Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine

Abstract

Objective: This study tested the hypothesis that brief cycles of iterative ischemia–reperfusion at onset of reperfusion (termed “postconditioning”, post-con) delays washout of intravascular adenosine and thereby increases endogenous adenosine receptor (AR) activation during the early moments of reperfusion (R).

Methods: Isolated mouse hearts were subjected to 20 min global ischemia (I) and 30 min R with or without post-con (3 or 6 cycles of 10 s R&I). Intravascular purines in coronary effluent were analyzed by HPLC. To assess the functional role of endogenous AR activation in post-con, an open-chest rat model of myocardial infarction was employed. Rats were randomly divided into 11 groups: control, no intervention at R; post-con, three cycles of 10 s R followed by 10 s LCA re-occlusion immediately upon R. In the following interventions, drugs (or vehicle) were administered 5 min before R in the absence or presence (±) of post-con. Vehicle (DMSO <300 μl/kg); 8-SPT (non-selective AR antagonist, 10 mg/kg) ± post-con; DPCPX (A_1AR antagonist, 0.1 mg/kg) ± post-con; ZM241385 (A_2AAR antagonist, 0.2 mg/kg) ± post-con; MRS1523 (A₃AR antagonist, 2 mg/kg) ± post-con.

Results: In isolated mouse hearts, post-con reduced diastolic pressure during both early (26 ± 3* vs. 37 ± 3 mmHg at 5 min) and late (22 ± 3* vs. 34 ± 3 mmHg at 30 min) R. Post-con also hastened the early recovery of contractile function (developed pressure 39 ± 6* vs. 16 ± 2 mmHg at 5 min R), although differences did not persist at 30 min R. Importantly, post-con was associated with reduced adenosine washout (58 ± 5* vs. 155 ± 16 nM/min/g) at 2 min R suggesting greater retention time of intravascular adenosine. In rats, post-con significantly attenuated infarct size compared to control (40 ± 3% vs. 53 ± 2%* in control), an effect that was unaltered by DPCPX (42 ± 2%) but was abrogated by 8-SPT (50 ± 2%), ZM241385 (49 ± 3%) or MRS1523 (52 ± 1%) (P<0.02).

Conclusion: These data suggest that post-con involves endogenous activation of A_2A and A₃ but not A₁AR subtypes. This activation may be linked to the delay in the washout of intravascular adenosine during the early minutes of R during which post-con is applied.

1. Introduction

The definitive treatment for myocardial ischemia is reperfusion. However, reperfusion has the potential to cause additional reversible and irreversible damage to the myocardium [1,2]. Reperfusion injury has been shown to be attenuated by pharmacological and mechanical interventions introduced at the onset of reflow. Accordingly, exerting control over the hydrodynamics of the early (i.e. first 30) minutes of reperfusion reduces post-ischemic injury in a canine model of acute coronary occlusion–reperfusion [3,4]. These studies not only supported the concept that reperfusion is associated with additional injury, but also provided the scientific basis for the more recently reported phenomenon of “postconditioning” (post-con) [5], which is a time-sensitive cardioprotective strategy induced by brief repetitive interruptions in blood flow applied immediately at the onset of reperfusion [5–8]. To date, cardioprotection by postconditioning has been reported by independent laboratories in several species (i.e. canine [5,7], rabbit [9], rat [6]), in isolated perfused hearts [10] and in in vivo and cell culture models [5,8]. In addition, it has been documented that postconditioning affords protection comparable to that of ischemic preconditioning (pre-con) [5,9,10], although there are mixed reports on whether these strategies are capable of invoking additive protection when combined [5,7,9,10]. Importantly, recent studies have provided direct evidence supporting a role for nitric oxide and the reperfusion injury salvage kinase (RISK) pathway [9,10]. However, there are no studies that have investigated potential upstream triggers of the signalling elements proposed.

