GF109203X

Coronary response to diadenosine tetraphosphate after ischemia–reperfusion in the isolated rat heart

Abstract

Diadenosine tetraphosphate (AP4A) is a vasoactive mediator that may be released from platelet granules and that may reach higher plasma concentrations during coronary ischemia–reperfusion. The objective of this study was to analyze its coronary effects in such conditions. To this, rat hearts were perfused in a Langendorff preparation and the coronary response to Ap4A (10−7–10−5 M) was recorded. In control hearts, Ap4A produced concentration-dependent vasodilatation both at the basal coronary resting tone and after precontracting
coronary vasculature with 11-dideoxy-1a,9a-epoxymethanoprostaglandin F2α (U46619), and this vasodilatation was reduced by reactive blue 2 (2 × 10−6 M), glibenclamide (10−5 M), H89 (10−6 M), U73122 (5 × 10−6 M) and endothelin-1 (10−9 M), but not by L-NAME (10−4 M), isatin (10−4 M), GF109203x (5 × 10−7 M), or wortmannin (5 × 10−7 M). After ischemia–reperfusion, the vasodilatation to Ap4A diminished, both in hearts
with basal or increased vascular tone, and in this case the relaxation to Ap4A was not modified by reactive blue 2, L-NAME, glibenclamide, isatin, H89, GF109203x or wortmannin, although it was reduced by U73122 and endothelin-1. UTP produced coronary relaxation that was also reduced after ischemia–reperfusion. These results suggest that the coronary relaxation to Ap4A is reduced after ischemia–reperfusion, and that this reduction may be due to impaired effects of KATP channels and to reduced response of purinergic P2Y receptors.

1. Introduction

Diadenosine polyphosphates (ApnAs) are molecules consisting of two adenosine moieties linked by a chain of two to six phosphate groups, and they may act as extracellular or intracellular mediators (Mclennan, 2000). These substances are present in human myocardial tissue (Luo et al., 1999, 2004), and they are also stored in and released from chromaffin cells (Castillo et al., 1992) and platelets (Flodggard and Klenow, 1982). There is evidence that they may have effects on myocardial function (Vahlensieck et al., 1999) and vascular smooth muscle. In the latter, the effect may vary depending on the particular ApnA in question as well as on the prior arterial tone. Indeed, in mesenteric arteries from humans (Steinmetz et al., 2002) or rats (Ralevic et al., 1995), in radial arteries and saphenous veins from humans (Conant et al., 2008) and in rat renal circulation (van der Giet et al., 1997), ApnAs produce vasoconstriction if the arteries are at basal resting tone, and vasodilatation if the vessel tone is raised. With regard to the coronary circulation, ApnAs produce vasodilation in rats (Pohl et al., 1991), guinea-pigs (Stavrou et al., 2001), pigs (Sumiyoshi et al., 1997) or dogs (Sugimura et al., 2000) when used at concentrations (nM to μM) that may exist in plasma under normal conditions (Flores et al., 1999).

Coronary ischemia–reperfusion is a frequent clinical event that may produce dysfunction of coronary vessels. Coronary vascular dysfunction after ischemia–reperfusion involves reduced vasodilatory and increased vasoconstrictor responses (Climent et al., 2005, 2006), and these alterations may underlie the clinical phenomenon of no- reflow, whereby coronary blood flow remains reduced after the reopening of the occluded artery (Reffelmann and Kloner, 2004). A previous study from our laboratory suggests that the coronary response to adenosine pentaphosphate (Ap5A) changes from pre- dominantly vasodilatation in control conditions to predominantly vasoconstriction after ischemia–reperfusion (García-Villalón et al., 2009), and as the concentration of ApnAs increases in coronary venous blood during ischemia (Kitakaze et al., 1995), these substances may be involved in the no-reflow phenomenon. As other ApnAs, such as diadenosine tetraphosphate (Ap4A) are present in myocardial tissue (Luo et al., 1999, 2004) and increase during ischemia (Kitakaze et al., 1995), they could also participate in the pathophysiology of ischemia– reperfusion. However, the effects of the diadenosines may differ depending on the length of the polyphosphate chain, and it is not known whether the response to Ap4A is affected after ischemia– reperfusion, as occurs with Ap5A. The hypothesis of the study is that the effects of Ap4A on the coronary circulation may be altered after myocardial ischemia–reperfusion, which might contribute to the alterations on coronary blood flow which occur in this condition. Therefore, the objective was to analyze the coronary effects of Ap4A during ischemia–reperfusion, the effects of this substance on coronary vasculature being recorded before and after ischemia–reperfusion of perfused rat hearts.

2. Materials and methods

2.1. Experimental set up

In this study, 71 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 compliance 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 anesthesia with i.p. pentobarbital sodium (40 mg/kg) and i.v. injection of heparin (1000 IU). 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, KH2PO4 1.2 mM, MgSO4 1.2 mM, CaCl2 2.5 mM, NaHCO3 25 mM and glucose 11 mM) equili- brated 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 approximately 70 mm Hg. Both the perfusion solution and the heart were maintained at 37 °C. 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 mm Hg, both connected to Statham transducers. Left ventricular developed pressure (systolic left ventricular pressure minus diastolic left ventricular pressure), the first derivate of the left ventricular 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).

