401
Purinergic Axis in Cardiac Blood Vessels
Agonist-Mediated Release of ATP From
Cardiac Endothelial Cells
Shumei Yang, Dennis J. Cheek, David P. Westfall, Iain L.O. Buxton
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Abstract Purified endothelial cells isolated from guinea pig
hearts by enzymatic perfusion were grown in monolayer
culture and used to test the ability of a variety of vasoactive
agents to stimulate ATP release from these cells. Stimulation
of endothelial cells with the peptide agonist bradykinin (1
nmol/L), acetylcholine (1 ,umol/L), serotonin (1 ,umol/L), or
adenosine 5Y-diphosphate (10 1mol/L) resulted in the rapid
appearance of ATP in the incubation medium determined
with the firefly luciferase assay for ATP. Addition of antagonists for muscarinic (atropine, 0.1 gmol/L) and purinergic
(suramin, 100 ,umol/L; reactive blue-2, 100 ,umol/L) receptors
suggested that ATP release from these cells was receptormediated. Bradykinin-induced release of ATP was rapid (peak
<30 seconds at 3 nmol/L bradykinin), dose-dependent (EC50,
0.18 nmol/L), and diminished with repeated administration of
agonist. Desensitization to bradykinin also affected the ability
of acetylcholine to induce release and was reversible when
cells were returned to growth conditions for short periods.
Measurement of released adenyl purines as their fluorescent
N6-ethenopurine derivatives by high-performance liquid chro-
matography revealed the origin of the purine released to be
ATP and confirmed its rapid dephosphorylation. Addition of
the purine nucleotide analogues 2-methylthio-ATP (2-methylS-ATP), ADP, and 18y-methylene ATP to endothelial cell
cultures resulted in a dose-dependent increase in the appearance of ATP measured in the medium bathing the cells at 30
seconds, suggesting the presence of ATP-induced ATP release. The rank-order of potency for ATP-induced ATP release (2-methyl-S-ATP>ADP> >f3y-methylene ATP-aj8methylene ATP) and its inhibition by suramin and reactive
blue-2 suggest a role for the P2y receptor in mediating the ATP
response. Incubation of cells with ['H]adenine to label the
cellular ATP pool available for release confirmed that addition
of ADP stimulated [3H]ATP release from the cells into the
medium in a manner blocked by addition of suramin. Our data
are discussed in the light of evidence for a purinergic axis in
cardiac blood vessels. (Circ Res. 1994;74:401-407.)
Key Words * ATP * ADP * adenosine * acetylcholine
* blood vessels * bradykinin * cardiac endothelial cells d
endothelial factors * purinergic receptors
T he autocrine and paracrine actions of adenyl
purines in the cardiovascular system have been
well recognized since the early description of
the hypotensive and bradycardic actions of adenosine by
Drury and his colleagues more than 60 years ago.12
Indeed, effects of adenosine acting through extracellularly directed receptors of the A1 and the A2 subtype of
the P1 subclass of purinergic receptor have been described in all organ systems and include regulation of
The acceptance of a role for the extracellular actions
of adenosine notwithstanding, there has been some
resistance to the notion that ATP is also released from
cells to act extracellularly as ATP per se. Despite the
existence of dogma predicting that healthy cells should
not release their energy currency, numerous reports
appearing over the past two decades have now firmly
established a number of diverse roles of extracellular
ATP, including neurotransmission,12 regulation of endothelium-derived relaxing factor release in blood vessels,13 and stimulation of cell growth.14"5 The origins of
extracellular ATP have also been suggested. There is
strong evidence in the peripheral nervous system that
ATP is coreleased along with norepinephrine from
sympathetic and with acetylcholine from parasympathetic nerves (for review see Reference 16). A role for
ATP as the principal neurotransmitter in nonadrenergic
noncholinergic nerves has also been proposed,17"18 and
evidence that ATP is a neurotransmitter in the central
nervous system19 has recently emerged.
