To the Editor:

Endogenous adenosine is an important mediator of ischaemic preconditioning and postconditioning. The protective effect of ischaemic preconditioning is dependent on activation of adenosine A₁ receptors in the first few minutes of reperfusion [1]. In contrast, the infarct size-limiting effect of ischaemic postconditioning is mediated by the activation of adenosine A_2A and A_2B receptors at the time of reperfusion [2, 3]. Paradoxically, although the cardioprotective effect of preconditioning and postconditioning are critically dependent on adenosine receptor stimulation immediately after reperfusion, several research groups could not demonstrate an infarct size-limiting effect of administration of exogenous adenosine at the moment of reperfusion [4‐6]. A possible explanation is that the pre- and postconditioning stimuli increase the sensitivity of a specific adenosine receptor sub-type. Indeed, this mechanism was recently proposed for the adenosine A_2B receptor [7]. The A_2B receptor has a K _m value for adenosine that is much higher than the endogenous adenosine concentration reached during ischaemia. Kuno et al have recently shown in the rabbit heart that brief preconditioning ischaemia markedly lowered the threshold for an adenosine A_2B agonist to increase the phosphorylation of the kinases, Akt and Erk1/2 [7].

In this study, we test the hypothesis that ischaemic preconditioning also increases the sensitivity of the adenosine A₁ receptor at the time of myocardial reperfusion, and that ischaemic postconditioning increases the sensitivity of the adenosine A_2A receptor, using an isolated perfused rat heart model. As adenosine A₁ receptor stimulation has a negative inotropic effect and adenosine A_2A receptor stimulation induces coronary vasodilation, we used heart rate and coronary flow as read-out parameters for adenosine A₁ and A_2A receptor sensitivity, respectively. Male Sprague-Dawley rats were obtained from the Biological Services Unit of University College London (United Kingdom). All experiments were conducted in accordance with UK Home Office Guide on the Operation of Animal (Scientific Procedures) Act of 1986. After anaesthesia with pentobarbital, hearts were excised and mounted on a constant pressure (80 mmHg) Langendorff-apparatus and perfused with modified Krebs–Henseleit bicarbonate buffer (gassed with 95% O₂ / 5% CO₂, pH 7.35–7.5). Heart rate and coronary flow were monitored at regular intervals. To study the sensitivity of the adenosine A₁ receptor, hearts were perfused with incremental concentrations of the selective A₁ receptor agonist 2-chloro-N6-cyclopentyl-adenosine (CCPA) (10⁻¹⁰ to 10⁻⁶ mmol/l) for 5 min per concentration. Heart rate was measured in the last minute of perfusion of each concentration and compared to the baseline heart rate. To study the sensitivity of the A_2A receptor, the hearts were perfused with the selective A_2A receptor agonist CGS21680 (10⁻¹⁰ to 10⁻⁶ mmol/l) and coronary flow rate was measured in the last minute of each concentration. These measurements were performed at baseline (after 30 min of equilibration), at 20 min reperfusion after 35 min of regional ischaemia in control hearts and hearts treated with a standard ischaemic preconditioning protocol (two cycles of 5 min of global ischaemia and reperfusion) or a standard ischaemic postconditioning protocol (6 cycles of 10 s of global ischaemia and reperfusion). The measurements were performed 20 min after reperfusion to allow the heart rate and coronary flow to return to baseline values after the ischaemic insult.

Perfusion with CCPA dose-dependently lowered heart rate (Fig. 1). This reduction in heart rate was similar at baseline and at 20 min myocardial reperfusion (data not shown), and was not affected by preconditioning (P = 0.9 and P = 0.4 for comparison of EC₅₀ and V_max values, respectively, univariate analysis of variance). Perfusion with CGS21680 dose-dependently increased coronary flow at baseline (Fig. 2). However, after ischaemia, this effect was blunted, and did not differ between the postconditioning and control groups (P = 0.8, ANOVA for repeated measures on absolute flow values).

In conclusion, the present study demonstrates that ischaemic preconditioning and postconditioning do not increase the sensitivity of the adenosine A₁ and A_2A receptor, respectively. Therefore, this mechanism does not explain the apparent discrepancy that the cardioprotective effect of ischaemic pre-, and postconditioning is critically dependent on adenosine receptor sub-type stimulation during reperfusion, whereas administration of exogenous adenosine during reperfusion does not reduce infarct size. An alternative explanation could relate to the role of the coronary endothelium in the metabolism of adenosine. As the endothelium acts as a strong metabolic barrier for circulating adenosine by its rapid uptake and intracellular degradation, intravascular administration of adenosine does not affect the myocardial interstitial adenosine concentration, whereas ischaemic pre- and postconditioning do also increase the interstitial concentration [8]. Additional studies are needed to test this hypothesis.