2.2. Experimental procedure

After a 15 min equilibration period with constant flow perfusion diadenosine tetraphosphate (Ap4A) 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 10−7, 10−6 and 10−5 M. After each concentration, Ap4A was washed out and the heart was allowed to recover before injecting the next concentration. In some experiments the coronary arteries were at their basal resting tone, and in others coronary arteries were precontracted by adding the thromboxane A2 ana- logU46619 to the perfusion solution 5 min before applying the diadenosine. Firstly U46619 10−8 M was applied, and the concentration
was increased progressively until the contractile tone developed was approximately 120–140 mm Hg. The concentrations of U46619 used to reach this tone were between 10−8and 3 ×10−8 M before ischemia and between 5 ×10−8 and 2 ×10−7 M after ischemia–reperfusion. After recording the response to Ap4A in control conditions, Ap4A and U46619 were washed out and the hearts were then exposed to global zero-flow ischemia for 30 min, followed by reperfusion for 15 min at the same flow rate as that before ischemia. After ending the reperfusion, the response to Ap4A was again recorded at the basal coronary vascular tone or after coronary precontraction with U46619 (Fig. 1). All the times used for ischemia and reperfusion were chosen on the basis of previous studies (García-Villalón et al., 2004, 2009), which showed that they produce decreases in the endothelium-dependent coronary relaxation without modifying endothelium-independent coronary relaxation. In time- control experiments, two successive injections of Ap4A (10−7–10−5 M) were administered, separated by 45 min of perfusion without ischemia. In these constant flow experiments the measurement of the perfusion pressure characterized the coronary perfusion resistance.

To analyze the mechanisms underlying the effects of Ap4A, the response to these substances was recorded after coronary precontraction with U46619 both before and after ischemia–reperfusion, in the presence and absence of the purinergic P2Y receptor antagonist reactive blue 2 (2 ×10−6 M), of the nitric oxide synthesis blocker N-omega-nitro-L- arginine methyl ester (L-NAME 10−4 M), the blocker of KATP channels glibenclamide (10−5 M), the blocker of atrial natriuretic peptide isatin (10−5 M), the blocker of protein kinase A (PKA) H89 (10−6 M), the
blocker of protein kinase C (PKC) GF109203x (5×10−7 M), the blocker of protein kinase PI3K wortmannin (5 ×10−7 M), the blocker of phospho- lipase C (PLC) U73122 (5×10−6 M), and the vasoconstrictor peptide endothelin-1 (10−9 M). These substances were infused 5 min before and
during infusion of Ap4A, but not during ischemia or the 15 min reperfusion period.

2.3. Statistical analysis

The coronary vascular response is expressed as the mean (±S.E.M.) change in perfusion pressure, and the hemodynamic parameters and the coronary response to Ap4A before and after ischemia–reperfusion were compared with repeated measures ANOVA followed by Bonferroni test. The responses to Ap4A in the presence of the antagonists or of endothelin-1 were compared with the responses in their absence using one-way ANOVA followed by Dunnett’s test. A probability of less than 0.05 was considered significant.

2.4. Drugs and chemicals used

The substances used were: P1,P4-di(adenosine-5′) tetrapho- sphate ammonium salt (Ap4A); 1-amino-4[[4-[[4-chloro-6-[[3- sulfophenyl]amino]-1,3,5-triazin-2-yl]amino]3-3sulfophenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid (reactive blue 2), 9,11-dideoxy-1a,9a-epoxymethanoprostaglandin F2α (U46619), N-omega-nitro-L-arginine methyl ester hydrochloride (L-NAME), 5- chloro-N-[4-(cyclohexylureidosulfonyl) phenethyl]-2-methoxybenza- mide; N-p-[2 (5-chloro-2-methoxy-benzamido) ethyl] benzenosulfonyl- nN′-cycloexylurea (glibenclamide, glyburide); isatin (2,3-Indolinedione), H89 (N[2-[bromocinnamylamino]ethyl]-5-isoquinolinesulfonamide dihydrochloride hydrate); GF109203x (bisindolylmaleimide 1 hydrochlo- ride); wortmannin (from Penicillium funiculosum); U73122 (1-[6 [[[17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl]amino]hexyl]-1,5- pyrrolidinedione); UTP (uridine 5′-triphosphate trisodium salt hydrate); all obtained from Sigma and endothelin-1 (human, porcine,…) acetate salt from Bachem, Switzerland.

Fig. 1. Diagram showing the experimental protocol, and the times at which the treatments were applied.

