Coronary response to diadenosine pentaphosphate after ischaemia-reperfusion in the isolated rat heart.
ABSTRACT Diadenosine polyphosphates are vasoactive mediators that may be released from platelet granules and which may be present at higher concentrations during coronary ischaemia-reperfusion. The objective of this study was to analyse their effects in such conditions.
Rat hearts were perfused in a Langendorff preparation and the response to diadenosine pentaphosphate (Ap5A, 10(-7)-10(-5) M) was recorded. In control hearts, Ap5A produced a small, transient coronary vasoconstriction followed by marked vasodilatation, as well as a reduction in the left ventricular developed pressure dP/dt and heart rate, both at the basal coronary resting tone or after pre-contracting coronary arteries with 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F2alpha (U46619). After ischaemia-reperfusion, the vasoconstriction in response to Ap5A was augmented and vasodilatation diminished, both in hearts with basal or increased vascular tone. The pyridoxal derivative P(2) purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 3 x 10(-6) M), inhibited this vasoconstriction, while the antagonist of purinergic P(2Y) receptors, Reactive Blue 2 (2 x 10(-6) M), inhibited the vasodilatation, both before and after ischaemia-reperfusion. The antagonist of nitric oxide synthesis N-omega-nitro-L- arginine methyl ester (L-NAME, 10(-4) M) did not modify the response to Ap5A, whereas the cyclooxygenase inhibitor, meclofenamate (2 x 10(-6) M), reduced contraction and increased the relaxation in response to Ap5A after ischaemia-reperfusion but not under control conditions.
Ischaemia-reperfusion reduces the vasodilatory response to Ap5A and increases the vasoconstriction provoked due to a reduced influence of purinergic P(2Y) receptors and/or to the production of vasoconstrictor prostanoids.
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ABSTRACT: Despite being known for over 30 years, the functions of the dinucleoside polyphosphates, such as diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A) and diadenosine 5',5"'-P(1), P(3)-triphosphate (Ap(3)A), are still unclear. On the one hand, they may have important signalling functions, both inside and outside the cell (friend), while on the other hand, they may simply be the unavoidable by-products of certain biochemical reactions, which, if allowed to accumulate, would be potentially toxic through their structural similarity to ATP and other essential mononucleotides (foe). Here, the occurrence, synthesis, degradation, and proposed functions of these compounds are briefly reviewed, along with some new data and recent evidence supporting roles for Ap(3)A and Ap(4)A in the cellular decision making processes leading to proliferation, quiescence, differentiation, and apoptosis. Hypotheses are forwarded for the involvement of Ap(4)A in the intra-S phase DNA damage checkpoint and for Ap(3)A and the pFhit (fragile histidine triad gene product) protein in tumour suppression. It is concluded that the roles of friend and foe are not incompatible, but are distinguished by the concentration range of nucleotide achieved under different circumstances.Pharmacology [?] Therapeutics 01/2000; 87(2-3):73-89. · 7.79 Impact Factor
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ABSTRACT: Diadenosine polyphosphates present at the cytosol can be transported to secretory granules allowing their exocytotic release. Extracellularly, they can act through specific metabotropic or ionotropic receptors, or as analogues of P2X and P2Y nucleotide receptors. The specific ionotropic receptor P4 is present in synaptic terminals, and modulated by protein kinases (PK) A and C and protein phosphatases. Activation of PKA or PKC, directly or through membrane receptors, results in a decrease of affinity or in reduction of the Ca2+ transient respectively. Adenosine and ATP, both products of the extracellular destruction of diadenosine polyphosphates, acting through A1 or P2Y receptors respectively, are important physiological modulators at the P4 receptor.FEBS Letters 07/1998; 430(1-2):78-82. · 3.58 Impact Factor
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ABSTRACT: Diadenosine polyphosphates (ApnA) (n = 3-6) induced vasoconstrictions in isolated human mesenteric resistance arteries (hMRAs) mounted in a microvessel myograph (rank order of potency: Ap5A > Ap6A > Ap4A > Ap3A). The contractile effects of ApnA in hMRA were similar to their effects in rat MRA investigated previously. ATP, ADP, AMP, and adenosine had less contractile potency than ApnA, suggesting that the observed effects were not induced by the degradation products of ApnA. Ap4A- and Ap5A-induced vasoconstriction was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (P2X purinoceptor antagonist) but not by ADP3'5' (P2Y purinoceptor antagonist). Thus, this purinergic vasoconstriction of hMRA seems to be P2X but not P2Y purinoceptor-mediated. In precontracted hMRA all ApnA caused vasorelaxations but (in contrast to rat MRA) the potencies of the ApnA did not differ significantly from each other. The ApnA