One likely candidate for a cardioprotective trigger stimulated by postconditioning is adenosine receptor activation. In the last 10 years, an abundance of studies have cited the cardioprotective effects of adenosine receptor activation during both ischemia and reperfusion [11–14]. Importantly, adenosine receptor activation during reperfusion appears to be of key importance during the first 30 min, exerting not only anti-neutrophil properties but also infarct-sparing effects [11,15]. Despite mixed support for a role for A₁ARs [16–19], ample evidence suggests that activation of A_2AAR and A₃ARs mediates protection exerted during reperfusion [15,20–27]. Accordingly, we hypothesised that AR activation by endogenous adenosine, in particular activation of the A_2AAR and A₃ARs, may be involved in mediating postconditioning cardioprotection. An additional aim of this study was to investigate whether the transient impediments to reflow during postconditioning alters intravascular adenosine concentrations during early reperfusion, thereby providing a rationale for adenosine receptor activation during the postconditioning phase.

2. Materials and methods

Investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publications No. 85-23, revised 1996).

2.1. In vivo rat surgical preparation

Ninety-five male Sprague–Dawley rats weighing 260–380 g were initially anesthetized via intraperitoneal administration of sodium pentobarbital (40 mg.kg⁻¹). A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent ventilator (Harvard Rodent Ventilator Model 683, Holliston, MA). Rats were ventilated with O₂-enriched room air at ∼30–50 breaths per minute, with tidal volumes set to 1.0 ml/100 mg body weight. Arterial pO₂, pCO₂ and pH were monitored (Stat Profile M, Nova Biomedical, Waltham, MA) during the experiment and values were maintained within a normal physiological range (pH 7.35–7.45; pCO₂, 25–40 mmHg; and pO₂, 80–110 mmHg) by adjusting the ventilatory rate and tidal volume, or by intravenous administration of sodium bicarbonate. Body temperature was kept at a constant 37 °C by using an adjustable heating pad.

The left carotid artery was cannulated with a 24-gauge angiocatheter connected to a fluid-filled pressure transducer to continuously monitor phasic and mean arterial pressure (MAP) and heart rate (HR) using the EMKA data acquisition and analysis system (EMKA Technologies Inc., Falls Church, VA). The right external jugular vein was cannulated for delivery of anesthesia and drug treatments. The chest was opened via a left thoracotomy through the fourth or fifth intercostal space, and the ribs were gently retracted to expose the heart. After pericardiotomy, a 6-0 proline (Ethicon, NJ) ligature was placed under the left coronary artery (LCA), and the ends of the suture were threaded through polyethylene tubing (PE-50) to form a snare for reversible LCA occlusion. Following surgical preparation (prior to LCA occlusion), a bolus dose of sodium heparin (100 U/kg) was administered to ensure heparinization throughout the experiment. The LCA was occluded by tightening the snare using a light weight hemostat. Ischemia was confirmed by transient decrease in blood pressure and cyanosis on the myocardial surface. Reperfusion was indicated by an epicardial hyperemic response and rapid disappearance of cyanosis.

2.2. Experimental protocol for in vivo studies

All rats were subjected to 30 min LCA occlusion, followed by 3 h reperfusion. In those hearts that received drug treatment, antagonists (or vehicle) were given as a bolus dose 5 min prior to reperfusion to ensure proper receptor blockade. Rats were randomly assigned to one of eleven groups (n=8–12 in each group, Fig. 1): 1: Control, no intervention either before or after LCA occlusion (n=12); 2: Vehicle control, DMSO (<300 μl/kg) alone (n=8); 3: Post-con, initiated immediately at the onset of reperfusion, 3 cycles of 10 s full reperfusion and 10 s re-occlusion (total intervention time of 1 min) (n=10); 4: 8-SPT (10 mg/kg) alone (n=9); 5: 8-SPT+Post-con, post-con stimulus invoked in the presence of antagonist 8-SPT (n=8); 6: A₁AR antagonist DPCPX (0.1 mg/kg) alone (n=8); 7: DPCPX+Post-con (n=8); 8: A_2AAR antagonist ZM241385 (0.2 mg/kg) alone (n=8); 9: ZM241385+Post-con (n=8); 10: A₃AR antagonist MRS1523 (2 mg/kg) alone (n=8) and 11: MRS1523+Post-con (n=8).