In the heart, the exact mechanism underlying the
moment-to-moment regulation of cardiac blood flow by
local mediators is not fully understood but appears to
involve ATP.2021 Although the notion that endothelial
cells can release ATP is not new,22 little has been done
to describe the regulation of endothelial cell purine
release since these earlier studies. Recent reports2324
suggest that shear stress can release ATP from endothelial cells and that hypoxia may alter the ratio of ATP
release to that of other mediators such as endothelin.
smooth muscle contraction, in which adenosine is
known to relax some smooth muscles, particularly those
of some blood vessels (for review see Reference 3), and
contract others.45 The vascular origins of extracellular
adenosine have also been described.6 Adenosine is
released from the placenta7 and affects the fetus,7'8 and
it is thought to be produced from the breakdown of
ATP, as in the case of the presynaptic actions of
adenosine produced after the breakdown of ATP released from nerve terminals along with neurotransmitters such as norepinephrine.9 In addition, adenosine is
known to be released from cardiac muscle cells that
become ischemic10 and may be present in the bloodstream after metabolism of purine nucleotides released
from platelets during the release reaction."
Received September 10, 1993; accepted December 13, 1993.
From the Department of Pharmacology, University of Nevada
School of Medicine, Reno, Nev.
Correspondence to Iain L.O. Buxton, University of Nevada,
Reno, School of Medicine, Department of Pharmacology/318,
Reno, NV 89557-0046.
402
Circulation Research Vol 74, No 3 March 1994
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The pharmacological regulation of ATP release and
rigorous studies of the origin of extracellular ATP,
however, have not been established.
The normal vascular response to released purine
nucleotide may be either vasodilation or vasoconstriction,21 25 depending on the blood vessel and the site of
nucleotide release. Direct application of ATP, for example, relaxes both guinea pig and rabbit coronary
artery segments in the presence or absence of the
endothelium,26 suggesting the presence of P2y receptors
on coronary smooth muscle. The ability of coronary
artery to relax to ATP in the absence of endothelium
appears to be a uniform response across species, if not
an entirely unique response. Thus, a common finding in
numerous blood vessels lacking endothelium is contraction1621 via actions of ATP at a P2x receptor, whereas
some vessels, once contracted, can relax to addition of
ATP, suggesting the presence of both P2x and P2y
receptors on the vascular smooth muscle.25,27,28
In addition to a role for luminal ATP in mediating
blood vessel tone, ATP released into the bloodstream is
thought to serve an antithrombogenic role, since it is an
antagonist of ADP at the platelet P2t receptor.20 Furthermore, metabolism of purine nucleotides to adenosine downstream from the site of ATP release by the
well-characterized ecto-5'-nucleotidase activity on the
endothelial cell29 would also be expected to inhibit
further platelet activity30 and promote dilation of arterioles. Indeed, adenosine is thought to salvage at-risk
myocardium in an affected region by increasing local
blood supply.'0 We suggest that the purinergic axis in
the blood vessel, including ATP release and metabolism
by endothelium, may serve to regulate blood flow in
nonpathological states both directly and by virtue of the
release of additional endothelial mediators such as
nitric oxide. In this report we build on the evidence for
endothelial release of ATP provided by ourselves3' and
others22~24 and describe studies designed to explore
agonist-mediated release of ATP from cardiac endothelial cells.
Experimental Procedures
Cardiac Endothelial Cell Isolation and Culture
Cardiac endothelial cells were isolated as previously described.3' Briefly, hearts from young adult guinea pigs of either
sex were rapidly removed after CO2 anesthesia and perfused via
the aorta in a nonrecirculating fashion with cold (10°C) Krebs
buffer containing no added Ca21 followed by collagenase (1.5
mg/mL) and trypsin (1 mg/mL) diluted in the same buffer at
32°C. Cells released during the perfusion were collected from
the perfusate (250g, 10 minutes), purified by centrifugation
(2000g 15 minutes; 1.04 g/L) on preformed 50% isotonic
Percoll (Pharmacia, Piscataway, NJ) gradients (25 00g, 50
minutes), resuspended in Dulbecco's modified Eagle's medium
with 10% fetal calf serum, and seeded in plastic 12-well culture
dishes (3.8 cm2 per well) precoated with ProNectin F. ProNectin
F was dissolved with the diluent provided (500 ,ug/mL), and 5
/L was added directly to coat the culture surface. After 90
minutes the dishes were rinsed with sterile phosphate-buffered
saline and stored dry until use. Cultures were maintained in a
standard tissue culture incubator at 95% humidity gassed with
95% 02/5% C02. The culture medium was changed 4 to 6 hours
after plating and again at 24 hours. When confluent (day 5 to 7),
endothelial cells grown in multiwell plates were used in release
experiments. After experiments, cell number was determined in
each well by removing cells with trypsin and counting with a
hemocytometer.