degradation products had less vasorelaxing potency than ApnA, demonstrating that the vasorelaxations can be ascribed to the ApnA themselves. Ap5A-induced vasorelaxation of hMRA could neither be inhibited with ADP3'5' nor with PPADS, which reveals a decisive difference to the rat MRA where the inhibitory profile demonstrated the importance of the P2Y purinoceptor for Ap5A-induced vasorelaxation. However, Ap4A-induced vasorelaxation in hMRA could be inhibited by ADP3'5'. These findings show that Ap4A-induced vasorelaxation in hMRA is due to P2Y purinoceptor activation, that Ap5A evokes vasorelaxation in hMRA via another mechanism than Ap4A, and that data derived from the animal model cannot be simply transferred to human conditions.Journal of Pharmacology and Experimental Therapeutics 08/2002; 302(2):787-94. · 3.89 Impact Factor
Coronary response to diadenosine pentaphosphate after
ischaemia–reperfusion in the isolated rat heart
A´ngel Luis Garcı ´a-Villalo ´n*, Luis Monge, Nuria Ferna ´ndez, Adely Salcedo,
Rau ´l Narva ´ez-Sa ´nchez, and Godofredo Die ´guez
Departamento de Fisiologı ´a, Facultad de Medicina, Universidad Auto ´noma de Madrid, Arzobispo Morcillo 2,
28029 Madrid, Spain
Received 27 March 2008; revised 13 November 2008; accepted 18 November 2008; online publish-ahead-of-print 24 November 2008
Time for primary review: 26 days
Aims Diadenosine polyphosphates are vasoactive mediators that may be released from platelet granules
and which may be present at higher concentrations during coronary ischaemia–reperfusion. The objec-
tive of this study was to analyse their effects in such conditions.
Methods and results Rat hearts were perfused in a Langendorff preparation and the response to diade-
nosine pentaphosphate (Ap5A, 1027–1025M) was recorded. In control hearts, Ap5A produced a small,
transient coronary vasoconstriction followed by marked vasodilatation, as well as a reduction in the
left ventricular developed pressure dP/dt and heart rate, both at the basal coronary resting tone or
after pre-contracting coronary arteries with 9,11-dideoxy-11a, 9a-epoxymethanoprostaglandin F2a
(U46619). After ischaemia–reperfusion, the vasoconstriction in response to Ap5A was augmented and
vasodilatation diminished, both in hearts with basal or increased vascular tone. The pyridoxal derivative
P2purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS, 3 ? 1026M),
inhibited this vasoconstriction, while the antagonist of purinergic P2Yreceptors, Reactive Blue 2 (2 ?
1026M), inhibited the vasodilatation, both before and after ischaemia–reperfusion. The antagonist of
nitric oxide synthesis N-v-nitro-L- arginine methyl ester (L-NAME, 1024M) did not modify the response
to Ap5A, whereas the cyclooxygenase inhibitor, meclofenamate (2 ? 1026M), reduced contraction and
increased the relaxation in response to Ap5A after ischaemia–reperfusion but not under control con-
Conclusion Ischaemia–reperfusion reduces the vasodilatory response to Ap5A and increases the vaso-
constriction provoked due to a reduced influence of purinergic P2Yreceptors and/or to the production
of vasoconstrictor prostanoids.
Purinergic P2x receptors;
Purinergic P2y receptors;
Diadenosine polyphosphates (ApnAs) are molecules consist-
ing of two adenosine moieties linked by a chain of two to
six phosphate groups, and they may act as extracellular or
intracellular mediators.1In the central nervous system
ApnAs may act as neurotransmitters,2and they are also
stored in and released from chromaffin cells3and platelets.4
These substances may produce vasodilatation or vasocon-
striction of blood vessels depending on the particular ApnA
in question and the prior tone of the arteries. Indeed, in
the human5or rat6mesenteric arteries, and in rat renal cir-
culation,7ApnAs produce vasoconstriction if the arteries are
at basal resting tone and vasodilatation if the vessel tone is
raised. In addition, vasodilation is weaker and vasoconstric-
tion stronger for ApnAs with longer phosphate chains.6In
coronary circulation, ApnAs produce vasodilation in rats,8
guinea-pigs,9pigs,10or dogs11when they are present at nM
to mM concentrations, as may exist in plasma under
Coronary ischaemia–reperfusion is a frequent clinical
event that may produce dysfunction of coronary vessels. Cor-
onary vascular dysfunction after ischaemia–reperfusion
involves reduced vasodilatory and increased vasoconstrictor
responses,13,14and these alterations may underlie the clini-
cal phenomenon of no-reflow, whereby coronary blood flow
remains reduced after the reopening of the occluded
artery.15In addition, platelet activation and platelet-
released substances, including ApnAs, may be involved in
the pathophysiology of ischaemia–reperfusion.16It has been
shown that the concentration of ApnAs increases in coronary
venous blood during ischaemia17and as they can produce
vasodilatation or vasoconstriction depending on the con-
dition of the blood vessels, these compounds might partici-
pate in the altered coronary regulation associated with this
condition. However, the coronary effects of ApnAs after
*Corresponding author. Tel: þ34 91 497 5412; fax: þ34 91 497 5478.