2.3. Determination of infarct size

Upon completion of the experiment, the LCA was re-occluded and the area at risk (AAR) was delineated by injecting 1.0 ml of 20% Unisperse blue dye via the external jugular vein. The heart was rapidly excised and placed into 0.9% saline. An individual blinded to the protocol subsequently separated the LV from remaining tissue and thinly (∼2 mm) cross-sectioned the LV before separating the AAR (unstained) from blue stained non-ischemic zone. The AAR was incubated for 10 min in a phosphate buffered 1% TTC solution at 37 °C, enabling a clear differentiation of necrotic tissue. Area of necrosis (AN) and AAR were determined by gravimetric analysis. AN was expressed as a percentage of the AAR (AN/AAR).

2.4. Plasma creatine kinase (CK) activity

Arterial blood (0.3 ml) was collected in EDTA tubes at baseline, end of ischemia and 3 h of reperfusion for CK quantitation to confirm morphologic injury (necrosis). Samples were centrifuged at 2500 × g for 10 min and stored at −70 °C until analyzed enzymatically (CK assay, Diagnostics Chemicals Limited, Oxford, CT). Plasma CK activity is expressed as IU/g protein.

2.5. Murine Langendorff buffer-perfused heart model

Twenty-three adult male C57/Bl6 mice (8–12 weeks age, 30 ± 0.5 g body weight, 130 ± 4 mg blotted heart weight) were anesthetized with sodium pentobarbital (50 mg.kg⁻¹). Hearts were isolated and prepared as described previously [28,29]. After 20 min stabilization hearts were switched to pacing (420 beats.min⁻¹) using silver electrodes (0.5 ms square pulses, 20% above threshold, typically 2–5 V) placed on the LV and a Grass S9 stimulator (Grass, Quincy, MA, USA). After a further 10 min equilibration, hearts were then subjected to 20 min global normothermic ischemia, followed by 30 min reperfusion. Pacing was terminated on initiation of ischemia and resumed after 2.5 min of reperfusion in all groups. The following post-con regimes were assessed: 1: Control, no post-con stimulus (n=7); 2: Post-con, initiated immediately at the onset of reperfusion, 3 cycles of 10 s full reperfusion and 10 s re-occlusion (total intervention time of 1 min) (n=7); 3: Post-con, 6 cycles of 10 s full reperfusion and 10 s re-occlusion (total intervention time of 2 min) (n=9).

2.6. Analysis of venous purine efflux

To determine if post-con-mediated protection involves modulation of extracellular purine concentrations, coronary perfusate flow was quantified using a cannulating Doppler flow-probe (Transonic Systems, Inc., Ithaca, NY, USA) in the aortic perfusion line and connected to a T206 flowmeter (Transonic Systems), and coronary venous effluent was sampled every minute for the first 5 min of reperfusion and then collected at 10, 20 and 30 min time points for the remainder of the reperfusion period. The samples were immediately frozen at −80 °C until analyzed by reverse-phase HPLC with PDA detection (Waters, NSW, Australia) for adenosine, inosine, hypoxanthine, xanthine and uric acid. Early (0–5 min) and total post-ischemic purine efflux values were calculated as the product of concentration reached in the reperfusion effluent (nmol/ml) × reperfusion volume (ml/min/g wet weight).

2.7. Chemicals

The A_2AAR antagonist ZM241385 was purchased from Tocris, Ellisville, MO. All other drugs were purchased from Sigma/RBI (Sigma, St. Louis, MO). 8-SPT was dissolved directly in 0.9% saline, whereas DPCPX, ZM24138 and MRS1523 were dissolved in <300 μl/kg DMSO. Vehicle studies showed that this concentration had no effect on hemodynamics or infarct size (Tables 1 and 2, Fig. 2).