3. Results

3.1. Effects of ischemia–reperfusion in perfused hearts

In hearts perfused at the basal coronary resting tone (n=5, Table 1), 30 min of ischemia and 15 of reperfusion did not change the coronary perfusion pressure and diminished the left ventricular developed pressure (Pb 0.05) and maximal dP/dt (Pb 0.01). In the hearts with coronary vasculature precontracted with U46619 prior to ischemia–reperfusion (n=5, Table 1), the coronary perfusion pressure was higher than in hearts perfused at resting pressure (Pb 0.01), while the left ventricular developed pressure, maximal dP/dt and heart rate remained similar. In these precontracted hearts, ischemia–reperfusion did not modify significantly the coronary perfusion pressure, left ventricular developed pressure, the maximal dP/dt and heart rate. In time-control hearts perfused at basal coronary resting tone (n=4, Table 1), coronary perfusion pressure, left ventricular developed pressure, maximal dP/dt and heart rate were similar at the beginning of the experiment and 45 min later.

3.2. Response to Ap4A in hearts perfused at basal coronary resting tone

At basal coronary tone before ischemia–reperfusion, injection of Ap4A (n= 5) (10−7–10−5 M) reduced perfusion pressure, whereas after ischemia–reperfusion this reduction was significantly lower, and in addition this response was preceded by a small transient vasoconstriction (Fig. 2). Ap4A produced small, non-significant increases or decreases in left ventricular developed pressure, maximal dP/dt and heart rate before and after ischemia–reperfusion (Table 2). In time-control hearts perfused at coronary basal tone (n= 4), a second injection of Ap4A (n= 4) 45 min after the first one produced similar effects to the first one in terms of perfusion pressure (Fig. 2), left ventricular developed pressure, maximal dP/dt and heart rate.

3.3. Response to Ap4A after coronary precontraction with U46619

In hearts after precontraction of coronary vasculature with U46619, without ischemia–reperfusion, Ap4A (n= 5) also decreased perfusion pressure, and this decrease was more marked than in the hearts that were with coronary vasculature at a basal tone both before and after ischemia–reperfusion. After ischemia–reperfusion, in the precontracted hearts, the decrease in perfusion pressure induced by Ap4A was lower than before ischemia–reperfusion, Ap4A causing also a small transient vasoconstriction before the vasodilatation (Fig. 3). The effects on left ventricular developed pressure, maximal dP/dt and heart rate produced by Ap4A in hearts during coronary precontraction were similar to those in the hearts perfused at basal tone, before and after ischemia–reperfusion (Table 2).

3.4. Response to Ap4A in hearts treated with reactive blue 2

Before ischemia–reperfusion when hearts were subjected to coronary precontraction with U46619 and treated with reactive blue 2 (2 × 10−6 M), Ap4A (n= 6) produced vasoconstriction which was followed by vasodilatation, the latter being lower than in hearts not treated with reactive blue 2. After ischemia–reperfusion, in precontracted hearts plus reactive blue 2 Ap4A caused a higher initial vasoconstriction and a similar relaxation to that in precontracted hearts after ischemia–reperfusion but not exposed to reactive blue 2 (Fig. 3). In the hearts precontracted and treated with reactive blue 2, both before and after ischemia–reperfusion, Ap4A reduced left ventricular developed pressure and maximal dP/dt without modifying and after ischemia, glibenclamide by itself did not modify coronary perfusion pressure, left ventricular developed pressure, maximal dP/ dt or heart rate compared to untreated hearts (Table 1).

3.7. Response to Ap4A in hearts treated with isatin

Isatin (10−4 M) did not modify the relaxation to Ap4A in hearts precontracted with U46619, before or after ischemia–reperfusion
(n= 5, Fig. 3). The effects on left ventricular developed pressure, maximal dP/dt and heart rate produced by Ap4A after precontraction with U46619 and treatment with isatin were similar to those precontracted and untreated, before and after ischemia–reperfusion (Table 2). Before and after ischemia isatin by itself did not modify coronary perfusion pressure, left ventricular developed pressure or heart rate compared with untreated hearts, whereas it reduced maximal dP/dt after but not before ischemia–reperfusion (Table 1).

3.8. Response to Ap4A in hearts treated with H89

In the hearts precontracted with U46619, H89 (10−6 M) reduced the relaxation to Ap4A before but not after ischemia–reperfusion
(n= 5, Fig. 3). Before and after ischemia, H89 by itself did not modify coronary perfusion pressure, left ventricular developed pressure, maximal dP/dt or heart rate compared to untreated hearts (Table 1). Before but not after ischemia–reperfusion, Ap4A in the presence of H89 reduced left ventricular developed pressure and maximal dP/dt without modifying heart rate (Table 2).

Fig. 2. Coronary dilation to diadenosine tetraphosphate (Ap4A; 10−7–10− 5 M) in rat perfused hearts. Measurements were taken repeatedly 45 min apart in control experiments (A), as well as before and after a 30 min total ischemia followed by 15 min of reperfusion (B). Values are the mean (±S.E.M.) of five experiments. Statistically significant (*Pb 0.05; **Pb 0.01) with respect to the control.