E-mail address: firstname.lastname@example.org
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
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Cardiovascular Research (2009) 81, 336–343
example, acidosis, which may occur in the tissue during
ischaemia, reduces the vasodilatory effect of diadenosine
tetraphosphate (Ap4A) and pentaphosphate (Ap5A).18
The objective of this study was to analyse the effects
of ApnAs on coronary blood vessels during ischaemia–
reperfusion. Accordingly, the effects of Ap5A on coronary
circulation were recorded before and after ischaemia–
reperfusion of perfused rat hearts. Ap5A was selected as
an ApnA with a long phosphate chain as it is more likely to
produce vasoconstriction and therefore, to be involved in
the coronary vasoconstriction that frequently occurs after
ischaemia–reperfusion. Moreover, Ap5A is produced in the
heart and its production increases during heart ischaemia.19
In this study, 75 male Sprague–Dawley rats (weight 300–350 g) were
used in experiments carried out in accordance with the US National
Institutes of Health Guide for the Care and Use of Laboratory
Animals (NIH Publication No. 85–23, revised 1996) and in compli-
ance with all applicable laws and regulations. The use of these
animals was also approved by the Institute’s Animal Care and Use
Committee. The hearts were obtained from the rats after anaesthe-
sia with pentobarbital sodium (40 mg/kg) and injection of heparin
(1000 UI). After their removal, the ascending aorta was cannulated
and the heart was subjected to retrograde perfusion with
Krebs-Henseleit buffer (NaCl 115 mM, KCl 4.6 mM, KH2PO41.2 mM,
MgSO41.2 mM, CaCl22.5 mM, NaHCO325 mM, and glucose 11 mM)
equilibrated with 95% oxygen and 5% carbon dioxide to a pH of
7.3–7.4. Perfusion was initiated in a non-recirculating Langendorff
heart perfusion apparatus at a constant flow of 11-15 mL/min in
order to reach a basal perfusion pressure of ?70 mmHg. Both the
perfusion solution and the heart were maintained at 378C. Perfusion
coronary pressure was measured through a lateral connection in the
perfusion cannula and the left ventricular pressure was measured
with a latex balloon inflated to a diastolic pressure of 5–
10 mmHg, both connected to Statham transducers. Left ventricular
developed pressure (systolic left ventricular pressure minus dias-
tolic left ventricular pressure), the first derivate of the left ventri-
cular pressure curve (dP/dt) and heart rate were obtained from the
left ventricular pressure curve. These parameters were recorded on
a Macintosh computer by use of Chart v 3.6/s software and a
MacLab/8e data acquisition system (ADInstruments).
After a 15 min equilibration period with constant flow perfusion,
diadenosine pentaphosphate (Ap5A) was injected into the perfusion
cannula with an infusion pump at a constant rate over 3 min in order
to reach a final concentration of 1027, 1026, and 1025M. In some
cases, the coronary arteries were at their basal resting tone and
in others, coronary arteries were pre-contracted by adding the
thromboxane A2analogue U46619 (1028–1027M) to the perfusion
solution 5 min before applying Ap5A. The concentration of U46619
was adjusted in each experiment to reach a perfusion pressure of
120–140 mmHg. After recording the response to Ap5A in control
conditions, Ap5A and U46619 were washed out and the hearts
were then exposed to global zero-flow ischaemia for 30 min, fol-
lowed by reperfusion for 15 min at the same flow rate as that
before ischaemia. After ischaemia–reperfusion, the response to
Ap5A was again recorded at the basal coronary vascular tone or
after coronary pre-contraction with U46619. All the times used for
ischaemia and reperfusion were chosen on the basis of previous
which showed they
endothelium-dependent coronary relaxation without modifying
endothelium-independent coronary relaxation. In time-control
experiments, two successive injections of Ap5A (1027–1025M)
were administered, separated by 45 min of perfusion without
ischaemia. In addition, in some experiments the Ap5A was only
injected after ischaemia–reperfusion in order to test whether the
injection before ischaemia modified the subsequent response. In
these constant flow experiments the measurement of the perfusion
pressure characterized the coronary perfusion resistance. The
response to the endothelium-independent vasodilator sodium nitro-
prusside (1028–1026M) was also recorded before and after ischae-
mia–reperfusion, both at basal vascular tone or after coronary
pre-contraction with U46619.