ZM241385 is a highly selective A_2AAR antagonist with a 1000-fold A_2AAR:A₁AR selectivity, 91-fold A_2AAR:A_2BAR selectivity and 500,000-fold A_2AAR:A₃AR selectivity [30] and has very little, if any, activity against responses mediated by A₁ or A₃ARs in vivo [31]. Furthermore, MRS1523 is a highly potent ligand at rat A₃ARs with selectivity of 140 and 18-fold vs. A₁ and A_2A receptors, respectively [32]. In addition, 2 mg/kg of MRS1523 has been reported to be the minimum dose required to inhibit A₃-dependent histamine release from 100 μg/kg CI-IB-MECA in mouse [33].

2.8. Statistical analysis

All values are reported as mean ± S.E.M. Data were analyzed using Statistica version 7 (StatSoft, Tulsa, OK). One-way analysis of variance (ANOVA) (i.e. AAR and infarct size data) and multi-way ANOVA with repeated measures (cardiodynamics and functional responses) were used as appropriate, with Student–Newman–Keuls post-hoc test when significant effects were detected. In all tests P<0.05 was considered indicative of statistical significance.

3. Results

3.1. Hemodynamic data

The hemodynamic data are summarized in Table 1. There were no significant differences observed between control and treatment groups for any variable [HR, MAP or rate-pressure product (RRP)] at any time point (baseline, end of ischemia, and 180 min of reperfusion). Thus, despite the fact that the postcon group resulted in a smaller infarct size, hemodynamic variables were not enhanced. Indeed, this apparent dissociation has been observed by others, notably Cohen et al. [34] in which infarct size was reduced by preconditioning by 80%, and regional wall motion was improved, but systemic hemodynamics were unaltered.

3.2. A_2A and A₃AR (but not A₁AR) antagonism abolishes the infarct-sparing effect of post-con

The area placed at risk by LCA occlusion, expressed as a percent of left ventricular mass (AAR/LV), was comparable among the 11 groups (∼30–33%) (Table 2). Infarct size, expressed as a percentage of area at risk (AN/AAR), was significantly reduced in the post-con group (40 ± 3%) compared with the control rats (53 ± 2%, P<0.01) (Fig. 2). Pretreatment with 8-SPT (non-selective AR antagonist, 10 mg/kg), DPCPX (selective A₁AR inhibitor, 0.1 mg/kg), ZM241385 (selective A_2AAR inhibitor, 0.2 mg/kg) or MRS1523 (selective A₃AR inhibitor, 2 mg/kg) alone had no effect on infarct size compared to control (Table 2, Fig. 2A and B). However, the infarct sparing effects of post-con were completely abolished by 8-SPT, ZM241385 and MRS1523 (50 ± 2%, 49 ± 3% and 52 ± 1%, respectively, P<0.02) (Fig. 2A and B). In contrast, the A₁AR inhibitor DPCPX was unable to modulate the cardioprotective effects of post-con (42 ± 2%).

Plasma CK activity was comparable among the 11 groups at the end of baseline and ischemia (data not shown). In the control group, plasma CK activity after 180 min of reperfusion was shown to increase by 3-fold in comparison to baseline, a response which was markedly attenuated by post-con (25 ± 6 vs. 46 ± 3 IU/g protein in control) (Fig. 3). Administration of drug treatments alone did not alter the elevations in CK activity relative to the control group. Importantly, DPCPX did not modify CK activity in post-con group (Fig. 3A), whereas 8-SPT and MRS1523 was associated with greater CK activity at 180 min of reperfusion (Fig. 3A and B). Interestingly, despite inhibition of the infarct-sparing effects of post-con by ZM241385, the reduction in CK activity observed in the post-con group was only partially reversed by selective A_2AAR antagonism (36 ± 3 vs. 26 ± 3 IU/g protein in post-con group, P<0.06) (Fig. 3B). Importantly, post-con in the presence of ZM241385 was not significantly different to ZM241385 alone (P<0.4) or control hearts (P<0.3). These data are largely consistent with the infarct size data.