3.5. Response to Ap4A in hearts treated with L-NAME

In the hearts precontracted and treated with L-NAME (10−4 M), the relaxation to Ap4A was increased, whereas after ischemia–
reperfusion the presence of L-NAME did not modify the relaxation although it increased the initial vasoconstriction produced by Ap4A (n= 5, Fig. 3). In the hearts precontracted and treated with L-NAME, Ap4A did not modify significatively the left ventricular developed pressure, maximal dP/dt or heart rate, before or after ischemia– reperfusion (Table 2). Before ischemia and after ischemia–reperfusion, L-NAME by itself increased coronary perfusion pressure (Pb 0.01), reduced left ventricular developed pressure and maximal dP/dt (P b 0.05) and did not modify heart rate compared to untreated hearts (Table 1).

3.6. Response to Ap4A in hearts treated with glibenclamide

When subjected to coronary precontraction with U46619, glib- enclamide (10−5 M) reduced the relaxation to Ap4A before but not after ischemia–reperfusion (n= 5, Fig. 3). In the hearts treated with U46619 and glibenclamide, Ap4A increased significatively the left
ventricular developed pressure and maximal dP/dt without modifying heart rate, before but not after ischemia–reperfusion, (Table 2). Before significatively the heart rate (Table 2). Before and after ischemia reactive blue 2 by itself did not modify coronary perfusion pressure, left ventricular developed pressure or heart rate compared with untreated hearts, whereas it reduced maximal dP/dt after but not before ischemia–reperfusion (Table 1).

3.9. Response to Ap4A in hearts treated with GF109203x

The PKC antagonist GF109203x (5 × 10−7 M) did not modify the relaxation to Ap4A in hearts precontracted with U46619, before or
after ischemia–reperfusion (n= 6, Fig. 3). Ap4A in the presence of GF109203x reduced left ventricular developed pressure and maximal dP/dt without modifying heart before but not after ischemia– reperfusion (Table 2). Before and after ischemia GF109203x by itself did not modify coronary perfusion pressure, left ventricular devel- oped pressure or heart rate compared with untreated hearts, whereas it reduced maximal dP/dt after but not before ischemia–reperfusion (Table 1).

3.10. Response to Ap4A in hearts treated with wortmannin

After treatment with wortmannin (5 × 10−7 M) the relaxation to Ap4A in hearts precontracted with U46619 was not modified compared with hearts precontracted and untreated, before or after ischemia–reperfusion (n= 5, Fig. 3). Before and after ischemia, wortmannin by itself did not modify coronary perfusion pressure, left ventricular developed pressure, maximal dP/dt or heart rate compared to untreated hearts (Table 1), and the effects on left ventricular developed pressure, maximal dP/dt and heart rate produced by Ap4A after precontraction with U46619 and treatment with wortmannin were similar to those precontracted and untreated, before and after ischemia–reperfusion (Table 2).

3.11. Response to Ap4A in hearts treated with U73122

Treatment with the PLC antagonist U73122 (5 × 10−6 M) reduced the relaxation to Ap4A in hearts precontracted with U46619, both
before and after ischemia–reperfusion (n= 6, Fig. 3) compared with untreated hearts. Before ischemia U73122 by itself did not modify coronary perfusion pressure, left ventricular developed pressure, maximal dP/dt or heart rate compared to untreated hearts (Table 1) but after ischemia–reperfusion reduced left ventricular developed pressure and maximal dP/dt. The effects on left ventricular developed pressure, maximal dP/dt and heart rate produced by Ap4A after precontraction with U46619 and treatment with U73122 were similar to those precontracted and untreated, before and after ischemia– reperfusion (Table 2).

3.12. Response to Ap4A in hearts treated with endothelin-1

Endothelin-1 (10−9 M) reduced the relaxation to Ap4A in hearts precontracted with U46619, both before and after ischemia–reperfusion
(n=6, Fig. 3) compared with untreated hearts. Before and after ischemia, endothelin-1 at the concentration used did not modify by itself coronary perfusion pressure or heart rate compared to untreated hearts, increased left ventricular developed pressure before ischemia and reduced both left ventricular developed pressure and maximal dP/dt after ischemia–reperfusion (Table 1). Ap4A in the presence of endothelin-1 increased left ventricular developed pressure and maximal dP/dt before but not after ischemia–reperfusion, and it did not modify heart rate (Table 2).

3.13. Response to UTP after coronary precontraction with U46619

In hearts after precontraction of coronary vasculature with U46619, UTP (n= 7) decreased perfusion pressure, and this decrease was reduced after ischemia–reperfusion (Fig. 4). Also, UTP reduced left ventricular developed pressure and maximal dP/dt, without modifying heart rate, before and after ischemia–reperfusion.

Fig. 3. Coronary vasodilatation (negative values) or vasoconstriction (positive values) to diadenosine tetraphosphate (Ap4A; 10−7–10−5 M) in rat perfused hearts during coronary precontraction with U46619, both before (A) and after (B) ischemia–reperfusion, in hearts untreated or treated with reactive blue 2 (2 ×10−6 M), L-NAME (10−4 M), glibenclamide (10−5 M). Isatin (10−4 M), H89 (10−6 M), GF109293x (5 ×10−7 M), wortmannin (5 ×10−7 M), U73122 (5×10−6 M), or endothelin-1 (10−9 M). Values are the mean (±S.E.M.) of 5–6 experiments and the statistical significance shown are between control and ischemic-reperfused hearts (*Pb 0.05; **P b 0.01) or between treated and untreated hearts (#P b 0.05; ##P b 0.01).