To analyse the mechanisms underlying the effects of Ap5A, the
response to this substance was recorded after coronary pre-
contraction with U46619 both before and after ischaemia–reperfu-
sion, in the presence and absence of the purinergic P2Xreceptor
antagonist, pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid
(PPADS, 3 ? 1026M), the purinergic P2Yreceptor antagonist, reac-
tive blue 2 (2 ? 1026M), of the nitric oxide synthesis blocker,
N-v-nitro-L- arginine methyl ester (L-NAME 1024M), or of the
L-NAME increased the perfusion pressure, at the concentration
used none of the other antagonists modified the perfusion pressure
or the heart functional parameters.
To analyse the effects of previous contractile tone on the relax-
ation of coronary circulation, the response to a single concentration
of Ap5A (1026M) or to sodium nitroprusside (1027M) was recorded
at four levels of perfusion pressure (approximately 40, 70, 110,
and 150 mmHg). The lowest pressure (40 mmHg) was attained by
lowering the perfusion flow rate (8–10 mL/min), the intermediate
pressure (70 mmHg) was reached by perfusing at the normal flow
rate (11–15 mL/min), and the two higher pressures (110 and
150 mmHg) were achieved by perfusing at the normal flow rate
with an intermediate (1028–1027M) and high (1026M) concen-
tration of U46619 in the perfusion solution, respectively. This
latter concentration (1026M) was maximal, as higher concen-
trations of U46619 did not increase the perfusion pressure further.
To analyse the effects of treatment with Ap5A on coronary blood
flow and myocardial function, hearts were perfused with Ap5A
during reperfusion after ischaemia. In these experiments, the
hearts were equilibrated by perfusing for 15 min, and they were
then subjected to 30 min of ischaemia and reperfused for a
further 15 min. Ap5A (1025M) was added at the beginning of the
reperfusion and it remained present throughout the reperfusion
period. Other protocols were also employed whereby the hearts
were perfused during reperfusion in the presence of both Ap5A
(1025M) and PPADS (1025M), where they were subjected to ischae-
mia–reperfusion without any prior exposure to drugs, or when they
were perfused with Ap5A (1025M) for 15 min after 45 min of per-
fusion without ischaemia. During all these experiments, the per-
fusion pressure was maintained at 70 mmHg to detect the changes
in coronary flow produced by Ap5A and/or ischaemia.
The coronary vascular response is expressed as the mean (+SEM)
change in perfusion pressure and the coronary responses before and
after ischaemia–reperfusion were compared with the paired Stu-
dent’s t-test. The responses to the antagonists were compared
with the responses in their absence using one-way ANOVA followed
by Dunnet’s test. A probability of ,0.05 was considered significant.
The substances used were: P1,P5,di(adenosine-50) pentaphosphate
pentaamonium salt (diadenosine pentaphosphate, Ap5A); pyridoxal
ymethanoprostaglandin F2a(U46619); N-v-nitro-L-arginine methyl
ester hydrochloride (L-NAME), meclofenamic acid sodium salt (meclo-
fenamate), all obtained from Sigma.
(2 ? 1026M).While
In hearts perfused at the basal coronary resting tone (n ¼
13, Figure 1) the coronary perfusion pressure did not
Diadenosine pentaphosphate and ischaemia
change, while after 30 min ischaemia and 15 min of reperfu-
sion the left ventricular developed pressure (P . 0.001),
maximal dP/dt (P , 0.001) and heart rate (P , 0.05) dimin-
ished. Following coronary pre-contraction with U46619 prior
to ischaemia–reperfusion (n ¼ 15, Figure 1), the coronary
perfusion pressure and the left ventricular developed
pressure was higher than in hearts perfused at resting
pressure, while the maximal dP/dt and heart rate remained
similar. After ischaemia–reperfusion, in the hearts during
coronary pre-contraction with U46619, coronary perfusion
pressure, left ventricular developed pressure, and heart
rate were not significantly different to that before ischae-
mia–reperfusion, although the maximal dP/dt was lower
(P , 0.05).
