L), it had no effect. AR-C67085 and
the nucleotidase apyrase increased cAMP per se, indicating a constant cAMP inhibitory effect of endogenous
extracellular ADP. 2-MeSADP reduced plasma ATP concentrations in an in vivo pig model. Our results indicate that
the ATP degradation product ADP inhibits ATP release by acting on the red blood cell P2Y13 receptor. This negative
feedback system could be important in the control of plasma ATP levels and tissue circulation. (Circ Res. 2005;96:189196.)
Key Words: ATP release 䡲 cAMP 䡲 P2 receptors 䡲 microdialysis 䡲 red blood cells
I
the number of unoccupied hemoglobin O2 binding sites.7,9
The released ATP then binds to P2Y receptors on the
endothelium and stimulates vasodilatation by the release of
nitric oxide (NO), prostaglandins,2 and endothelium-derived
hyperpolarizing factor (EDHF).15,16 Thus, the RBC functions
as an O2 sensor, contributing to the regulation of blood flow
and O2 delivery by releasing ATP depending on the oxygenation state of hemoglobin.
Physiologically important signaling systems are usually
regulated by negative feedback systems, for example, noradrenaline and ATP release from sympathetic nerves is
inhibited by presynaptic ␣2 and P1 (A1 subtype) receptors.17
We hypothesized that ATP release from RBCs is regulated by
a P2 receptor–mediated negative feedback pathway.
t has become increasingly clear that, in addition to functioning as an intracellular energy source, ATP and ADP
can serve as important extracellular signaling molecules.1,2
Extracellular ATP in the circulation is rapidly degraded into
ADP, AMP, and adenosine by ectonucleotidases.3 ATP and
ADP activate P2 receptors on endothelium, platelets,4,5 and
other blood cells,6 regulating several physiological responses
including vascular tone,7 platelet aggregation, and the release
of endothelial factors. At least 15 nucleotide-activated cell
surface receptors (7 of P2X and 8 of P2Y) have been found
in man, with remarkably broad and varied physiological
responses.
The matching of oxygen supply with demand requires a
mechanism that increases blood flow in response to decreased
tissue oxygen levels. Several reports suggest that the red
blood cell (RBC) acts as a sensor for hypoxia, and different
mechanisms have been suggested by which the deoxygenated
RBC stimulates vasodilatation.7–10 RBCs contain millimolar
amounts of ATP and possess the membrane-bound glycolytic
enzymes necessary for its production.11–13 ATP is released in
response to reductions in oxygen tension and pH.7,9 It has
been shown in vitro that the vessels dilate in response to low
O2 levels only when blood vessels are perfused with RBCs.14
Recently, in vivo studies in man demonstrated that ATP is
released in working skeletal muscle circulation depending on
Materials and Methods
The studies were approved by the local Ethics Committee of the
Lund University and were conducted according to the principles of
the Declaration of Helsinki. All participants gave written consent for
the study.
Preparation of RBCs and Reticulocytes
Human RBCs were collected as described.12 The packed RBCs were
resuspended and washed three times in a physiological salt solution
(PSS; in mmol/L: 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 140.5 NaCl, 21.0
Tris, and 11.1 dextrose; pH 7.4). The oxygen tension was 85 to
90 mm Hg. RBCs were prepared on the day of use. Previous
Original received May 12, 2004; resubmission received August 30, 2004; revised resubmission received December 2, 2004; accepted December 6,
2004.
From the Department of Cardiology (L.W., G.O., M.G., D.E.), Transfusion Medicine (M.L.O.), and Cell and Molecular Biology (M.D.W.), Lund
University Hospital, Lund, Sweden.
Correspondence to Dr David Erlinge, Department of Cardiology, Lund University Hospital, S-221 85 Lund, Sweden. E-mail [email protected]
© 2005 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000153670.07559.E4
189
190
Circulation Research
February 4, 2005
Figure 1. Relative P2 gene expression in
RBCs and reticulocytes. Bar graphs show
expression of P2 receptors normalized to
GAPDH in RBCs (n⫽5, A) and reticulocytes
(n⫽4, B). ***P⬍0.001 compared with other P2
receptors.