Proof of origin of the isolated cells in our cultures was
routinely performed by assaying for clotting factor VIII antigen with a monoclonal antibody conjugated to fluorescein
isothiocyanate (Atlantic Antibodies, Scarborough, Me) followed by visual inspection with a fluorescent microscope.
Nonendothelial cells (NIH 3T3 cells) served as a negative
control.
Measurement of Adenyl Purine Release by
High-Performance Liquid Chromatography
Cell cultures were removed from the incubator and washed
two times with warm (37°C) oxygenated incubation buffer of
the following composition (in mmol/L): NaCI 118, KCl 4.7,
KH2PO4 0.6, NaH2P(4 0.6, MgCl2 1.2, dextrose 20, CaCl2 1,
HEPES 25; pH 7.4. Culture plates were then placed on a
warming pad (37°C) and equilibrated for 30 minutes, during
which time the buffer (1.0 mL) was changed again. Experiments were initiated 15 minutes later by withdrawing 500 ,L
and replacing it with the same buffer (37°C) to which drug had
been added at twice the desired final concentration. Samples
(500 ,uL) were withdrawn at the desired time, immediately
placed on ice, and spiked with EDTA (3 mmol/L) to chelate
Ca 2+ and Mg 2+. This sample was concentrated by freezing in
liquid nitrogen and lyophilizing to dryness. Samples were then
resuspended in water and assayed for adenyl purine content by
C18 reverse-phase high-performance liquid chromatography
(HPLC) with precolumn derivitization (800C, 10 minutes) as
we have previously described,32 in which process chloroacetaldehyde is used to form the fluorescent 1,N6-ethenopurine (eg,
E-ATP), which is then separated by a linear 50 mmol/L KHP04
(pH 6.0)/methanol gradient (0 to 30% methanol) on C18
reverse-phase HPLC for 20 minutes (0.75 mL/min) and returned to starting conditions over the following 10 minutes.
The fluorescent e-purines were detected by excitation at 227
nm and emission at 416 nm. Under these conditions e-adenosine and the e-nucleotides, E-ATP, c-ADP, and e-AMP, are
well resolved at 13, 4.5, 5.6, and 11 minutes, respectively. The
limit of detection of e-purines when generated from a series of
purine standards is 0.75 fmol for adenosine and 10 fmol for
each of the nucleotides. The production of E-purines from
purine standards, which was 98% complete at 10 minutes, was
used to generate a standard curve from which sample e-purine
was calculated. Data are expressed as pmol e-purine/106 cells.
Measurement of ATP Release
by Chemiluminescence
In experiments in which ATP congeners such as /,y-methylene ATP were added as agonists to study adenine nucleotide-induced ATP release, an assay using the luciferin-luciferase reaction was employed. In this procedure, only ATP is
readily detected, since the enzymatic reaction of firefly luciferase to oxidize luciferin is specific for natural ATP. Controls showed that ADP did not support the same signal in the
assay; thus, ADP was controlled for with an ADP blank. The
assay uses an ATP bioluminescence assay kit (Sigma Chemical,
St Louis, Mo) containing ATP assay mixtures and ATP
standards; ATP assay mix dilution buffer contains 2 mmol/L
Tris-HCl (pH 7.8), 200 ,umol/L EDTA, 1 mmol/L MgSO4, and
0.1 % bovine serum albumin. Samples, diluted as necessary,
are injected (100 ,uL) into 100 ,L assay mixture, and recordings are made in a Turner luminometer over a 10-second
period. The response in a given sample or standard is quantified as area under the peak of the response and averaged for
triplicate determinations. Data are expressed as moles ATP
based on standards determined under the same conditions
with each experiment. Control experiments revealed that
addition of suramin (100 ,amol/L) to Krebs buffer blocked the
chemiluminescence signal to ATP standards in the luciferinluciferase assay. Filtration of the suramin/ATP sample
through a 0.45-,m nylon HPLC sample preparation filter
(Cole Palmer, Chicago, Ill) before introduction into the assay
Yang et al Purinergic Axis in Cardiac Vessels
removed the blockade of luciferase, resulting in ATP standard
curves similar to those generated in the absence of suramin.
