INTRODUCTION

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ISCHEMIC PRECONDITIONING is not organ specific because it has not only been demonstrated for the heart (17), but also for the kidneys, liver, brain, skeletal muscle, and lung (6). Przyklenk et al. (19) expanded the concept of ischemic preconditioning from intraregional to interregional myocardial protection by showing that a brief coronary artery occlusion (CAO) not only preconditioned the myocardium nourished by that coronary artery but also protected the adjacent virgin myocardium. Gho et al. (10) subsequently showed that 15 min of small intestinal or renal ischemia preceding a 60-min CAO by 10 min was also capable of limiting myocardial infarct size. This interorgan preconditioning (IOPC, remote myocardial preconditioning; preconditioning at a distance) of the heart by preceding transient ischemia in remote organs has been confirmed for the small intestine (24, 28), kidney (8, 18, 23), and skeletal muscle (1). However, not all remote organs may be able to protect the myocardium because De Zeeuw et al. (5) did not find any infarct size limitation when a 60-min CAO was preceded by global cerebral ischemia. Although not yet extensively studied, IOPC of the heart also appears to involve a delayed phase (24, 28).

The mechanism underlying classical ischemic myocardial preconditioning is still incompletely understood, but there is now consensus that it involves the release of a number of local mediators such as adenosine, norepinephrine, and bradykinin during the preconditioning stimulus, which, most likely via different signal transduction pathways (3), activate the mitochondrial ATP-sensitive K⁺ (K) channels (11). The mechanism underlying IOPC is less clear. Gho et al. (10) showed the involvement of a neurogenic pathway in IOPC by preceding small intestinal ischemia as pretreatment with the ganglion blocker hexamethonium abolished the cardioprotection. In that study it was also shown that reperfusion of the occluded mesenteric artery, responsible for small intestinal ischemia, was mandatory [an observation confirmed for the renal bed (23)]. The latter suggests that activation of the neurogenic pathway occurs upon reperfusion (10) or that reperfusion facilitates the transfer of hormonal preconditioning factors from the ischemic organ to the heart (7). Bradykinin (21) and adenosine (18, 23) may also be involved in IOPC of the heart, but the site(s) of action of these mediators, i.e., the ischemic organ or the heart or both, has not been investigated. Takaoka et al. (23) suggested that myocardial adenosine receptors might be involved because the adenosine concentrations in the carotid artery were 10 times higher after renal ischemia than after regional myocardial ischemia. However, based on the hypotension following renal artery reperfusion, Pell et al. (18) proposed that adenosine release from the kidney was insufficient to produce cardioprotection via the circulation. These investigators suggested, based on the observations by Gho et al. (10), that adenosine released in the ischemic kidney stimulated the afferent renal nerves and thereby protected the myocardium. The latter hypothesis was recently confirmed by Ding et al. (8) in anesthetized rabbits.

To further elucidate the mechanism of IOPC, we not only investigated whether activation of adenosine receptors (and which of its subtypes) is involved in the protection by small intestinal ischemia, but we also determined the location(s) (myocardium and/or small intestine) of the adenosine receptors involved. Studies were performed in anesthetized rats. Until recently, it has been assumed that adenosine is not involved in ischemic preconditioning in the rat (9, 14), despite its capability to limit infarct size in this species (27). We have recently shown, however, that adenosine is involved in classical ischemic preconditioning in the rat, but that its role depends critically on the duration of the preconditioning stimulus (15). Capitalizing on our earlier observation that hexamethonium abolished the cardioprotection by small intestinal ischemia (10), we hypothesized that adenosine locally released during small intestinal ischemia stimulates afferent nerves within the mesenteric bed, which via a neurogenic pathway leads to activation of myocardial adenosine receptors before the sustained CAO, thereby preconditioning the heart. This hypothesis also implies that once the myocardial adenosine receptors have been activated, blockade of the neurogenic pathway will not abolish the cardioprotection by small intestinal ischemia.

	METHODS

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Experiments were performed in 228 ad libitum-fed male Wistar rats (280-360 g) in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23, Revised 1996) and with approval of the Erasmus University Rotterdam Animal Care Committee.