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experiments using real-time PCR have demonstrated high levels of
P2Y12 in platelets,4 P2Y11 in buffy coat,4 and P2X4 receptors in
monocytes and lymphocytes.18 Very low levels of mRNA for these
receptors (Figure 1) rule out contamination of other circulating cells.
Human reticulocytes were prepared from peripheral blood samples. A dry reticulocyte-enriched cell pellet was obtained by separation of the sample on a Lymphoprep gradient. The cells present in
the liquid phase below the lymphocytic layer were harvested, washed
repeatedly in PBS, and frozen at ⫺80°C.19
mg I⫺/mL (Nycomed) to ensure positioning of the catheter. Through
a catheter, the microdialysis probe (CMA70) was inserted and the
microdialysis tip was placed in the coronary sinus. A 6F introducer
sheath was inserted into the surgically exposed left carotid artery.
Through the left carotid artery, a catheter was placed in the left
anterior descending artery (LAD). Finally, 2 mL of 2-MeSADP
(10 ␮mol/L) was infused in the LAD for 1 minute. Samples were
collected by microdialysis (5 ␮L/min) from the coronary sinus every
5 minutes.
Incubating RBCs and RBC Ghosts In Vitro With
Microdialysis Equipment
Measurement of ATP
Washed RBCs were brought to a 20% hematocrit in PSS buffer.
RBC ghosts were prepared as described20 from washed RBCs with
20% hematocrit, resuspended into the original volume in PSS buffer.
The RBCs or RBC ghosts were kept in an incubation chamber (2.5
mL, 37°C). Microdialysis probes (CMA20; Microdialysis AB,
Sweden) were inserted into the solution and perfused at a rate of 5
␮L/min with PSS. Samples were balanced for 30 minutes before
pharmacological agents were added. RBCs and RBC ghosts were
incubated with different pharmacological agents for 10 minutes, if
not otherwise indicated, and samples were collected during the final
5 minutes for ATP quantification.
Our microdialysis method eliminates the need for centrifugation
and pipetting of RBCs that is known to cause nonspecific release of
ATP. Furthermore, it enables the investigation of real-time changes
of ATP release during sequential addition of drugs. When defined
concentrations of ATP (10⫺5 to 10⫺8 mol/L) were added in the
incubation chamber without RBCs, the dialysate ATP concentrations
had a linear relationship compared with the added ATP concentrations (r2⫽1.0, P⬍0.0001). Relative recovery (RR) was 7.6⫾0.5%,
determined as follows: RR⫽cd/cs, with cd being the concentration of
ATP in a dialysate fraction and cs its known concentration of ATP
within a sample solution.21 To further verify the specificity of ATP
assays, defined concentrations of ATP were incubated with
2-MeSADP (1 ␮mol/L), MRS2179 (10 ␮mol/L), adrenaline
(1 ␮mol/L), and AR-C67085 (30 ␮mol/L), which did not interfere
with the results of ATP measurements (data not shown).
In Vivo Pig Model With Microdialysis for
Determining ATP Levels in Blood
Healthy domestic pigs (Larsson, Lund, Sweden) of both sexes
weighing 25 kg were premedicated with azaperone (Stresnil Vet), 2
mg/kg. After induction of anesthesia with thiopental 5 to 25 mg/kg,
the animals were orally intubated and ventilated. A slow infusion of
1.25 ␮L/mL Fentanyl (Pharmalink AB) in Ringer-acetate was started
and adjusted as needed. 10 000 IU Heparin were given. An 8F
introducer (Onset, Cordis Co) was inserted into the right internal
jugular vein, and an 8F AL 1.0 was inserted into the coronary sinus.
A venography was obtained using contrast medium Omnipaque 300
ATP was measured by ATP Bioluminescent assay kit (Sigma)
according to the supplier’s instructions.