Although reactive blue-2 did not act as an inhibitor in the
luminometer assay, insoluble material was present in solution.
Thus, both putative P2 receptor antagonists, suramin and
reactive blue-2, were routinely filtered before use. Although
cells exposed to suramin and reactive blue-2 were not used
further in experiments, neither agent appeared to damage cells
as determined by visual inspection for exclusion of vital dye in
cells washed to remove inhibitor and returned to growth
conditions for 15 minutes.
Radioactive Measurement of ATP Release
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Confluent cultures of endothelial cells were prelabeled
overnight with [3H]adenine (100 ,uCi/mL) to label ATP pools
within the cells. [3H]Adenosine, often used to label cells, was
avoided because it is an agonist at purine receptors of the PI
subclass. Cells were washed with incubation buffer to remove
unincorporated label, and [3H]ATP release was measured
after equilibration of the cells as described above. Samples of
incubation buffer were acidified with HCIO4 (2% vol/vol),
clarified, neutralized with KHCO3, filtered, and injected onto
a strong anion-exchange HPLC column.
The labeled adenyl purines were separated by use of a
two-buffer system consisting of H20 and 2 mol/L ammonium
phosphate (NH4H2PO4) run over a strong anion-exchange
column (Whatman Partisil 5 SAX; 10 cm). Radioactivity was
detected with a flow scintillation counter ([3H] efficiency,
26%). Separation of [3H]adenyl purines began with a 10minute isocratic gradient of 100% H20, followed by a linear
gradient running from 100% H20 to 100% NH4H2PO4 over a
period of 30 minutes (flow rate, 1 mL/min). The column was
returned to its original conditions over the next 18 minutes.
Under these conditions, labeled purine standards (Dupont
NEN, Boston, Mass) were well resolved, and their elution
times were used to identify the presence of these compounds
in samples applied to the column. Data are expressed as area
under the peak in counts per minute.
Drugs
Acetylcholine, bradykinin, serotonin, ATP, ADP, a,3-methylene ATP, ,8y-methylene ATP, EDTA, reactive blue-2
(basilen blue E-3G), and all buffers and general chemicals
were obtained from Sigma Chemical Co, St Louis, Mo. The
ATP analogue 2-methyl-S-ATP was obtained from Research
Biochemicals International, Natick, Mass. The trypanocide
and putative P2 receptor antagonist suramin, [sym-bis(m-
aminobenzoyl-m-amino-p-methylbenzoyl-1-naphthylamino4,6,8-trisulfonate)] carbamide: hexasodium salt, was a gift
from Bayer AG, Germany.
Results
In control experiments designed to test the effects of
purine nucleotide agonists and putative antagonists in
the luciferin-luciferase assay, we discovered that 100
,gmol/L suramin, often used to test the specificity of P2
receptor-mediated responses, blocked the ATP signal.
This effect of suramin was not a P2 effect, since it
occurred when suramin was added directly along with
ATP in the determination of standards (data not
shown). When the freshly prepared suramin/ATP solution was first filtered through an HPLC limit filter (0.45
,um nylon) and then added to the assay, suramin no
longer blocked the luciferin-luciferase reaction.
To assess the role of endothelial cell ATP release in
the effects of agonists known to dilate blood vessels and
that may be expected to act on endothelial cells in
vivo,33 we exposed cultured endothelial cells to several
agonists for short periods and measured the ATP
E
100
403
|
6040-
20
0
CON
ACH
5HT
ADP
BK
FIG 1. Bar graph showing that various agonists cause ATP
release from cardiac endothelial cells. Endothelial cells were
grown in primary culture in plastic dishes and stimulated with
acetylcholine (ACH, 1 ,umol/L), serotonin (5HT, 1 1rmol/L), ADP
(10 ,umol/L), or 1 nmol/L bradykinin (BK) for 2 minutes as
described in the text. ATP released into the buffer bathing the
cells was determined in the luciferin-luciferase assay at the end
of the stimulation period. ATP release, normalized to cell number, is expressed as total ATP measured after the 2-minute
incubation. Data, plotted as mean+SEM, are from duplicate
determinations in three to five experiments of cells from 12 to 20
animals. Responses to ACH were not seen in passaged cells.