Experimental Procedures

Rats that fibrillated during occlusion or reperfusion were allowed to complete the protocol when conversion to normal sinus rhythm occurred spontaneously within 1 min or when resuscitation by gently thumping on the thorax or defibrillation with a modified battery of 9 V was successful within 2 min after onset of fibrillation. Occlusion and reperfusion were visually verified by appearance and disappearance of myocardial cyanosis.

Experimental Design

Protocol I: Choice of IOPC stimulus. Using the identical protocol of Li and Kloner (14), we recently observed (15) that three cycles of 3-min CAO interspersed by 5 min of reperfusion (3-CAO3) provided a greater degree of myocardial protection than a 15-min CAO (CAO15, Fig. 1). Therefore, we first investigated the cardioprotection afforded by IOPC elicited by three cycles of 3-min MAO interspersed by 5 min of reperfusion (3-MAO3, group 1) and compared this with the protection afforded by a single 15-min MAO followed by 10 min of reperfusion [MAO15, historic data (10)]. Because 3-MAO3 failed to afford cardioprotection (see RESULTS), we selected MAO15 as the IOPC stimulus.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Schematic overview of experimental protocols I, II, and III. See text for details and abbreviations.

Protocol II: Involvement of adenosine receptor stimulation in IOPC. To investigate the role of adenosine in IOPC, four groups of rats were studied (Fig. 1). The effect of a single MAO15 on infarct size by 60-min CAO was determined in the absence (Sham, group 2; MAO15, group 3) and presence (50SPT + Sham, group 4; 50SPT + MAO15, group 5) of a nonselective dose (2 × 25 mg/kg iv, 50SPT) of the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (8-SPT). Because we established the involvement of adenosine in this model of IOPC (see RESULTS), we further investigated whether the A₁- and A₃-receptor subtypes are involved. For this purpose, Sham and IOPC animals were pretreated with either an A₁-selective dose (2 × 5 mg/kg iv) of 8-SPT (10SPT, groups 6 and 7) or an A₃-selective dose (3.3 mg/kg iv) of the adenosine receptor antagonist MRS-1191 (MRS, groups 8 and 9) (15). Finally, IOPC animals were pretreated with a combination of 10SPT and MRS (group 10).

Protocol III: Effect of small intestinal adenosine receptor stimulation on myocardial infarct size. To investigate whether activation of adenosine receptors in the small intestine contributes to IOPC, we employed intramesenteric artery infusions of vehicle (50 µl/min, saline IMA, group 11) and adenosine (10 µg/min, ADO IMA, group 12). This dose of adenosine has been shown to stimulate intestinal afferent nerves (12). The involvement of the neurogenic pathway in the cardioprotection by intramesenteric adenosine was verified by pretreating animals with hexamethonium (20 mg/kg iv, Hex + ADO IMA, group 13). To exclude that the intramesenteric artery infusion of adenosine protected the myocardium by direct stimulation of myocardial adenosine receptors (after recirculation), we also infused this dose of adenosine intravenously (ADO IV, group 14). Finally, to exclude that stimulation of the afferent nerves in the liver contributed to the cardioprotection, we also studied the effect of infusion of 10 µg/min adenosine into the portal vein (ADO IPV, group 15).