Measurement of cAMP
Washed RBCs were brought to a 20% hematocrit in PSS buffer and
incubated with different pharmacological agents for 10 minutes if not
otherwise indicated. After the incubation, the samples were treated as
described.12 The concentration of cAMP was determined in duplicate
using the cAMP EIA System (Amersham Biosciences)
Quantitative TaqMan Real-Time Reverse
Transcription Polymerase Chain Reaction Assay
Total cellular RNAs were extracted as described.4,5,18,22,23 The
TaqMan Reverse Transcription (RT) Reagents Kit was used to
transcribe mRNA into cDNA. Real-time polymerase chain reaction
(PCR) was performed by means of a PRISM 7700 Sequence
Detector as described.4,23 Oligonucleotide primers and TaqMan
probes were designed by using Primer Express, based on sequences
from the GenBank database.4,23 Constitutively expressed glyceraldehyde phosphodehydrogenase (GAPDH) was selected as external
endogenous control genes to correct for any potential variation in
RNA loading, cDNA synthesis, or efficiency of the amplification
reaction. The threshold cycle (CT) is defined as the fractional cycle
number at which the reporter fluorescence reaches a certain level (set
to 10 times the SD of the baseline). The target gene normalized to
GAPDH was expressed as ⌬CT (CT of target gene minus CT of
GAPDH). The normalized value is given by the equation 2-⌬Ct. To
further verify the specificity of the PCR assays, the PCR was
performed with non–reverse-transcribed total cellular RNA and
samples lacking the DNA template. No significant amplifications
were obtained in any of these samples (data not shown).
SDS-PAGE and Western Blot
RBC membranes24 and platelets4,5 were isolated. The resultant
pellets were dissolved in RIPA buffer and frozen at ⫺20°C.
SDS-PAGE and Western blot were performed as described.4,23
Membranes were incubated with anti-P2Y1 (Alomone) and antiP2Y12 (AstraZeneca) antibodies. P2Y1 and P2Y12 antibodies were
preabsorbed with specific antigen peptides to examine specificity.
Wang et al
P2Y13 Receptor Inhibition of ATP Release From RBCs
191
The anti-P2Y1 antibody was also tested in transfected astrocytoma
cells (hP2Y1-1321N1), indicating specificity of the antibody.22 Our
previous studies of tissues known to express P2Y1 receptors (platelets and endothelium) have also shown 180-kDa bands.4,22,23 The
anti-P2Y12 antibodies were also tested in P2Y12 transfected cells and
shown to represent specific binding compared with nontransfected
cells by AstraZeneca, Sweden. Then membranes were reprobed with
the anti-GAPDH antibody (Chemicon) as control.22
Statistical Methods
Data are expressed as mean⫾SEM. n indicates the number of
subjects tested. Statistical analysis of the normalized CT values (⌬CT)
was performed with a one-way ANOVA, followed by a multiple
comparison post test (Tukey’s test).4,22,23 For other comparisons, the
Student t test was used. Differences were considered significant at
*P⬍0.05, **P⬍0.01, and ***P⬍0.001 (two-tailed test), compared
with control if not indicated.
Results
mRNA Quantification in RBCs and Reticulocytes
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In RBCs, P2Y13 was the most abundantly expressed mRNA
(P⬎0.0001, compared with other P2 receptors), whereas
P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2X1, P2X4, and P2X7
had extremely low expression levels (Figure 1A). The expression pattern was strikingly different compared with our
previous mRNA quantifications of P2 receptors in vascular
smooth muscle cells, endothelial cells,20 platelets,4 monocytes, lymphocytes, and CD34⫹ stem and progenitor cells.18
To illustrate expression of the P2 receptors relative to each
other, the GAPDH was used as endogenous control gene, ie,
the other receptors were expressed as a ratio of the GAPDH.
For verifying the mRNA expressions, ADP receptors in reticulocytes were also detected and the P2Y13 receptor was expressed
at a strikingly higher level than the other known ADP receptors
(P2Y1 and P2Y12) (Figure 1B). A preparation of reticulocytes
(purity ⬎95%) was also isolated using FACS (cells from a
peripheral blood sample were labeled with FITC- and PEconjugated monoclonal antibodies against surface markers
CD71 and glycophorin A, and double positive cells subsequently
sorted directly to PCR tubes for real-time quantitative PCR). In
this sample, P2Y13 had markedly higher levels than P2Y1 (2.1%)
and P2Y12 (2.8% in % of P2Y13 100%).
Western Blotting for ADP Receptors in RBCs
P2Y13 is one of three ADP receptors in man; the others are
P2Y1 and P2Y12. To confirm expression on the protein level,
Western blotting analysis was performed. Unfortunately, no
antibodies against the P2Y13 receptor are available. However,
the other ADP receptor subtypes P2Y1 and P2Y12 were not
expressed in erythrocytes, yet expressed in platelets as expected.