In time-control hearts perfused at basal coronary resting
tone (n ¼ 5, Figure 1), coronary perfusion pressure, left ven-
tricular developed pressure, maximal dP/dt, and heart rate
were similar at the beginning of the experiment and 45 min
At basal coronary tone before ischaemia–reperfusion,
injection of Ap5A (1027–1025M, n ¼ 5) into the hearts pro-
duced a small, transient increase in perfusion pressure
that was followed by a marked reduction in perfusion
pressure (Figure 2), as well as a reduction in left ventricular
time of the first and the second infusion of diadenosine pentaphosphate. CCP, coronary perfusion pressure; LVDP, left ventricle developed pressure; MdP/dt,
maximal dP/dt; and HR, heart rate.
Diagram showing the experimental protocol, the times at which the infusions were applied, and the values of the haemodynamic parameters at the
more slowly developing coronary vasorelaxation (negative values) are
shown as a function of the concentration of diadenosine pentaphosphate
(1027–1025M) applied to the coronary perfusion solution. Measurements
were taken repeatedly 45 min apart in control experiments (top panel), as
well as before and after a 30 min total ischaemia followed by 15 min of reper-
fusion (bottom panel, left and right, respectively). Values are the mean (+
SEM) of five experiments. *; ** Statistically significant (*P , 0.05; **P , 0.01)
with respect to the control.
Maximum initial coronary contraction (positive values) and the
A ´.L. Garcı ´a-Villalo ´n et al.
(Table 1). After ischaemia–reperfusion, the vasoconstriction
in response to Ap5A increased while vasodilatation dimin-
ished in comparison to the response before ischaemia–
reperfusion (Figure 2). In contrast, the reductions in left
ventricular developed pressure, maximal dP/dt, and heart
rate were similar (Table 1).
In time-control hearts perfused at coronary basal tone
(n ¼ 5), a second injection of Ap5A 45 min after the first
one produced similar effects to the first one in terms of per-
fusion pressure (Figure 2), left ventricular developed
pressure, maximal dP/dt, and heart rate. When Ap5A was
administered after ischaemia–reperfusion without having
injected it into the same heart prior to ischaemia, the
effects were similar to those after ischaemia–reperfusion
when it had also been injected prior to ischaemia (for
Ap5A 1027, 1026, and 1025M, vasoconstriction 14+3,
30+2, and 54+7 mmHg; and vasodilatation 0, 29+4,
and 23+2 mmHg, respectively, n ¼ 4).
When pre-contracted before ischaemia–reperfusion with
U46619 (n ¼ 5), Ap5A also produced transient vasoconstric-
tion followed by a marked relaxation, the time course,
and extent of which was not markedly different to that in
the hearts that were at a basal tone before ischaemia–
reperfusion. After ischaemia–reperfusion, in these pre-
contracted coronary systems, the initial vasoconstrictor
effect of Ap5A augmented and the vasodilatation was
weaker (Figures 3 and 4) when compared with the effects
before ischaemia–reperfusion. The reduction in left ventri-
cular developed pressure, maximal dP/dt, and heart rate
produced by Ap5A in hearts during coronary pre-contraction
were similar to those in the hearts perfused at basal tone,
before, and after ischaemia–reperfusion.
When hearts were pre-contracted with U46619 and
treated with PPADS (n ¼ 5), the contraction in response to
Ap5A was less than in pre-contracted hearts not exposed
to PPADS, both before and after ischaemia, whereas relax-
ation was not modified. After ischaemia–reperfusion in the
presence of PPADS the relaxation was weaker and the con-
traction was stronger than before ischaemia–reperfusion in
the presence of PPADS (Figure 4).
When hearts were subjected to coronary pre-contraction
with U46619 and treated with reactive blue 2 (n ¼ 5), the
contraction to Ap5A was stronger than in hearts pre-
contracted but not exposed to reactive blue 2. In addition,
relaxation was nearly abolished both before and after ischae-
mia. After ischaemia–reperfusion in the presence of reactive
blue 2, the contraction was higher than that before ischae-
mia–reperfusion in the presence of reactive blue 2 for Ap5A
1027M, lower for 1025M, and similar for 1026M (Figure 4).
was not significantly modified by exposing hearts pre-
contracted with U46619 to L-NAME (n ¼ 5, Figure 4).
When subjected to coronary pre-contraction with U46619,
meclofenamate treatment before ischaemia–reperfusion
(n ¼ 6) did not modify contraction or relaxation in response
to Ap5A, although after ischaemia–reperfusion it diminished
the contraction and increased the relaxation to this diade-
nosine (Figure 4).