Fig. 4. Coronary dilation to uridine triphosphate (UTP; 10−7–10−5 M) in rat perfused hearts, before and after a 30 min total ischemia followed by 15 min of reperfusion. Values are the mean (±S.E.M.) of seven experiments. Statistically significant (*; Pb 0.01) with respect to the control.

4. Discussion

The results we present here suggest that the diadenosine polypho- sphate Ap4A may have coronary vasoactive effects, and that these effects may be altered after ischemia–reperfusion. In normal conditions, Ap4A produced coronary vasodilatation both when the coronary arteries were at basal and increased tone. This is in accordance with studies of Ap4A, which produces coronary vasodilatation in the canine heart (Sugimura et al., 2000), of Ap3A, Ap4A, Ap5A and Ap6A that produce coronary vasodilatation in the guinea pig heart (Stavrou et al., 2001), and of Ap4A and Ap5A that produce vasodilatation in pig coronary arteries (Sumiyoshi et al., 1997) and in rabbit coronary circulation (Pohl et al., 1991). Our results in the perfused heart also agree with studies in vivo which show that injection in vivo of Ap4A reduces systemic vascular resistance in rats (Khattab et al., 1998) and increase myocardial blood flow in dogs (Kikuta et al., 1994) or pigs (Nakae et al., 1996). Therefore, our model in the perfused heart reproduces the effects of Ap4A in vivo in control conditions, and may be useful to analyze these effects after ischemia–reperfusion which may be more difficult to produce in the rat in vivo.

In a previous study from our laboratory (García-Villalón et al., 2009) using the same preparation as in the present one, we found that Ap5A produced a biphasic response, with vasoconstriction followed by vasodilatation. In the present study, Ap4A did induce only vasodilatation in control hearts, but after inhibition of purinergic P2Y receptors with reactive blue 2 it produced also a transient vasoconstriction. It has been proposed that diadenosines with a longer phosphate chain tend to produce vasoconstriction, whereas vasodilatation predominates in diadenosines with a shorter chain (Ralevic et al., 1995). Our present and previous studies agree with that, as Ap5A produced vasoconstric- tion in basal conditions (García-Villalón et al., 2009) and Ap4A only after inhibition of purinergic receptors. Ap4A metabolism by myocardial tissue is relatively slow (Hoyle et al., 1996), therefore breakdown of this substance probably plays a minor role in these effects. The relaxation to Ap4A may be due to stimulation of purinergic P2Y receptors as it was reduced by reactive blue 2, whereas nitric oxide may not be involved in this relaxation as it was not reduced but increased by L-NAME. This increase in the relaxation in the presence of L-NAME may be probably related to the greater contractile tone produced by U46619 after inhibition of nitric oxide, as it has been found that changes in the level of tone may modify the vascular relaxation in the perfused heart (García- Villalón et al., 2009). Nitric oxide does also not participate in the relaxation to Ap5A in perfused rat hearts (García-Villalón et al., 2009) and to Ap4A in human mesenteric arteries (Steinmetz et al., 2002). Also, participation of nitric oxide in response to APnAs may vary with the animal species used, since while nitric oxide might mediate the vasodilatation provoked by Ap4A in the canine heart (Sugimura et al., 2000), it may not be involved in the vasomotor effects of Ap3A, Ap4A, Ap5A and Ap6A in the guinea-pig coronary circulation (Stavrou et al., 2001), and it may participate in the relaxation to Ap4A but not that to Ap3A in the rabbit coronary circulation (Pohl et al., 1991). L-NAME increased coronary basal perfusion pressure, which may be due to inhibition of basal nitric oxide release in the coronary circulation (Garcia et al., 1992), and also reduced myocardial contractility, which may be due to coronary perfusion being impaired by the inhibition of nitric oxide. However, this change in myocardial contractility may not reduce unspecifically the coronary vasodilatation, as the relaxation to Ap4A was not reduced in these conditions.

The role of potassium channels in the coronary response to diadenosine polyphosphates may also vary depending on the length of the phosphate chain. Ap5A may inhibit KATP potassium channels (Jovanovic et al., 1996), which may contribute to its vasoconstrictor effect. On the contrary, our present results suggest that Ap4A may also activate these potassium channels and produce vasodilatation, as the vasodilator response to this substance was reduced by glibenclamide. Ap4A also produces activation of KATP potassium channels in pig coronary circulation (Nakae et al., 1996).