GAPDH was expressed at similar levels in erythrocytes and
platelets (Figure 2A and 2B). This indicates that the effects of
ADP on erythrocytes are mediated by P2Y13 receptors.
Regulation of ATP Release From RBCs
In the present assay, baseline ATP levels outside erythrocytes
were 0.76⫾0.12 ␮mol/L, n⫽12. Incubation of RBCs with the
more stable ADP analogue 2-MeSADP, dose-dependently
reduced extracellular ATP concentrations with a maximum
reduction of 48% after 10 minutes (Figure 3A).
During the incubation of intact RBCs, the ATP levels were
unaltered over time, indicating that a constant release of ATP
Figure 2. Lack of protein expression of P2Y1 and P2Y12 receptors in RBCs. Western blotting analysis was performed with an
anti-P2Y1 antibody (A) or an anti-P2Y12 antibody (B). Membranes
were reprobed with the anti-GAPDH antibody as control. Experiments were repeated at least three times and the results were
highly reproducible. Control experiments with antigenic peptides
(AP) of each receptor are shown.
from RBCs is necessary for the maintenance of extracellular
ATP levels (Figure 3D). This balance was affected by
2-MeSADP (1 ␮mol/L), causing a reduction in extracellular
ATP concentrations, which were reduced to 83% after 5
minutes and to 75% after 10 minutes (Figure 3D).
There is no selective P2Y13 antagonist, but AR-C67085, an
antagonist of P2Y12 receptors in nanomolar concentrations, is
also an antagonist of P2Y13 receptors in micromolar concentration.25 In the presence of 30 ␮mol/L of AR-C67085 to
block both P2Y13 and P2Y12 receptors, the ATP reducing
effect of 2-MeSADP was abolished (Figure 3C). In contrast,
no effect of AR-C67085 was seen in concentrations where it
only blocks P2Y12 receptors (0.1 ␮mol/L). MRS2179, is an
antagonist of the P2Y1 receptor, another ADP receptor known
to be present on the endothelium and on platelets.26 MRS2179
did not have any effect on the RBCs and did not block the
inhibition of ATP release by 2-MeSADP. Furthermore,
30 ␮mol/L of AR-C67085 raised extracellular ATP levels
(Figure 3B) and the effect was additive to adrenaline (6B).
After the decrease of extracellular ATP levels with
2-MeSADP, adrenaline caused a stepwise increase of ATP
levels (Figure 4A). Adrenaline increases ATP release and this
effect was inhibited by 2-MeSADP (Figure 4B). The
␤-adrenergic antagonist propranolol (1 ␮mol/L) but not the
␣-adrenergic antagonist phentolamine (100 ␮mol/L), blocked
the effect of adrenaline (Figure 6B).
Regulation of Plasma ATP Concentrations in an
In Vivo Pig Model
After intracoronary injection of 2-MeSADP, samples were
obtained from the venous effluent of the heart in the coronary
sinus with a microdialysis probe. Analysis showed decreased
concentrations of plasma ATP after intracoronary injection of
2-MeSADP (Figure 4C).
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Figure 3. Effects of agonists and antagonists of ADP receptors on ATP release
from RBCs in vitro. A, ADP receptor agonist 2-MeSADP dose-dependently inhibited the release of ATP (Emax⫽48.6⫾2.4%,
EC50⫽9.15⫻10⫺7); n⫽4. B, AR-C67085
(30 ␮mol/L), as an antagonist of both
P2Y13 and P2Y12 receptors, increased
ATP release. AR-C67085 (0.1 ␮mol/L), as
a selective P2Y12 receptor, had no effect.