At basal tone, injection of sodium nitroprusside (1028–
1026M) into the hearts produced vasodilatation, which was
significantly reduced after ischaemia–reperfusion (1028M,
2+1 vs. 7+2 mmHg; 1027M, 6+1 vs. 28+5 mmHg, P ,
0.05; 1026M, 10+2 mmHg vs. 32+6, P , 0.05; n ¼ 4).
Moreover, the relaxation induced by sodium nitroprusside
was similar before and after ischaemia–reperfusion in hearts
pre-contracted with U46619 (1028M, 14+4 vs. 13+2;
1027M,36+7vs.53+10;1026M,51+7vs.58+11;n ¼ 6).
When hearts were perfused at different pressures, the
relaxation to Ap5A increased with the perfusion pressure
150 mmHg). However, the initial contraction to Ap5A
increased with perfusion pressures in the range 37–
114 mmHg while it fell at the highest perfusion pressure
examined (150 mmHg, Table 2). The relaxation to sodium
nitroprusside increased at perfusion pressures between 40
and 113 mmHg, although the relaxation at 143 mmHg was
similar to that at 113 mmHg (Table 2).
coronary tone by injection of diadenosine phentaphosphate (102721025M) before (control) and after ischaemia–reperfusion. Values are
means+SEM of 5–6 experiments
Reduction (%) of left ventricular developed pressure, systolic dP/dt, and heart rate produced in rat hearts perfused at basal
1027M 1026M 1025M 1027M 1026M 1025M
Left ventricular developed pressure
sphate (102721025M) on the coronary perfusion pressure in rat hearts
during coronary pre-contraction with U46619, both before (control) and
Representative recordings of the effects of diadenosine pentapho-
Diadenosine pentaphosphate and ischaemia
At a constant perfusion pressure of 70 mmHg, during the
first 5 min of reperfusion after ischaemia (n ¼ 5) there was
less coronary flow in the hearts treated with Ap5A (1025M)
than in untreated ones (4.1+0.6 vs.7.1+0.7 mL/min, n ¼
5, P , 0.05), whereas after 10 and 15 min of reperfusion cor-
onary flow was similar in both groups (7.2+0.6 vs. 7.9+0.7
and 8.1+0.4 vs. 7.7+0.6 mL/min, respectively). The hearts
treated with both Ap5A and PPADS during reperfusion (n ¼ 5)
had a similar coronary flow to untreated hearts at 5, 10, and
15 min (7.6+0.9, 8.0+0.8, and 7.9+0.7 mL/min for 5,10,
and 15 min). In addition, after ischaemia–reperfusion the
left ventricular developed pressure (37+1 mmHg, P ,
0.05), dP/dt (508+42 mmHg/s, P , 0.05), and heart rate
(163+28 b.p.m., P , 0.05) in untreated hearts was lower
than before ischaemia, and these parameters were no
different in treated and untreated ischaemic hearts. Finally,
the coronary flow in non-ischaemic hearts treated with Ap5A
(22.9+3.8, 25.5+3.7, and 25.9+3.5 mL/min at 5, 10,
and 15 min after beginning Ap5A injection, n ¼ 4) was
greater than before injection or than in treated or untreated
The results we present here suggest that Ap5A may have
vasoactive effects on coronary circulation and that these
effects may be altered after ischaemia–reperfusion. Thus,
in normal conditions, the main effect of Ap5A on coronary
circulation is likely to be vasodilatory, although it can
cause a small transient contraction both when hearts are
denosine pentaphosphate (102721025M) during coronary precontraction with U46619 (102821027M), before (control), after ischaemia–reperfusion, and in the
presence or absence (untreated) of PPADS (3 ? 1026M), reactive blue 2 (2 ? 1026M), L-NAME (1024M), or meclofenamate (2 ? 1026M). Values are the mean
(+SEM) of 5–6 experiments and the statistical significance shown is between control and ischaemic–reperfused hearts (*P , 0.05; **P , 0.01) or between
treated and untreated hearts (†P , 0.05;††P , 0.01).
The extent of the initial contraction (positive values) and of the more slowly developing relaxation (negative values) in rat hearts in response to dia-
nitroprusside (1027M) produced in rat hearts perfused at different initial perfusion pressures
Maximums of coronary initial contraction and slow relaxation to diadenosine pentaphosphate (1026M) and relaxation to sodium
Diadenosine pentaphosphate Sodium nitroprusside
Initial perfusion pressure (mmHg)
Values are means+SEM of five experiments.