Moreover, the effects of Ap4A may be mediated by PKA and PLC, as these effects were reduced by H89 and U73122, respectively. PLC is stimulated by Ap4A in human astrocytoma cells (Lazarowski et al., 1995) due to activation of PY2-purinoceptors (Nicholas et al., 1996), and PKA mediates the relaxation to P2Y purinoceptors stimulation in urinary bladder smooth muscle (McMurray et al., 1998). It may be hypothesized that stimulation of P2Y receptors in coronary arteries results in the activation of PLC and PKA pathways, which activates KATP potassium channels and produces vasodilatation. Our results partly agree with those of Yuan et al. (2007), who found that the negative inotropic effect of Ap4A in isolated rat atria was mediated by activation of PLC and KATP potassium channels. However, in rat atria this negative inotropic effect of Ap4A was also mediated by the release of atrial natriuretic peptide and activation of PKC (Yuan et al., 2007), but these mechanisms may not play an important role in the effects of Ap4A in the coronary circulation, as these effects were not modified by isatin or GF109203x. This discrepancy may be due to different parameters analyzed in both studies (myocardial contractility vs. coronary vasodilatation) and different receptor types involved in these responses (A1 vs. P2Y). Moreover, atrial natriuretic peptide release may be lower in the ventricular than in the atrial tissue (Casserly et al., 2010).

Diadenosine polyphosphates may have effects on myocardial contractility and heart rate, but these effects may vary. Ap3A, Ap4A and Ap5A may increase or decrease myocardial contractility, depending on the animal species and the previous condition of myocardium (Vahlensieck et al., 1999; Hoyle et al., 1996). We have found that in untreated rat hearts Ap4A sometimes produced small increases and sometimes small decreases in contractility, measured as changes in left ventricle developed pressure and maximal dP/dt, but no consistent responses were observed. However, after inhibition of purinergic receptors with reactive blue 2 this substance decreased contractility, and after blockade of KATP potassium channels it increased contractility.

This suggests that Ap4A may have positive inotropic effects mediated by purinergic receptors, as described for Ap4A in human (Vahlensieck et al., 1999), and also negative inotropic effects mediated by KATP potassium channels, and that these opposite effects cancel each other in untreated hearts. In the presence of H89 and GF109203x, Ap4A also reduced contractility, which suggests that the effects of Ap4A in myocardium may be mediated by PKA and/or PKC. There is evidence that purinergic receptors have positive inotropic effects in the heart (see Erlinge and Burnstock, 2008), however, purinergic agonists such as ATP or UTP may have both positive and negative inotropic effects (Froldi et al., 2001), which may account for the negative inotropic effect of UTP observed in the present study.

After ischemia–reperfusion, the coronary relaxation to Ap4A was reduced compared to that observed in control conditions, and Ap4A also produced a small initial vasoconstriction. This agrees with previous results using Ap5A, in which we found that after ischemia–reperfusion the vasodilatation to this substance was reduced and the vasoconstric- tion was increased (García-Villalón et al., 2009). Therefore, ischemia– reperfusion may produce impairment of the vasodilating response and enhancement of the vasoconstrictor effect of diadenosine polypho- sphates, although the relative importance of the vasodilatation and the vasoconstriction after ischemia–reperfusion may vary with the length of the phosphate chain. Also, these changes may not be due to unspecific changes in the vasodilating and vasoconstricting capacity of the vascular smooth muscle, as in previous studies we have found that the vasodilatation to sodium nitroprusside was not reduced (García- Villalón et al., 2009), and the vasoconstriction to endothelin-1 or U46619 was not increased (García-Villalón et al., 2005), in the perfused heart of rats after ischemia–reperfusion of similar duration as that used in the present study.

The reduction in the relaxation to Ap4A after ischemia–reperfusion may be due to an impaired response of purinergic P2Y receptors, as reactive blue 2 reduced the relaxation to Ap4A in control conditions but not after ischemia–reperfusion. Moreover, the relaxation to the agonist of purinergic P2Y receptors UTP (Matsumoto et al., 1997) was also reduced after ischemia–reperfusion, suggesting impairment of these receptors. Likewise, glibenclamide reduced the relaxation to Ap4A in control conditions but not after ischemia–reperfusion, therefore ischemia–reperfusion may also produce impairment of KATP potassium channels in the present study. Ischemia–reperfusion reduces the coronary vasodilatation to the KATP opener pinacidil in the perfused rat heart (Maczewski and Beresewicz, 1997), and reduction of the purinergic P2Y response may also be involved in the impairment of the vasodilatation to Ap5A in this condition (García-Villalón et al., 2009). On the contrary, nitric oxide may not mediate the relaxation to Ap4A, and the reduction of nitric oxide may not be involved in the diminished response to this substance after ischemia–reperfusion. We do not know the mechanisms of these impaired purinergic P2Y and potassium channel responses after ischemia–reperfusion. The reperfusion-induced injury signaling kinase (RISK) pathway is known to be activated in this situation (Davidson et al., 2006), but wortmannin, an inhibitor of PI3K which belongs to this pathway, did not modify the effects of ischemia– reperfusion, therefore this kinase may not be involved in the effects of ischemia–reperfusion in the present experimental set-up. On the other hand, the coronary relaxation to Ap4A was reduced by treatment with endothelin-1, and there is evidence that endothelin-1 concentration in the heart tissue increases after ischemia–reperfusion (Tonnessen et al., 1993). Therefore this peptide might be involved in the impaired response to Ap4A in this condition, however, more studies are necessary to clarify this point.