P2Y1 receptor antagonist MRS2179
(10 ␮mol/L) had no effect. n⫽6. C,
2-MeSADP–mediated inhibition of ATP
release was antagonized by 30 ␮mol/L of
AR-C67085, but not by 0.1 ␮mol/L of
AR-C67085 or 10 ␮mol/L of MRS2179;
n⫽8. D, Incubation of RBCs with
2-MeSADP (1 ␮mol/L) reduced the extracellular ATP concentrations, but no
changes occurred without 2-MeSADP
(PSS buffer group); n⫽6. E, During incubation of RBC ghosts with ATP (1 ␮mol/
L), the ATP levels were reduced rapidly
(t1/2⫽2.3 minutes, created by one phase
exponential decay, R2⫽0.9997), but no
changes occurred without RBC ghosts
(only buffer). Degradation rate was not
changed by coincubation with
2-MeSADP or ARC67085 (P⬎0.05).
There was a significant difference
between “no RBC ghosts” and others
(P⬍0.01); n⫽4 to 8.
Effect of Ectonucleotidases in RBCs
When a certain amount of ATP (1 ␮mol/L) was added to the
RBC ghost solution, the ATP level was reduced to 22% after
5 minutes and to 9% after 10 minutes (Figure 3E). No
changes were found without RBC ghosts. The rate of ATP
degradation was not changed by coincubation with
2-MeSADP or AR-C67085 (Figure 3E).
Participation of cAMP-Regulating ATP Release
Incubation of erythrocytes with 2-MeSADP decreased intracellular cAMP levels (Figure 5A) and this change was
dose-dependent (Figure 5C). Adrenaline caused an increase
of cAMP levels both alone and after a decrease of cAMP
levels by 2-MeSADP (Figure 5B). The ␤-adrenergic antagonist propranolol (1 ␮mol/L) but not the ␣-adrenergic antag-
Figure 4. Effects of adrenaline on ATP
release in vitro and effects of agonists of
ADP receptors on plasma ATP levels in
vivo. A, Decrease in extracellular ATP
levels by 2-MeSADP (1 ␮mol/L) was
reversed by the addition of adrenaline
(1 ␮mol/L); n⫽5. B, Increase in extracellular ATP levels by adrenaline (1 ␮mol/L)
was reversed by 2-MeSADP (1 ␮mol/L);
n⫽6. C, Effects of 2-MeSADP on plasma
ATP concentrations in an in vivo pig
model. Bar graphs show plasma ATP
concentrations sampled from the venous
outflow of the heart with a microdialysis
probe in the coronary sinus 5 to 10 minutes after intracoronary injection of
2-MeSADP (10 ␮mol/L, 2 mL); n⫽8.
Wang et al
P2Y13 Receptor Inhibition of ATP Release From RBCs
193
40% RBCs, containing a 1000-fold higher ATP concentration
than plasma (mmol/L versus ␮mol/L), even a minor release
of ATP from the high intracellular concentrations could have
major circulatory effects. A negative system may therefore be
of great physiological importance to mitigate ATP release.
Furthermore, the previous view of the RBC as a “passive bag
that transports oxygen” is challenged. It now turns out that it
releases ATP in response to stimuli and as with most
important signaling systems, it has a negative feedback
system to terminate its release.
Selective Expression of the P2Y13 Receptor
in RBCs
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Figure 5. Effects of agonists and antagonists of ADP receptor
on cAMP levels in RBCs. A, 2-MeSADP (1 ␮mol/L) decreased
intracellular cAMP levels. P2Y13 receptor antagonist AR-C67085
(30 ␮mol/L) increased cAMP levels per se and blocked the
effect of 2-MeSADP; n⫽4. B, Decrease in intracellular cAMP levels by 2-MeSADP was reversed by adrenaline (1 ␮mol/L).
Adrenaline alone caused an increase of cAMP levels (n⫽4, A
and B were incubated for 10 minutes). C, 2-MeSADP dosedependently decreased intracellular cAMP levels for 20 minutes;
n⫽4. D, ATP and ADP degrading enzyme apyrase (1 U/mL,
grade I; Sigma) increased cAMP levels per se; n⫽10.
onist phentolamine (100 ␮mol/L) blocked the effect of
adrenaline on cAMP (Figure 6A). Incubation of erythrocytes
with 30 ␮mol/L of AR-C67085 increased intracellular cAMP
levels (Figure 5A) and the effect was additive to adrenaline
(Figure 6A). The ATP degrading enzyme apyrase was used to
eliminate ATP and ADP from the media, which lead to
markedly increased cAMP levels (Figure 5D).