CON indicates control.
content of the incubation medium using the luciferinluciferase assay. Addition of acetylcholine, bradykinin,
serotonin (5 -HT), and ADP to endothelial cell cultures
at concentrations routinely used for the study of the
action of these agonists on endothelium led to the
appearance within 2 minutes of ATP in the incubation
medium (Fig 1). A background level of ATP (Fig 1,
control) could be detected in the absence of agonist but
was independent of time before sampling, suggesting
basal release of ATP and subsequent metabolism in the
absence of agonist. ATP levels in the incubation buffer
were elevated fivefold by 1 nmol/L bradykinin. The
bradykinin-induced release of ATP was rapid and dose
dependent (Fig 2, inset), consistent with the notion that
the release measured was a result of the action of
bradykinin at the endothelial cell B2 receptor.34 The
appearance of ADP, AMP, and adenosine in the incubation buffer lagged successively behind that of ATP
and suggested that these purines were generated extracellularly after release of ATP (Fig 2). Stimulation of
cells with agonists leading to the release of ATP was not
the result of cell damage, because if returned to growth
conditions after treatment, cells continued to exclude
vital dye and could be stimulated again to release ATP.
In separate experiments, addition of 1 nmol/L bradykinin to duplicate cultures for 2 minutes (S1) followed
by 2 minutes in the absence of bradykinin and then
reintroduction of bradykinin (S2 through S4) resulted in
release that was diminished significantly only after the
third such challenge (Fig 3). This desensitization by
bradykinin also affected acetylcholine-induced release,
suggesting that the desensitization was heterologous,
involving the releasable ATP pool (Fig 3, legend). When
bradykinin-desensitized cells were returned to growth
conditions for 15 minutes, subsequent challenge with 1
nmol/L bradykinin (S5) resulted in ATP release that
was the same as cells that had not previously been
exposed to agonist (Fig 3).
Because ATP released from endothelial cells might
reasonably be expected to act as ATP (or ADP) at P2y
404
Circulation Research Vol 74, No 3 March 1994
50O
120
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Time (min)
2.
FIG
Graph showing bradykinin (BK)-stimulated ATP release
and metabolism by cardiac endothelial cells. Endothelial cells
were stimulated by addition of BK (3 nmol/L) to replicate culture
wells, and the buffer bathing the cells was removed at the times
indicated and derivitized, as described in the text, to form the
etheno (E)-purines, e-ATP (e), E-ADP (-), E-AMP (A), and e-adenosine (o). Samples were separated by reverse-phase highperformance liquid chromatography and detected by fluorescence as described. Data are the mean±SEM of duplicate
determinations in three separate experiments using cells from 12
animals and are plotted as picomoles normalized to cell number.
The release of ATP (measured as E-ATP) to increasing concentrations of BK (EC50, 0.18 nmol/L) was measured in replicate
cultures at 2 minutes and is presented in the inset as the mean
(n=2) fit to a sigmoid curve.
on the same cells or endothelial cells35 immediately adjacent to the site of release in vivo, and since
endothelial cells respond to addition of ATP by release
of vascular mediators,33 including nitric oxide,'3 we
receptors
120
--.n
c
E
100
U,
80
c-
60
e-
40
Sl
FIG 3.
32
33
34
35
Bar graph showing that ATP release from endothelial
cells desensitizes to repeated addition of agonist. Cardiac en-
dothelial cells were stimulated by addition of 1 nmol/L bradykinin
to
duplicate
culture
wells for 2
minutes
(Si)
followed
by 2
minutes in the absence of bradykinin and then reintroduction of
bradykinin for 2 minutes, and the process was repeated
through
S4).