Protocol IV: Involvement of myocardial adenosine receptor stimulation in IOPC. It is well established that adenosine receptor blockade between the preconditioning stimulus and the sustained CAO abolishes the cardioprotection in ischemic myocardial preconditioning (25). We therefore followed two approaches to study the involvement of myocardial adenosine receptors in IOPC (Fig. 2). First, we established that the neurogenic pathway (required for IOPC of the heart; Hex + MAO15, see Ref. 10) was no longer required for IOPC after 5 min of mesenteric artery reperfusion (MAO15 + Hex, group 16), indicating that the neurogenic pathway had already activated cardiac adenosine receptors. Subsequently, we determined the role of myocardial adenosine receptors by administering 50SPT at 5 min of mesenteric artery reperfusion (MAO15 + 50SPT, group 17). The second approach involved reocclusion of the mesenteric artery 5 min before the 60-min CAO to prevent the subsequently administered 8-SPT would reach the small intestine. To exclude that 8-SPT would reach the small intestine via collaterals, we also occluded in a number of animals the collaterals in the mesenteric vascular bed (16). We first confirmed that a permanent MAO (MAO-P, group 18) was not cardioprotective (10) and then studied IOPC produced by MAO15 + MAO-P (group 19). These experiments revealed a potentation of the cardioprotection by MAO15 + MAO-P (see RESULTS). To establish whether this potentiated cardioprotection also involved the neurogenic pathway, we determined the effects of treatment with hexamethonium administered before MAO15 (Hex + MAO15 + MAO-P, group 20) and more importantly when administered after 5 min of mesenteric artery reperfusion just before MAO-P (MAO15 + Hex + MAO-P, group 21). We then studied the effect of 8-SPT administered after reocclusion of the mesenteric artery (MAO15 + MAO-P + 50SPT, group 22) to obtain further evidence for the involvement of myocardial adenosine receptor activation in IOPC. Finally, we studied the role of myocardial A₁ (MAO15 + MAO-P + 10SPT, group 23) and A₃ (MAO15 + MAO-P + MRS, group 24) adenosine receptors in IOPC.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Schematic overview of experimental protocol IV. See text for details and abbreviations.

Small Intestinal Collateral Blood Flow

Data Analysis and Presentation

	RESULTS

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Mortality

Area At Risk

Infarct Size

Protocol I: Choice of IOPC stimulus. Figure 3 shows that MAO15 and CAO15 afford similar cardioprotection. However, in contrast to 3-CAO3, 3-MAO3 failed to protect the myocardium. Consequently, MAO15 was selected as the stimulus for IOPC.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Cardioprotection by classical and interorgan preconditioning (IOPC). Note that whereas 3-CAO3 was more cardioprotective than CAO15 in classical preconditioning, 3-MAO3 was ineffective in IOPC. Historic data have been presented for Sham, CAO15, and 3-CAO3 (15), and for MAO15 (10). * P < 0.05 vs. Sham; P < 0.05 vs. corresponding 15-min stimulus. See text for details and abbreviations.

Protocol II: Involvement of adenosine receptor stimulation in IOPC. Pretreatment with a nonselective dose of 8-SPT (50SPT) abolished the cardioprotection by MAO15 (Fig. 4). The A₁-selective dose of 8-SPT (10SPT) and the A₃-selective dose of MRS-1191 each attenuated the cardioprotection by MAO15 by 65%. Combined pretreatment with the A₁- and A₃-selective doses of these antagonists (10SPT + MRS) abolished the cardioprotection.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Involvement of adenosine receptor stimulation and of A1- and A3-receptor subtypes in IOPC by MAO15. * P < 0.05 vs. corresponding Sham; P < 0.05 vs. MAO15.

Protocol III: Effect of small intestinal adenosine receptor stimulation on myocardial infarct size. Intramesenteric artery infusion of 10 µg/min adenosine (ADO IMA), but not of its vehicle, limited infarct size (47 ± 4%) to the same extent as MAO15 (Figs. 4 and 5). Pretreatment with hexamethonium abolished the cardioprotection by ADO IMA (Hex + ADO IMA), implying involvement of a neurogenic pathway in the cardioprotection by locally administered adenosine. This is also confirmed by the lack of effect of intravenous adenosine infusion (ADO IV), excluding that recirculation of adenosine during ADO IMA stimulated cardiac adenosine receptors directly. Finally, infusion of adenosine into the portal vein (ADO IPV) did not limit myocardial infarct size, excluding that spillover of adenosine during ADO IMA stimulated adenosine receptors in the liver and contributed to the cardioprotection by ADO IMA.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Involvement of small intestinal adenosine receptor stimulation in IOPC. Intramesenteric artery infusion of 10 µg/min adenosine (ADO IMA) limited infarct size to the same extent as MAO15 (see Fig. 4). The protection by ADO IMA is under neurogenic control because pretreatment with hexamethonium (Hex + ADO IMA) abolished the protection and intravenous adenosine infusion (ADO IV) was ineffective. Stimulation of adenosine receptors in the liver by spillover of adenosine during ADO IMA does not contribute to the protection by ADO IMA, because adenosine infusion into the portal vein (ADO IPV) was ineffective. * P < 0.05 vs. Sham. P < 0.05 Hex + ADO IMA vs. ADO IMA. See text for details and abbreviations.