These results may have biological relevance, as diadenosine polyphosphates are endogenous substances which are present in the heart tissue in vivo (Luo et al., 2004), therefore the effects observed in the present study may also occur in vivo and be altered after ischemia– reperfusion. A frequent complication of clinical coronary ischemia– 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 produced by substances that affect the coronary vasculature during ischemia. As diadenosine polypho- sphates may be involved in the regulation of coronary circulation and their release may be increased after ischemia–reperfusion, the present results by showing that the effects of these substances may be disrupted after ischemia–reperfusion, suggest that these substances may contrib- ute to the reduction in coronary flow in these conditions.

In summary, the present study shows that after ischemia–reperfusion the coronary vasodilatory effect of AP4A is impaired, which may be due to reduced response of purinergic P2Y receptors be involved in the coronary pathophysiology of this condition.

Acknowledgments

We are indebted to María Ester Martínez and Hortensia Fernández- Lomana for their invaluable technical assistance.This work was financed by Fondo de Investigaciones Sanitarias (PS09/00394), and Fundación Mutua Madrileña (AP57242009).

References

Casserly, B., Pietras, L., Schuyler, J., Wang, R., Hill, N.S., Klinger, J.R., 2010. Cardiac atria are the primary source of ANP release in hypoxia adapted rats. Life Sci. 87, 382–389. Castillo, C.J., Moro, M.A., Del Valle, M., Sillero, A., Garcia, A.G., Sillero, M.A., 1992.
Diadenosine tetraphosphate is co-released with ATP and catecholamines from bovine adrenal medulla. J. Neurochem. 59, 723–732.
Climent, B., Fernández, N., Sanz, E., Sánchez, A., Monge, L., García-Villalón, A.L., Diéguez, G., 2005. Enhanced response of pig coronary arteries to endothelin-1 after ischemia–reperfusion. Role of endothelin receptors, nitric oxide and prostanoids. Eur. J. Pharmacol. 524, 102–110.
Climent, B., Fernández, N., García-Villalón, A.L., Monge, L., Sánchez, A., Diéguez, G., 2006. Effects of antagonists for ETA and ETB receptors on coronary endothelial and myocardial function after ischemia–reperfusion in anaesthetized goats. Vasc. Parmacol. 44, 384–390.
Conant, A.R., Theologou, T., Dihmis, W.C., Simpson, A.W., 2008. Diadenosine polypho- sphates are selective vasoconstrictors in human coronary artery bypass grafts. Vascul. Pharmacol. 48, 157–164.
Davidson, S.M., Hausenloy, D., Duchen, M.R., Yellon, D.M., 2006. Signaling via the reperfusion injury signalling kinase (RISK) pathway links closure of the mitochondrial permeability transition pore to cardioprotection. Int. J. Biochem. Cell Biol. 38, 414–419. Erlinge, D., Burnstock, G., 2008. P2 receptors in cardiovascular regulation and disease.
Purinergic Signal. 4, 1–20.
Flodggard, H., Klenow, H., 1982. Abundant amounts of diadenosine 5′,5″-P1, P4- tetraphosphate are present and releasable, but metabolically inactive, in human platelets. Biochem. J. 208, 737–742.
Flores, N.A., Stavrou, B.M., Sheridan, D.J., 1999. The effects of diadenosine polypho- sphates on the cardiovascular system. Cardiovasc. Res. 42, 15–26.
Froldi, G., Ragazzi, E., Caparrotta, L., 2001. Do ATP and UTP involve cGMP in positive inotropism on rat atria? Comp. Biochem. Physiol. 128, 165–274.
Garcia, J.L., Fernandez, N., Garcia-Villalon, A.L., Monge, L., Gomez, B., Dieguez, G., 1992. Effects of nitric oxide synthesis inhibition on the goat coronary circulation under basal conditions and after vasodilator stimulation. Br. J. Pharmacol. 106, 563–567. García-Villalón, A.L., Sanz, E., Monge, L., Fernández, N., Climent, B., Diéguez, G., 2004.
Urocortin protects coronary endothelial function during ischemia–reperfusion: a brief communication. Exp. Biol. Med. 229, 118–120.
García-Villalón, A.L., Amezquita, Y.M., Monge, L., Fernández, N., Climent, B., Sánchez, A., Diéguez, G., 2005. Effect of ischemia duration and nitric oxide on coronary vasoconstriction after ischemia–reperfusion. Eur. J. Pharmacol. 509, 165–170.
García-Villalón, A.L., Monge, L., Fernández, N., Salcedo, A., Narváez-Sánchez, R., Diéguez, G., 2009. Coronary response to diadenosine pentaphosphate after ischemia– reperfusion in the isolated rat heart. Cardiovasc. Res. 81, 336–343.