3.3. Post-con delays washout of intravascular adenosine during early reperfusion and improves contractile recovery in buffer-perfused hearts

In control hearts, post-ischemic contractile function failed to recover to pre-ischemic levels following 20 min ischemia and 30 min reperfusion. End-diastolic pressure (EDP) was significantly elevated (∼30 mmHg), and LV developed pressure (LVDP) and rate-pressure product (RPP) recovered to approximately 45% of pre-ischemic levels. Coronary perfusate flow rate was not significantly different to pre-ischemic values (data not shown). Interestingly, while the post-con algorithm of three cycles of 10 s reperfusion and 10 s ischemia was unable to modify contractile function in hearts, lengthening the duration of the algorithm to six cycles significantly improved post-ischemic systolic and diastolic function of hearts (Fig. 4). Relative to controls, EDP was significantly attenuated during early (5 min, 26 ± 3 vs. 37 ± 3 mmHg in control, P<0.05) and late (30 min, 22 ± 3 vs. 34 ± 3 mmHg in control, P<0.05) reperfusion (Fig. 4A). However, post-ischemic LVDP was significantly greater with 6 cycles of post-con during early (39 ± 6 vs. 16 ± 2 mmHg in control, P<0.05) but not during late reperfusion (82 ± 5 vs. 66 ± 3 mmHg in control) (Fig. 4B). Interestingly, RPP was significantly greater in comparison to control hearts during 5, 10 and 20 min of reperfusion, but not 30 min of reperfusion (Fig. 4C). Importantly, improvement in early reperfusion contractile recovery observed in these post-con hearts was also associated with greater retention time of intravascular adenosine during early reperfusion (58 ± 6 vs. 155 ± 16 nM/min/g in control hearts, P<0.05) (Fig. 5A). At 1 min of reperfusion, the effluent adenosine concentration with the 10 s algorithm (3 or 6 cycles) of post-con was significantly lower than in controls with unbridled reperfusion. However at 2 min of reperfusion, 1 min after the three cycle algorithm had ended, the effluent adenosine concentration returned to control levels in these hearts. In contrast, the effluent adenosine concentration with the six cycle algorithm remained lower at 2 min of reperfusion (i.e. until the end of post-con stimuli), and was significantly higher than controls at 3 min of reperfusion. Post-con did not modify total intravascular adenosine (Fig. 5B) or purine (Fig. 5C) concentration, suggesting that post-con results in the retention of adenosine rather than altering the metabolism of adenosine or its metabolites.

4. Discussion

Data from the present study suggest that cardioprotection invoked by postconditioning involves endogenous activation of adenosine receptors. Our results support a key role for both A_2A and A₃AR subtypes, but not the A₁AR, in the infarct-sparing effects of postconditioning. In addition, postconditioning results in a greater retention time of extracellular (i.e. intravascular) adenosine during the period of postconditioning in isolated hearts, which was also associated with improved recovery of contractile function. These novel data therefore demonstrate that the infarct sparing effects of postconditioning can be abrogated by blocking adenosine receptor activation, suggesting functional involvement of endogenously released adenosine. Moreover, delay in washout of intravascular adenosine with postconditioning may accordingly increase the presence and concentration of adenosine in the vascular (and interstitial) space. Indeed, the time frame of delay in immediate adenosine washout is consistent with the critical timing of the postconditioning intervention [6]. Thus, adenosine receptor activation may provide the trigger for activation of downstream signaling mechanisms involved in cardioprotection.

4.1. Postconditioning mediated cardioprotection involves endogenous activation of adenosine receptors during early reperfusion

To assess the role of endogenous adenosine receptor activation in postconditioning mediated protection, we studied the infarct-sparing effects of postconditioning in the absence and presence of non-selective adenosine receptor antagonist, 8-SPT, and subtype specific adenosine receptor antagonists, DPCPX (A₁AR inhibitor), ZM241385 (A_2AAR inhibitor) and MRS1523 (A₃AR inhibitor). The protective effects of postconditioning were abolished to a comparable degree by non-selective adenosine receptor antagonism, and by specific A_2A and A₃AR blockade (Fig. 2B). In contrast, we found no role for activation of A₁ARs in postconditioning mediated protection (Fig. 2A). These data suggest that endogenous activation of A_2A and A₃ARs during reperfusion play a key role in triggering the infarct-sparing effects of postconditioning. Similarly, CK activity closely matched changes in infarct size (Fig. 3A and B).