A ´.L. Garcı ´a-Villalo ´n et al.
perfused at their basal coronary resting tone and when cor-
onary arteries are pre-contracted. This is in accordance with
studies of Ap4A, which produces vasodilatation in the canine
heart,11of Ap3A, Ap4A, Ap5A, and Ap6A that produce vaso-
dilatation in the guinea pig heart,9and of Ap4A and Ap5A
that produce vasodilatation in pig coronary arteries10and
in rabbit coronary circulation.8However, the effect of
ApnA on blood vessels may depend on their previous con-
tractile tone. Indeed, Ap5A in human renal arteries,21and
Ap3A, Ap4A, Ap5A, and Ap6A in rat mesenteric arteries,22
produced vasoconstriction when the arteries were at the
basal tone and vasodilation when they were pre-contracted.
In contrast, we found that the response was predominantly
vasodilation in both cases. This may reflect the differences
in experimental design, since the coronary arteries of the
hearts perfused under basal conditions in our preparations
may have some degree of tone. Conversely, in isolated vas-
cular segments basal contractile tone is likely to be very low
and under these latter conditions, the vasoconstrictor
effects of APnA will probably be more evident.23We have
seen that isolated segments of the anterior descending cor-
onary artery of the rat did not relax on exposure to Ap5A
unless they were pre-contracted (unpublished results). In
addition, Ap5A also reduced myocardial contractility and
heart rate, in agreement with the negative inotropic
effects of Ap4A found in the dog24and guinea-pig25heart,
and of Ap3A, Ap4A, and Ap5A in guinea-pig hearts.26
After ischaemia–reperfusion, the relaxation provoked by
Ap5A was milder and vasoconstriction augmented, both in
hearts at the basal tone and in those with increased vascular
tone. At basal tone, the reduced vasodilation in the heart
may in part be non-specific, as the relaxation produced by
sodium nitroprusside was also milder. These reductions
may be due to differences in the contractile tone of the cor-
onary vasculature, particularly since our results suggest that
the increase in contractile tone enhances, and that a
reduction in contractile tone impairs, the relaxation in
response to both Ap5A and sodium nitroprusside. In the
hearts perfused at basal resting tone, there may be
changes in the contractile status of the coronary vessels
that reduce the amount of tone available for relaxation,
which could be due to metabolites released from the
working or hypoxic myocardium. However, when vascular
tone was increased with U46619, the relaxation produced
by sodium nitroprusside was not altered after ischaemia–
reperfusion, while the relaxation to Ap5A was reduced and
contraction increased. Pre-contraction with U46619 may
increase coronary tone and eliminate any possible differ-
ences in tone between control and ischaemia–reperfusion,
thereby abolishing the non-specific constraint on relaxation
to sodium nitroprusside but not the specific impairment in
relaxation to Ap5A. Thus, the coronary effects of AP5P
may change after ischaemia–reperfusion, increasing the
contractile effects while impeding its vasodilatory effects.
The coronary constrictor and dilatory effects of Ap5A may
gic receptors of the P2Xsubtype, PPADS, reduced contraction
while leaving relaxation unaffected, whereas the antagonist
ation and augmented contraction. Therefore, coronary vaso-
constriction in response to Ap5A may be mediated by
purinergic P2Xreceptors and coronary vasodilatation may be
mediated by purinergic P2Y receptors, in agreement with
other studies. For example, in rat mesenteric arteries the
vasodilator effect of Ap2A and Ap3Awas mediated by puriner-
gic P2Yreceptors, and the vasoconstrictor effect of Ap4A,
teric arteries the vasoconstriction in response to Ap4A and
Ap5A was controlled by purinergic P2Xreceptors, and the
relaxation to Ap4A was mediated by P2Yreceptors but not
that in response to Ap5A.5In rat renal circulation, P2Xrecep-
tors mediate vasoconstriction in response to Ap4A, Ap5A,
and Ap6A.7Moreover, Ap5A inhibits ATP-sensitive potassium
channels27and adenylate kinase activity,28which could also
contribute to Ap5A vasoconstriction.
In the hearts treated with PPADS, the relaxation after
ischaemia–reperfusion remained low when compared with
that observed in the same hearts before ischaemia.
However, in the hearts treated with reactive blue 2, only
contraction in response to the lower concentration of
Ap5A increased after ischaemia–reperfusion, whereas the
contraction in untreated hearts in response to all the con-
centrations studied increased after ischaemia–reperfusion.