(S2
ATP released during the stimulation period was
measured in the luminometer flash assay as described in the
text. Significant desensitization of ATP release occurred after the
third challenge with agonist (S4; P<.01). The same cells were
returned to growth conditions for 15 minutes and rechallenged
with 1 nmol/L bradykinin (S5), resulting in ATP release that was
not
signifilcantly
--
-8
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Log [agonist], (M)
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different from that seen after the original agonist
stimulation (Si). Data are the mean±SEM of duplicate determinations in three separate experiments.
FIG 4. Graph showing P2 receptor-stimulated ATP release from
cardiac endothelial cells. Cardiac endothelial cells were stimulated with increasing concentrations of purinergic receptor agonists for 30 seconds as described in the text, and ATP in the
buffer bathing the cells was measured in the luciferin-luciferase
assay. The apparent rank order of potency/efficacy, 2-methyiSATP (EC50, 0.36 prmol/L; *)>ADP (EC50, 9.3 ,lmol/L; u)>>,ymethylene ATP (A), and the lack of effect of a,8-methylene ATP
(o) suggest that these P2 agonists act at a P2y receptor to
stimulate ATP release. Data are the mean+SEM of duplicate
determinations in three experiments normalized to cell number.
reasoned that P2 receptor agonists might stimulate ATP
release (Fig 2). Study of this phenomenon is not trivial.
Any assay likely to detect ATP release by addition of
ATP or an ATP congener must be able to distinguish
the added agonist from the released purine. We have
approached this question with the knowledge from
control experiments that congeners of ATP, such as
2-methyl-S-ATP, that are P2y receptor agonists do not
react significantly in the luminometer flash assay.
Addition of 2-methyl-S-ATP to endothelial cell cultures resulted in a dose-dependent increase in the
appearance of ATP measured in the medium bathing
the cells at 30 seconds (Fig 4), as did addition of ADP.
The methyl isomer of ATP, P3y-methylene ATP, was
considerably less potent and less efficacious than
2-methyl-S-ATP or ADP at the P2y receptor, and a,methylene ATP did not stimulate release. The apparent
rank-order of potency for adenine nucleotide-evoked
ATP release, 2-methyl-S-ATP>ADP>>/,y-methylene
ATP, together with the failure of aP,-methylene ATP to
serve as an agonist, is generally consistent with the
action of these compounds at a P2y receptor (Fig 4). The
role of the P2y receptor in mediating release was further
supported by addition of P2 receptor antagonists. Addition of either suramin or reactive blue-2 resulted in a
dose-dependent inhibition of ADP-stimulated ATP release (Fig 5).
The actual release of ATP from within the endothelial cell versus extracellular metabolism of the added
purine substrate to a form capable of supporting the
oxidation of luciferin in the luminometer flash assay was
addressed by prelabeling cells with ['H]adenine. Control experiments revealed that the cellular pool of ATP
released to the addition of agonist was not in simple
equilibrium with the total cellular ATP pool since,
under our conditions, cellular ATP could be significantly labeled in as little as 2 hours, whereas labeling
the releasable pool required overnight incubation with
the radioactive precursor (data not shown). When ADP
Yang et al Purinergic Axis in Cardiac Vessels
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incubated with labeled cells ft )r 30 seconds,
['H]ATP was found in the medium (Fiig 6) along with
evidence that the ATP released is metsabolized rapidly
to AMP. In addition, the appearance of ['H]ATP in the
medium was blocked by suramin (100 ptmol/L), consistent with the notion that the cells were r eleasing ATP in
response to stimulation of a P2 receptoir.
was
Discussion
We have demonstrated that endothe lial cells in culture respond to the addition of seve ral agonists by
4.5
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X 2.7
1.8
p + SUIfif
M ADP
Basal
(c0 1
as
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o.o
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10
20
Time (min)
30
releasing ATP into the medium surrounding the cells.
This phenomenon has several features that are of
interest in understanding the role of the endothelium in
the regulation of vascular function and suggests that
adenyl purines play an important role in the myocardial
circulation.21,26,36
we have
Log [Suramin], (M)
Log [RB2], (M)
FIG 5. Graphs showing that suramin and reactive blue-2 (RB2)
inhibit ADP-induced ATP release from cardiac, endothelial cells.