Protocol IV: Involvement of myocardial adenosine receptor stimulation in IOPC. Figure 6 shows that whereas pretreatment with hexamethonium abolished IOPC (Hex + MAO15, infarct size 74 ± 2%), hexamethonium did not abrogate IOPC when administered at 5 min of mesenteric artery reperfusion (MAO15 + Hex, infarct size 54 ± 2%). In contrast, both pretreatment (50SPT + MAO15, infarct size 74 ± 1%) and treatment at 5 min of mesenteric reperfusion with 8-SPT (MAO15 + 50SPT, infarct size 67 ± 3%) abolished IOPC. Figure 6 also confirms that permanent occlusion of the MAO (MAO-P) did not confer significant cardioprotection (infarct size 63 ± 2%). However, MAO-P potentiated the cardioprotection by MAO15 because infarct size was only 30 ± 5% after MAO15 + MAO-P versus 49 ± 3% after MAO15 (P < 0.05). This enhanced protection was also mediated via a neurogenic pathway as 1) pretreatment with hexamethonium (Hex + MAO15 + MAO-P) completely abrogated all cardioprotection and 2) posttreatment selectively abolished the potentation leaving the protection by MAO15 unperturbed (MAO15 + MAO-P + Hex). Administration of 8-SPT after the mesenteric artery was reoccluded following MAO15 (MAO15 + MAO-P + 50SPT) entirely abolished the protection. In several of these experiments, the collaterals of the mesenteric vascular bed were also occluded to minimize the adenosine antagonist from entering the mesenteric bed via these collaterals and blocking the adenosine receptors in the small intestine (Fig. 6). Both selective A₁- and A₃-receptor blockade blunted the cardioprotection by MAO15 + MAO-P because infarct size was 61 ± 2% after 10SPT and 53 ± 3% after MRS, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Involvement of myocardial adenosine receptor stimulation in IOPC by MAO15. Notice that, whereas posttreatment with hexamethonium (MAO15 + Hex) had no effect on IOPC, posttreatment with 8-SPT (MAO15 + 50SPT) abolished IOPC. To prevent that 8-SPT would reach the adenosine receptors in the small intestine, 8-SPT (50 mg/kg iv) was administered after the mesenteric artery was permanently occluded (MAO-P) following the 15-min MAO (MAO15 + MAO-P + 50SPT). Also shown is that MAO-P potentiated the cardioprotection by MAO15 and that this enhanced protection was also under neurogenic control (MAO15 + Hex + MAO-P). IOPC involved myocardial adenosine receptor stimulation because cardioprotection was abolished in MAO15 + MAO-P + 50SPT. Infarct size was not different in animals in which the small intestine collateral vessels were occluded (animals indicated by triangles). * P < 0.05 vs. Sham; P < 0.05 vs. MAO15; P < 0.05 vs. MAO15 + MAO-P. See text for details and abbreviations.