Discussion
The main finding of the study is that ADP activates a negative
feedback pathway for ATP release from human RBCs via
P2Y13 receptors. Because blood consists of approximately
We have recently developed a sensitive quantitative mRNA
assay for most of the P2 receptors and demonstrated that it
was possible to quantify mRNA levels in platelets even
though they only contain minute levels of mRNA.4,22,23 The
RBC is also an anucleated cell with very low mRNA levels.
However, it was possible to extract and quantify mRNA for
P2 receptors. Surprisingly, the P2Y13 receptor was found to be
by far the highest expressed P2 receptor in RBCs. To verify
the mRNA expressions, ADP receptors in reticulocytes were
detected. It was found that the P2Y13 receptor had markedly
higher expression than P2Y1 and P2Y12 receptors. A role for
the P2Y13 receptor has not been clearly shown in any cell type
and its distribution has been assumed to be mainly in cells of
the immune system based on the observation of high mRNA
levels in the spleen and bone marrow.27,28 In light of our
findings, this mRNA is more likely to represent RBC mRNA.
It is unlikely that our finding should represent contamination
from other circulating cells because platelets do not express
P2Y13, and analysis of buffy coat mRNA revealed a completely different P2 receptor expression profile.4
In Western blotting, the other ADP receptor subtypes P2Y1
and P2Y12 were not expressed in RBCs, but in platelets as
expected. This gives support on the protein level that the
effects of ADP on RBCs are mediated by P2Y13 receptors.
The P2Y13 receptor is coupled to Gi proteins mediating
inhibition of intracellular cAMP levels.27 Previous reports
have shown that increased cAMP levels in RBCs lead to
release of ATP.12 It is therefore possible that ADP acting on
P2Y13 receptors could represent a negative feedback mechanism for ATP release by inhibition of intracellular cAMP
levels.
ATP Release From RBCs Affected by P2Y13
Receptor Activation
To study ATP release from RBCs, we first tried to separate
the extracellular buffer solution from RBCs by centrifugation
or dilution as suggested in previous studies.12,13 The problem
is that large amounts of ATP are released on centrifugation
and handling of samples and this caused a large variation of
the results. We therefore used a small microdialysis probe
with an external inlet and outlet, placed in the RBC solution.
This resulted in stable values with low variability. Furthermore, it made it possible to perform continuous registration of
extracellular ATP levels during subsequent in vitro additions
of different compounds.
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Circulation Research
February 4, 2005
Figure 6. Adrenaline increased intracellular cAMP levels (A, incubated for 20 minutes) and ATP release (B). Both were
potentiated by AR-C67085. ␤-Adrenergic
antagonist propranolol (1 ␮mol/L), but
not the ␣-adrenergic antagonist phentolamine (100 ␮mol/L), blocked the effect of
adrenaline (n⫽4).
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Incubation of RBCs with the stable ADP analogue
2-MeSADP, dose-dependently reduced extracellular ATP
concentrations. We interpret this as a reduced ATP release
from RBCs. A constant release of ATP is necessary to
maintain extracellular levels because of the rapid degradation
of extracellular ATP by ectonucleotidases on the RBC membrane.29 The RBC ghosts are deprived of their intracellular
ATP thus lacking the possibility to release ATP and can
therefore only alter the extracellular ATP levels by degradation via membrane-bound ectonucleotidases. When a certain
amount of ATP was added to the RBC ghost solution, the
ATP level was reduced (Figure 3D and 3E). No changes were
found without RBC ghosts, demonstrating rapid ATP degradation by RBC ectonucleotidases. The rate of ATP degradation was not changed by coincubation with 2-MeSADP or
AR-C67085, which indicates that the changes in extracellular
ATP levels were not caused by a direct effect of 2-MeSADP
or AR-C67085 on ectonucleotidases. During the incubation
of intact RBCs, the ATP levels were unaltered over time,
indicating that a constant release of ATP from RBCs is
necessary for the maintenance of extracellular ATP levels.
This balance is affected by 2-MeSADP, causing a reduction
in extracellular ATP concentrations. Thus, the degradation of
ATP by ectonucleotidases and the release of ATP from RBCs
appear to be key points in the regulation of the extracellular
ATP levels surrounding RBCs.