Endothelial cells were stimulated by addition a)f ADP (30 gmol/L)
to the buffer bathing the cells for 30 seconds irn the absence and
presence of increasing concentrations of the p utative P2 receptor
antagonists suramin (100 ,umol/L) and RB2 (1 100 ,umol/L). Sampies were withdrawn and ATP was determine d in the luminometer assay as described in the text. Solution.s of both suramin
and RB2 were filtered as described in the text before addition to
cell cultures. Data are the mean±SEM in four experiments
performed in duplicate.
=
405
40
FIG 6. Graph showing nucleotide-induced ATFP release from cardiac endothelial cells. Endothelial cells were pr relabeled overnight
with [3H1adenine as described in the text. L abeled cells were
washed and equilibrated in buffer for 15 mir nutes, followed by
addition of fresh buffer at the start of the experim lent (see text). After
30 seconds in the presence (ADP 30 gmol/L) or
agonist, the incubation buffer was removed and lassayed bystrong
anion-exchange high-performance liquid chror natography for the
presence of labeled adenine nucleotides. AD P stimulation was
repeated in replicate dishes in the simultan eous presence of
suramin (100 1imol/L). Radioactive peaks in the order of elution are
[3H]adenine/adenosine, [3H]adenosine 5'-moni ophosphate, [3H]adenosine 5-diphosphate, and [3H]adenosine 5' '-triphosphate. Data
plotted are the radioactive tracing in counts pe brminute produced
by the radioactive flow detector and show rep)resentative results
from experiments repeated three to six times.
shown that endothelial cells release
First,
purine rapidly as ATP per se. Second, the release of
ATP was a common receptor-mediated response to
several agonists, all known to be present in the bloodstream and to cause endothelium-dependent relaxation.
Third, as expected, the ATP released was metabolized
to ADP, AMP, and adenosine. This action of the
endothelial cell predicts that ADP will be present near
the site of ATP release to act at endothelial P2y
receptors, providing for the amplification of endothelial
ATP release
..
.
and,' thus,' the
amplification of local vaso-
dilation. Since we measured adenosine formation within
a short time after the release of ATP by endothelial
cells, it is also reasonable to assume that by the time
adenosine is formed in vivo,36 it is able to exert a
vasodilatory signal in small arterioles and/or venules,
leading to increased blood flow in the region of release.
In addition to the release of ATP from endothelial
cells shown here, agonists used in this study cause the
release of additional vascular mediators, including nitric
oxide.33 This effect is also produced by ATP and ADP
acting at an endothelial cell P2 receptor.13 The dual
action of agents like 5 -HT and bradykinin to stimulate
both ATP release and nitric oxide release and the action
of ATP to stimulate nitric oxide release as well as
additional ATP release provide for an amplification of
the vasodilatory action of mediators such as bradykinin
that are rapidly metabolized in the bloodstream and
may provide for vasodilation proceeding beyond the
lifetime of the original agonist (eg, bradykinin). This
amplification and sustaining of the initial effects of an
ATP-releasing agonist are also carried out by adenosine, which has now been shown to stimulate endothelial nitric oxide release in the heart.37
Our conclusion that ADP and other ATP analogues
stimulate the release of ATP from endothelial cells via
stimulation of the P2y receptor is supported by the
action of suramin and reactive blue-2 to block ATP
release. The presence of ATP-evoked ATP release may
be a phenomenon common to P2 responses. Recent
work by Katsuragi38 demonstrated the presence of ATPevoked ATP release in smooth muscle, and Westfall et
and Sedaa et a139 have shown that ATP is released
al12
from nonneuronal sites after corelease of ATP from
sympathetic nerves.
Interpretation of the action of suramin in support of
P2 responses is complicated by the fact that the compound was an inhibitor in the ATP assay we used and
may be viewed as an inhibitor of numerous ATPutilizing enzymes rather than a specific P2 receptor
antagonist.40 This result notwithstanding, when we filtered the suramin solution, we removed the ability of
suramin to block the luciferase enzyme while retaining
the ability of suramin to antagonize P2 receptor-mediated ATP release by endothelial cells. This result should
caution the routine use of suramin as a P2 receptor
antagonist when uncharacterized solutions are used.