PPADS or reactive blue 2 did not modify the coronary per-
fusion pressure, suggesting that changes in basal tone are
not involved in the effects of these antagonists on the
response to Ap5A. Therefore, the changes in the response
to Ap5A after ischaemia–reperfusion may be due to a dam-
pened response to the activation of purinergic P2Yreceptors
and not to an increase in the response to purinergic P2X
receptor activation, particularly since blockage of P2Y
receptors reduced these changes after ischaemia–reperfu-
sion but not blockage of P2X. During ischaemia–reperfusion,
the receptors in all cell types of the heart could to some
extent be altered by degradation or changes in their
expression. Also, ATP or ADP, which may be released from
damaged post-ischaemic heart tissue, may desensitize puri-
It is known that ischaemia–reperfusion may produce endo-
thelial dysfunction and therefore reduce the endothelium-
dependent relaxation mediated by nitric oxide.31However,
the relaxation induced by Ap5A in our experimental con-
ditions may not be mediated by nitric oxide as it was not
affected by L-NAME. Thus, nitric oxide reduction may not
be involved in the impaired relaxation to AP5P observed
after ischaemia-relaxation. Participation of nitric oxide in
response to APnAs may vary, since while it might mediate
the vasodilatation provoked by Ap4A in the canine heart,11
it may not be involved in the vasomotor effects of Ap3A,
Ap4A, Ap5A, and Ap6A in the guinea-pig coronary circula-
tion.9Indeed, in the rabbit heart nitric oxide may partici-
pate in the relaxation to Ap4A but not that to Ap3A.8
In control conditions, prostanoids do not seem to be
involved in the effects of Ap5A in coronary circulation,
because the cyclooxygenase inhibitor meclofenamate does
not modify vasodilatation or vasoconstriction in response
to this substance. However, the inhibition of prostanoid syn-
thesis with meclofenamate after ischaemia–reperfusion
increased relaxation and reduced contraction. Hence,
after ischaemia–reperfusion there appears to be some pro-
duction of vasoconstrictor prostanoids that may contribute
to the increased vasoconstriction and impaired vasodilation
produced by Ap5A. In coronary ischaemia–reperfusion a
change in arachidonic acid metabolism may provoke vaso-
constrictor prostanoid production, as observed in goat32or
Diadenosine pentaphosphate and ischaemia
A frequent complication of clinical coronary ischaemia–
reperfusion is the phenomenon of no-reflow, by which the
reduction in coronary blood flow persists after reperfusion.
This phenomenon may be related to impaired vasodilatation
and/or to increased vasoconstriction produced by sub-
stances that affect the coronary vasculature during ischae-
mia. While we found that Ap5A predominantly induces
vasodilatation in control conditions, this response switches
to vasoconstriction after ischaemia–reperfusion. If perfusion
pressure remains constant in non-ischaemic hearts, treat-
ment with Ap5A markedly increases coronary blood flow, in
accordance with the hypothesis that this substance produces
mainly vasodilatation in control conditions. However, after
ischaemia treatment with Ap5A reduced rather than
increase coronary flow during the first minutes of reperfu-
sion. Therefore, Ap5A released during reperfusion may
have a harmful effect on cardiac recovery by further redu-
cing coronary blood flow. Conversely, treatment with
PPADS improved coronary flow in ischaemic–reperfused
hearts treated with Ap5A and thus, blockage of purinergic
P2X receptors may serve as a therapeutic strategy to
improve coronary blood flow during reperfusion after ischae-
mia. However, the experimental model used in the present
study has limitations for the extrapolation of the results to
the in vivo situation, as in the present study the hearts
were perfused with a cristaloid solution and no platelets
were present. In these experimental conditions, the
release of endogenous diadenosines is probably very low
compared with the in vivo situation, and Ap5A has to be
The effects of Ap5A observed here were observed at con-
centrations that might be present in the plasma during
ischaemia–reperfusion. Indeed, ApnA concentrations may
be even higher locally, in the proximity of activated plate-
lets, and thus, Ap5A may indeed be involved in the increased
vasoconstriction associated with this condition. We also
found that the coronary vasoconstrictor response to Ap5A
after ischaemia–reperfusion may be impaired by antagonists
of purinergic P2Xreceptors or cyclo-oxygenase inhibitors,
suggesting that these agents could be useful in the treat-
ment of alterations to coronary perfusion in this condition.
Ferna ´ndez-Lomana for their invaluable technical assistance.
areindebtedto Marı ´aEsterMartı ´nez and Hortensia
Conflict of interest: none declared.
This work was financed by a grant from the Ministerio de Educacio ´n y
Ciencia (BSA 2004-04054).
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Diadenosine pentaphosphate and ischaemia