There is no selective P2Y13 antagonist, but AR-C67085 (an
antagonist of P2Y12 receptors in nanomolar concentrations) is
also an antagonist of P2Y13 receptors in micromolar concentrations.25 In the presence of 30 ␮mol/L of AR-C67085 to block
both P2Y13 and P2Y12 receptors, the ATP reducing effect of
2-MeSADP was abolished. In contrast, no effect of AR-C67085
was seen in concentrations where it only blocks P2Y12 receptors
(0.1 ␮mol/L). MRS2179, an antagonist of the P2Y1 receptor,26 did
not have any effect on the RBCs and did not block the inhibition of
ATP release by 2-MeSADP. This indicates that among the ADP
receptors, P2Y1, P2Y12, and P2Y13, only the P2Y13 receptor contributes to the regulation of ATP release in RBCs.
Interestingly, 30 ␮mol/L of AR-C67085 raised extracellular ATP levels per se, indicating antagonism of endogenous
extracellular ADP. Thus, the extracellular ATP level is
constantly regulated by ADP. A rapid loop must exist in
which ATP is secreted, subsequentially degraded to ADP,
AMP, and adenosine followed by intracellular reuptake and
phosphorylation back to ATP. The extracellular concentration
of ATP is dependent on its release from RBCs. The release of
ATP may be controlled by negative feedback inhibition mediated by ADP acting on the P2Y13 receptor. Although less potent
as an agonist of P2Y13 compared with ADP, ATP may have an
effect in high concentrations and inhibit its own release.27,28
Adrenaline increases ATP release and 2-MeSADP inhibits
this effect. Adrenaline stimulates ␤-adrenergic receptors on
the RBC membrane that increase intracellular cAMP by
coupling to Gs.24 After decrease of extracellular ATP levels
with 2-MeSADP, adrenaline increased ATP levels. This
indicates that the external ATP levels could be both raised or
lowered by receptor agonists.
In an in vivo pig model, plasma ATP concentrations were
decreased after injecting 2-MeSADP, indicating that
2-MeSADP could regulate plasma ATP concentrations by stimulation of P2Y13 receptors on RBCs in vivo. These findings may
be affected by other mechanisms. Vasodilatation by 2-MeSADP
may increase the oxygenation rate of hemoglobin and thereby
reduce ATP release from RBCs. On the other hand, platelet
activation by 2-MeSADP may cause ATP release.
Participation of cAMP in the P2Y13–Activated
Signal Transduction Pathway That Regulates
ATP Release
The P2Y13 receptor is coupled to Gi proteins mediating
inhibition of intracellular cAMP levels.27 Incubation of RBCs
Wang et al
P2Y13 Receptor Inhibition of ATP Release From RBCs
195
Figure 7. Schematic picture of a negative feedback pathway of ATP release
from RBCs mediated by ADP activation
of P2Y13 receptors (see Discussion).
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with 2-MeSADP decreased intracellular cAMP levels. The
inhibition of cAMP by 2-MeSADP could be reversed by
incubation of RBCs with adrenaline. Incubation of RBCs
with 30 ␮mol/L of AR-C67085 not only resulted in ATP
release, but also increased intracellular cAMP levels per se.
This indicates that AR-C67085 can block the P2Y13 activation
by endogenous ADP. To further test if endogenous ADP
mediates a constant inhibitory effect on cAMP levels, the
ATP degrading enzyme apyrase was used to eliminate ATP
and ADP from the media. This led to a marked increase in
cAMP levels. Thus, endogenous ADP may control ATP
levels outside the RBC by inhibition of intracellular cAMP
levels via activation of P2Y13 receptors (Figure 7). It is an
interesting parallel that the ␣2-receptor mediated negative
feedback pathway of noradrenaline release in nerves also acts
via Gi and cAMP inhibition. However, a recent article30
demonstrated that direct Gi-stimulation with mastoparan increased ATP release. In contrast to stimulation of Gi via the
P2Y13 receptor (and in contrast to the usual cAMP inhibition
of Gi proteins), they found increased cAMP levels in response
to mastoparan. Thus, the link between cAMP levels and ATP
release is similar, but differences depending on Gi protein
activation or subtypes may exist.