Furthermore, suramin has been described as both a P,x
and a P2y antagonist41 and thus may not be a selective
406
Circulation Research Vol 74, No 3 March 1994
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inhibitor of the extracellular actions of ATP. In our
experience, suramin may possess partial P2 agonist
properties, since addition of suramin to endothelial cells
in the absence of a P2 agonist results in modest but
repeatable increases in ATP release (data not shown).
Our data provide for the view that ATP, ADP, and
adenosine constitute both an autocrine and paracrine
axis in the blood vessel that may be responsible, in part,
for the moment-to-moment regulation of blood flow in
the heart. This view is further supported by the knowledge that ADP, along with 5-HT, is released in large
quantities when platelets begin to aggregate, an event
that takes place on or in intimate proximity to endothelium.42 The action of both 5-HT and ADP released from
the platelet and acting at the endothelial surface of a
normal vessel will be release of endothelial cell ATP
and blood vessel dilation. This action would appear to
be antithrombogenic, since ATP is the only known
antagonist of the platelet receptor responsible for amplification and synergism of the platelet release reaction, the P2t receptor.42 This antithrombotic action of
ATP release from the cardiac endothelial cell is carried
downstream where adenosine, formed from ATP by the
action of endothelial ectonucleotidases,29 is also an
antagonist of platelet aggregation at a P1 receptor.42
The region above the endothelium in a blood vessel is
not in immediate equilibrium with the majority of liquid
flowing in the vessel.43 The presence of this laminar flow
layer above endothelial cells suggests that the release of
ATP, its direct actions, and its metabolism take place in
a relatively cell-free environment exclusive of high concentrations of red blood cells and the significant mixing
occurring in the lumen of the vessel.44 These factors are
likely to contribute significantly to the ability of a
purinergic axis to operate in the blood vessel by maintaining the local concentration of purines available to
act at both P2 receptors as well as ectonucleoside/
nucleotide-metabolizing enzymes on the same and adjacent cells.
Actions of adenyl purines in addition to regulation of
blood vessel tone and platelet aggregation may also
accrue after ATP release from endothelial cells. It is
possible that endothelial cell-released ATP has trophic
actions on endothelium. Indeed, Van Daele and colleagues15 have recently demonstrated a stimulatory
action of ATP, ADP, and adenosine on endothelial cell
DNA synthesis. This action of the adenyl purines on
bovine aortic endothelial cells in culture did not result
in increased cell number in the cultures exposed. Nonetheless, this action of ATP and its breakdown products
could be related to those factors required for angiogenesis and maintenance of endothelial integrity. This is
particularly interesting in light of the fact that many of
the mediators thought to release ATP from endothelial
cells have the opposite effect on blood vessel tone in the
absence of the endothelium.21 Indeed, this is also true
for ATP, as described above. Although we have not
performed studies of the effects of purines on cell
growth, it is possible that purines may have more
profound effects on endothelial cells from different
regions, perhaps those from the microcirculation of the
heart rather than aorta. Whatever the explanation for
the induction of DNA synthesis in endothelial cells, it is
clear that these effects of ATP, ADP, and adenosine on
endothelium
are
consistent with autocrine and para-
crine actions of ATP and further support the notion of
purinergic axis in cardiac blood vessels.
We conclude that endothelial cells isolated from
guinea pig heart and maintained in culture are able to
release ATP in a fashion that suggests that these cells
do so in vivo in response to various agonists, including
ATP itself, and that this response takes place under the
same conditions that regulate the release of nitric oxide
and other vascular mediators from the same cells. Our
data support the notion that adenyl purines are involved
in the regulation of blood flow in the heart and constitute a purinergic axis in cardiac blood vessels.
a
Acknowledgments
This work was supported by National Institutes of Health
grants HD-26227 to Dr Buxton, HL-38126 to Dr Westfall, and
a Fellowship grant from the Nevada Affiliate of the American
Heart Association to Dr Yang. The authors are grateful for the
technical contributions of Leta Artega and James Walther.
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Purinergic axis in cardiac blood vessels. Agonist-mediated release of ATP from cardiac
endothelial cells.
S Yang, D J Cheek, D P Westfall and I L Buxton
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Circ Res. 1994;74:401-407
doi: 10.1161/01.RES.74.3.401
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