A recent study by Boucher et al. demonstrated that administration of A_2AAR agonist, CGS21680, starting 5 min prior to reperfusion resulted in a significant reduction in infarct size, whereas the same regimen initiated 5 min after the onset of reperfusion was ineffective [27]. These studies are not only consistent with our finding that A_2AARs are involved in triggering postconditioning mediated protection, but also reinforces the concept that timing of the intervention (i.e. early moments of reperfusion) is critical in initiating protection during reperfusion [6]. Moreover, previous studies suggest that the effects of adenosine during ischemia are mediated by A₁ and A₃ARs [18,29,35–40], whereas effects during reperfusion are largely ascribed to A_2A and A₃ARs [15,20–27], which is consistent with our findings that A_2A and A₃ARs (but not A₁ARs) play a significant role in postconditioning mediated protection. Indeed, despite some support for endogenous activation of A₁ARs reducing post-ischemic contractile dysfunction [17,18], administration of A₁AR agonists during reperfusion alone have been largely unsuccessful in limiting infarct size [16,19,41].

Interestingly, there is little evidence supporting cardioprotection by intrinsic activation of A₃ARs during reperfusion. Indeed, the beneficial effects of A₃ARs are reported to be dependent on exogenous activation [21,24,25,40], suggesting that interstitial adenosine concentrations during reperfusion are not sufficient to cause physiologically effective activation of A₃ARs. This is of particular interest since the present study showed that activation of both A_2A and A₃ARs by endogenous adenosine is involved in triggering the cardioprotection of postconditioning. More importantly, the results from the present study raise the possibility that endogenous adenosine concentrations are modulated by the postconditioning intervention, positioning adenosine as a stimulus or trigger of cardioprotection. Indeed, interstitial and extracellular adenosine concentrations have been previously reported to be ∼5 μM and 2 μM, respectively, during the first 5 min of reperfusion in the rat [18,42], which is well in excess of the adenosine affinities for the A₁ and A₃ARs of 10 nM vs. >1 μM, respectively, in rat [43]. However, while interstitial adenosine concentrations closely match intravascular levels under basal conditions [42], Lasley et al. demonstrated that washout of interstitial adenosine lags behind that of coronary venous adenosine levels following prolonged coronary artery occlusion [44]. These observations not only suggest that intravascular washout precedes interstitial washout within the first 5 min, but indicate that the rate of intravascular washout is relatively high, which emphasizes that adenosine concentrations in these spaces are rapidly changing during the crucial first minutes of reperfusion when postconditioning may be applied.

In light of this, we assessed the hypothesis that postconditioning delays the washout of endogenous adenosine, ostensibly by transiently impeding blood flow, thereby increasing intravascular adenosine concentrations during the critical early moments of reperfusion. Accordingly, our study revealed that postconditioning decreased coronary effluent adenosine during the first minute of reperfusion previously found to be critical for postconditioning [6] (Fig. 5A), which presumably implies a greater retention of the purine in the intravascular space. Indeed, analysis of purine efflux collected at 1 min intervals during early reperfusion revealed that postconditioning increased retention time of extracellular adenosine expressed as reduced effluent concentrations during the first 2 min of reperfusion (i.e. during the postconditioning stimulus) and increased concentrations between 2 and 4 min when hearts experienced uncontrolled reflow (Fig. 5A). While simultaneous measurement of intravascular adenosine from effluent and interstitial adenosine using microdialysis are necessary to confirm this hypothesis, this remains a significant challenge since the rate and volume of interstitial samples is much smaller than that which can be collected over the same time frame. Alternatively, the transient ischemic periods could have increased adenosine production, thereby contributing to the intravascular concentration. However, total adenosine and purine efflux were unaltered by post-conditioning, which argues against this possibility (Fig. 5B and C). The delayed washout of intravascular adenosine during reperfusion was also associated with improved early recovery of contractile function in our buffer-perfused hearts (Fig. 4). Therefore, the present study demonstrates that postconditioning 1) can protect against myocardial stunning, and 2) protection can be demonstrated in cell-free (asanguinous) system as reported previously [8]. These data suggest that protection exerted by postconditioning is not solely dependent on the presence of inflammatory cells or other blood-borne elements, but may also modify injury processes directly in the extravascular compartment.