Several mechanisms of ATP release outside the cell have
been suggested, including ATP binding cassette (ABC) transporters, connexin hemichannels, mitochondrial porins, and
stretch activated channels (See Lazarowski et al31 for review).
In RBCs, evidence suggests ATP release through an anion
channel, possibly the cystic fibrosis transmembrane conductance regulator (CFTR), which is known to be regulated by
cAMP.32
A Negative Feedback Pathway for ATP Release
From Human RBCs by P2Y13 Receptors
Our finding of a negative feedback system for ADP release
fits well with previous evidence of ATP as an extracellular
transmitter, as first proposed by Burnstock,1 and it is possible
that this negative feedback system plays an important role in
the control of peripheral circulation. In underperfused organs,
hypoxia stimulates an increase in intracellular cAMP, which
leads to the release of ATP to the blood plasma. This increase
of cAMP could be enhanced by adrenaline, that is in itself
elevated during hypoxia.33 Extracellular ATP stimulates endothelial cells to release dilatory factors such as NO, mediating vasodilatation and improved oxygen delivery to the
organ. To shut down the ATP release on the venous side
(where the blood is still deoxygenated), and to avoid unnecessary ATP release, the degradation product ADP acting on
P2Y13 receptors could inhibit cAMP and decrease ATP
release from RBCs. ATP and ADP are then degraded by
ectonucleotidases to adenosine, which is quickly taken up in
the RBCs where it can be recycled to ATP by the glycolytic
pathway. The described negative feedback pathway may be
important to avoid high extracellular concentrations of ATP.
At levels above 100 ␮mol/L, ATP concentrations may exceed
the catalytic capacity of ectonucleotidases and, could in fact,
stimulate ATP release by increasing permeability of the
RBC.34 At high concentrations of ATP, a self sustaining
process may thus be instigated, which may contribute to the
irreversible stage of circulatory shock that can develop
rapidly in severely ill patients.
According to one study, raising the plasma ATP levels by
expanding the intracellular ATP concentrations of the RBC
pool results in anticancer activities.35 It could be therefore be
speculated that a P2Y13 receptor antagonist, in addition to
infusions of ATP, could be of value in the treatment of cancer
by increasing ATP delivery to the tissues.
It is possible that in extreme conditions such as circulatory
or septic shock with acidosis and hypoxia, high ATP levels
could be deleterious, leading to a drop in blood pressure.
However, in most situations increased ATP levels ought to
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February 4, 2005
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have positive effects by increasing tissue perfusion. ATP
stimulates NO release from the endothelium and might be
beneficial in situations of endothelium dysfunction, such as
atherosclerosis, congestive heart failure, and diabetes. ATP,
in contrast to adenosine, has recently been demonstrated to be
sympatholytic, ie, counteracts sympathetic vasoconstriction.36 Increased ATP levels may therefore reduce hypertension and decrease peripheral vascular resistance in, for
example, congestive heart failure. Patients with primary
pulmonary hypertension have an impaired ATP release from
RBCs that may be a pathogenic factor for the etiology of their
pulmonary hypertension.13 Thus, a P2Y13 receptor antagonist
that increases ATP levels extracellularly by inhibiting the
negative feedback pathway may be valuable in congestive
heart failure, hypertension, pulmonary hypertension, atherosclerosis, and diabetes.
In conclusion, we have shown that the ATP degradation
product ADP inhibits ATP release by acting on the RBC
P2Y13 receptor. This negative feedback system could be
important in the control of plasma ATP levels and tissue
circulation.
Acknowledgments
The study was supported by the Swedish Heart and Lung, Bergqvist,
Wiberg, Bergwall, Zoegas, Netpharma, and Nilsson Foundations and
Swedish Medical Research Council 13130. We thank Randy S.
Sprague for methodological advice and AstraZeneca for the gift of
AR-C67085 and the P2Y12 antibody.
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ADP Acting on P2Y13 Receptors Is a Negative Feedback Pathway for ATP Release From
Human Red Blood Cells
Lingwei Wang, Göran Olivecrona, Matthias Götberg, Martin L. Olsson, Maria Sörhede Winzell
and David Erlinge
Circ Res. 2005;96:189-196; originally published online December 16, 2004;
doi: 10.1161/01.RES.0000153670.07559.E4
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