Heart Rate and Blood Pressure

Small Intestinal Collateral Blood Flow

	DISCUSSION

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The major findings of the present study are the following. First, in contrast to classical ischemic myocardial preconditioning in which 3-CAO3 provided greater cardioprotection than CAO15, 3-MAO3 was unable to limit myocardial infarct size. Second, pretreatment with a nonselective dose of the adenosine receptor antagonist 8-SPT abolished the cardioprotection by MAO15, whereas pretreatment with an A₁-selective dose of 8-SPT or the A₃-selective antagonist MRS-1191 attenuated cardioprotection by MAO15. Third, intramesenteric artery infusion of a dose of adenosine, which was ineffective when infused into either the portal or inferior caval vein, mimics the IOPC by MAO15, which was abolished by pretreatment with hexamethonium. Fourth, hexamethonium abolished IOPC by MAO15 but only when administered before the MAO and not when administered after 5-min of mesenteric artery reperfusion. In contrast, 8-SPT abolished IOPC also when administered after 5-min of mesenteric artery reperfusion. Fifth, whereas a permanent MAO was not cardioprotective by itself, it potentiated the protection by MAO15; this enhanced protection was also abolished by ganglion blockade. Finally, after the myocardium was preconditioned by MAO15, administration of 8-SPT, at a time point that the mesenteric artery was permanently reoccluded to prevent 8-SPT to reach the small intestine, abolished all cardioprotection by IOPC.

IOPC Stimulus

That MAO15 does not yet provide optimal protection was shown when we reoccluded the mesenteric artery permanently after the MAO15. Thus, whereas MAO-P alone was not cardioprotective, we observed that MAO-P potentiated the cardioprotection by MAO15. The reason for the activation of the neurogenic pathway during MAO-P by the preceding of MAO15 is not clear. However, it is likely that the MAO15 not only preconditioned the heart but also preconditioned the small intestine itself possibly via adenosine, calcitonin gene-related peptide, or endogenous opioids (2, 4). It may then be postulated that, whereas the virgin small intestine is unable to activate the neurogenic pathway during MAO and requires reperfusion (10), the preconditioned small intestine is capable of activating the neurogenic pathway during occlusion and thereby enhances the protection by the MAO15. Future studies involving measurement of interstitial adenosine concentrations in the small intestine and discharge rate of the small intestinal afferent nerves are needed to test this hypothesis.

Involvement of Adenosine Receptor Stimulation

Myocardial adenosine receptors. Because in protocol II the adenosine receptor antagonists were administered intravenously and before both MAO15 and 60-min CAO, the data in Fig. 4 do not reveal the site of action of adenosine. To investigate whether the myocardial adenosine receptors were involved, we administered 8-SPT 1) after 5 min of mesenteric artery reperfusion at a time when the cardioprotective mechanism no longer required continued activation of the neurogenic pathway, and 2) after the mesenteric artery had been reoccluded following MAO15 to prevent 8-SPT from reaching the small intestine. In several animals, we additionally ligated the collaterals in the mesenteric vascular bed (16) to exclude that 8-SPT would still reach the small intestine via these vessels during MAO-P in sufficient amounts to prevent activation of the neurogenic pathway. In this situation (MAO15 + MAO-P + 50SPT), 8-SPT abolished all cardioprotection by IOPC (and not only the potentation by MAO-P), implying that the myocardial adenosine receptors must be involved in the mechanism underlying this phenomenon. The myocardial adenosine receptors involved are of both the A₁- and A₃-subtypes. This observation is in agreement with earlier studies in which a role for both receptor subtypes was established when CAO15 was used to precondition the myocardium (15).

Small intestinal adenosine receptors. Intramesenteric infusion of 10 µg/min of adenosine limited myocardial infarct size to the same extent as MAO15. Moreover, because infusion of the same dose of adenosine in the portal vein or inferior caval vein did not protect the myocardium, adenosine must have acted in the small intestine. The action of locally administered adenosine was abolished when rats were pretreated with hexamethonium, which implies that the neurogenic pathway was also involved in this model of pharmacological IOPC. The intramesenteric artery infusion rate of adenosine was chosen such that only the adenosine receptors in the small intestine were activated. However, we do not know how the adenosine concentrations in the small intestine achieved by this route of administration compare with the adenosine concentrations that are achieved during MAO15. It cannot be excluded that in the portal vein higher concentrations were reached upon reperfusion of the ischemic small intestine than during the direct infusion of adenosine into the portal vein. If true, these higher concentrations might have been sufficient to stimulate afferent nerves in the liver and thereby have contributed to the cardioprotection by small intestine ischemia. That the concentrations during intramesenteric adenosine infusion and MAO15 were similar is suggested by the observation that the protection by the intramesenteric artery infusion of adenosine was very similar to the protection by MAO15.