Hoyle, C.H.V., Ziganshin, A.U., Pintor, J., Burnstock, G., 1996. The activation of P1- and P2-purinoceptors in the guinea-pig left atrium by diadenosine polyphosphates. Br. J. Pharmacol. 118, 1294–1300.
Jovanovic, A., Alekseev, A.E., Terzic, A., 1996. Cardiac ATP-sensitive K+ channel: a target for diadenosine 5′,5″-P1, P5-pentaphosphate. Naunyn Schmiedeberg’s Arch. Pharmacol. 353, 241–244.
Luo, J., Jankowski, V., Güngär, N., Neumann, J., Schmitz, W., Zidek, W., Schlüter, H., Jankowski, J., 2004. Endogenous diadenosine tetraphosphate, diadenosine penta- phosphate, and diadenosine hexaphosphate in human myocardial tissue. Hyper- tension 43, 1055–1059.
Maczewski, M., Beresewicz, A., 1997. Inhibitors of nitric oxide synthesis and ischemia/ reperfusion attenuate coronary vasodilator response to pinacidil in isolated rat heart. Br. J. Pharmacol. 48, 737–749.
Matsumoto, T., Nakane, T., Chiba, S., 1997. UTP induced vascular responses in the isolated and perfused canine epicardial coronary artery via UTP-preferring P2Y receptors. Br. J. Pharmacol. 122, 1625–1632.
Mclennan, A.G., 2000. Dinucleoside polyphosphates-friend or foe? Pharmacol. Ther. 87, 73–89.
McMurray, G., Dass, N., Brading, A.F., 1998. Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle. Br. J. Pharmacol. 123, 1579–1586.
Nakae, I., Takahashi, M., Takaoka, A., Liu, Q., Matsumoto, T., Amano, N., Sekine, A., Nakajima, H., Kinoshita, M., 1996. Coronary effects of diadenosine tetraphosphate resemble those of adenosine in anesthetized pigs: involvement of ATP-sensitive potassium channels. J. Cardiovasc. Pharmacol. 28, 124–133.
Nicholas, R.A., Lazarowski, E.R., Watt, W.C., Li, Q., Boyer, J., Harden, T.K., 1996. Pharmacological and second messenger signaling selectivities of cloned P2Y receptors. J. Auton. Pharmacol. 16, 319–323.
Pohl, U., Ogilvie, A., Lamontagne, D., Busse, R., 1991. Potent effects of Ap3A and Ap4A on coronary resistance and autacoid release of intact rabbit hearts. Am. J. Physiol. 260, H1692–H1697.
Ralevic, V., Hoyle, C.H.V., Burnstock, G., 1995. Pivotal role of phosphate chain length in vasoconstrictor versus vasodilator actions of adenosine dinucleotides in rat mesenteric arteries. J. Physiol. 483, 703–713.
Reffelmann, T., Kloner, R.A., 2004. Microvascular alterations after temporary coronary occlusion: the no-reflow phenomenon. J. Cardiovasc. Pharmacol. Ther. 9, 163–172 2004.
Stavrou, B.M., Sheridan, D.J., Flores, N.A., 2001. Contribution of nitric oxide and prostanoids to the cardiac electrophysiological and coronary vasomotor effects of diadenosine polyphosphates. J. Pharmacol. Exp. Ther. 298, 531–538.
Steinmetz, M., Janssen, A.K., Pelster, F., Rahn, K.H., Schlatter, E., 2002. Vasoactivity of diadenosine polyphosphates in human small mesenteric resistance arteries. J. Pharmacol. Exp. Ther. 302, 787–794.
Sugimura, A., Kanatsuka, H., Tanikawa, T., Ong, B.H., Shirato, K., 2000. Effect of diadenosine tetraphosphate (Ap4A) on coronary arterial microvessels in the beating canine heart. Jpn. Circ. J. 64, 868–875.
Sumiyoshi, R., Nishimura, J., Kawasaki, J., Kobayashi, S., Takahashi, S., 1997. Diadenosine polyphosphates directly relax porcine coronary arterial smooth muscle. J. Pharmacol. Exp. Ther. 283, 548–556.
Tonnessen, T., Naess, P.A., Kirkeboen, K., Offstad, J., Ilebekk, A., Christensen, G., 1993. Endothelin is released from the porcine coronary circulation after short-term ischemia. J. Cardiovasc. Pharmacol. 22 (Suppl. 8), S313–S316.
Vahlensieck, U., Bokník, P., Gombosová, I., Huke, S., Knapp, J., Linck, B., Lüss, H., Müller, H.L., Neumann, J., Deng, M.C., Scheld, H.H., Jankowski, H., Schlüter, H., Zidek, W., Zimmermann, N., Schmitz, W., 1999. Inotropic effects of diadenosine tetrapho- sphate (Ap4A) in human and animal cardiac preparations. J. Pharmacol. Exp. Ther. 288, 805–813.
Van der Giet, M., Khattab, M., Börgel, J., Schlüter, H., Zidek, W., 1997. Differential effects of diadenosine phosphates on purinoceptors in the rat isolated perfused kidney. Br. J. Pharmacol. 120, 1453–1460.
Yuan, K., Cao, C., Bai, G.Y., Kim, S.Z., Kim, S.H., 2007. Diadenosine tetraphosphate stimulates atrial ANP release via A1 receptor: involvement of KATP channel and PKC. Peptides 28, 1397–1405.