4.2. Cardioprotection triggered by A_2A and A₃ adenosine receptor activation

The observation that blockade of either A_2A or A₃ARs abrogated postconditioning mediated protection to a similar extent (i.e. infarct size comparable to control heart) suggests that A_2A and A₃ARs may be acting on a common end effector or that an “all or none” response by endogenous adenosine on the target effector is involved, whereby both receptors are required to be activated to manifest the response (i.e. one subtype alone cannot sufficiently modify signaling to produce the phenotypic change). Involvement of A_2A and A₃AR activation triggered by accumulating adenosine during postconditioning is consistent with their ability to inhibit inflammatory-related processes [2,15,20–22,45,46], which are manifested as a reduction in coronary artery endothelial dysfunction, neutrophil accumulation, generation of reactive oxygen species and their lipid peroxide products in myocardium protected by postconditioning [5–7,20]. Indeed, previous studies have shown that decreases in infarct size exerted by A_2A and A₃AR agonism involved reductions in neutrophil adherence and/or infiltration [20,21,47] and that adenosinergic cardioprotection elicited by activation of A_2AARs is also associated with reductions in ROS generation and endothelial adherence of neutrophils [20,46]. Additionally, inhibition of the coronary vascular endothelium by retained intravascular adenosine may attenuate the subsequent PMN-endothelial cell interactions, ROS generation and cytokine release that are observed during early reperfusion. Thus A_2A and A₃AR activation may modify a range of inflammatory processes contributing not only to direct intravascular protection, but also the infarct-sparing effects observed in the present study. However, as mentioned above, other mechanisms not involving a cell-mediated inflammatory response to reperfusion are likely engaged by postconditioning.

Interestingly, activation of the A_2A and A₃AR (but not A₁AR) subtypes during reperfusion may stimulate downstream signaling elements leading to cardioprotection relevant to postconditioning. Indeed, it has been demonstrated that the beneficial effects of A_2AAR activation during early reperfusion are dependent on phosphatidylinositol 3-kinase (PI3-K) [27]. Similarly, activation of A₃ARs has been linked to a reduction in apoptosis via the PI3-K pathway [48]. In contrast, however, there appears to be no role for the PI3-K/Akt pathway in A₁ mediated protection [49,50]. Thus, the very recent report by Yang et al. [51] that postconditioning was dependent on both endogenous adenosine receptor activation and PI3-K pathway, together with our findings that A_2A and A₃AR activation trigger postconditioning, is consistent with the hypothesis that adenosine receptor activation by endogenous adenosine provides a proximal trigger that can activate reperfusion injury survival kinases. However, we did not investigate the signaling pathways involved in A_2A or A₃ receptor activation by endogenous adenosine during postconditioning in the present study.

4.3. Conclusions

In conclusion, the current study demonstrates that endogenous activation of adenosine receptors, in particular the A_2A and A₃AR subtype, is involved in postconditioning-mediated cardioprotection in a rat model of myocardial infarction. In addition, our data suggest that postconditioning delays washout of intravascular adenosine during the critical first minutes of reperfusion, ostensibly increasing intravascular concentrations of endogenous adenosine during the postconditioning “stimulus”. We speculate that retention of intravascular adenosine may act either as a trigger of more distal cardioprotective signals, such as survival kinases, nitric oxide, and K_ATP channels previously shown to be involved in postconditioning-mediated cardioprotection [9,10] or by directly modulating inflammatory-related processes. Collectively, these data imply that postconditioning results in an enhanced level of endogenous adenosine during the early moments of reperfusion, which is capable of activating adenosine A_2A and A₃ receptor subtypes to elicit protection against myocardial infarction.

Acknowledgements

This work was supported in part by NIH grant # HL069487 and HL64886. Amanda J. Zatta (nee Flood) was the recipient of a Postgraduate Research Scholarship from the National Heart Foundation of Australia and is a recipient of a Postdoctoral Fellowship from the American Heart Association.

References

Author notes