Cyclooxygenase-2 does not mediate late preconditioning induced by activation of adenosine A₁ or A₃ receptors

Experimental Research Laboratory, Division of Cardiology, University of Louisville and Jewish Heart and Lung Institute, Louisville, Kentucky 40292

	ABSTRACT

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Recent studies have demonstrated that the adenosine A₁ receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) and the adenosine A₃ receptor agonist N⁶-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) produce a delayed phase of protection against infarction similar to the late phase of ischemic preconditioning (PC). However, the mechanism for adenosine A₁ or A₃ receptor-induced late PC remains unknown. The goal of this study was to determine whether the delayed cardioprotective effects of adenosine A₁ or A₃ receptors are mediated by cyclooxygenase-2 (COX-2), which is an obligatory mediator of ischemic PC. We found that COX-2 protein expression (Western blotting) did not increase 24 h after the administration of either CCPA (100 µg/kg iv) or IB-MECA (300 µg/kg iv) compared with controls. To probe the role of constitutive COX-2 expression, conscious rabbits were subjected to 30-min coronary occlusion followed by 72-h reperfusion. Twenty-four hours before the occlusion, the rabbits were pretreated with CCPA (100 µg/kg iv) or IB-MECA (300 µg/kg iv). Both CCPA and IB-MECA resulted in a marked (~47%) reduction in infarct size vs. controls [36.2 ± 4.0% of the risk region (n = 9), 31.2 ± 4.7% (n = 9), and 59.5 ± 3.8% (n = 9), respectively; P < 0.05], similar to that induced by the late phase of ischemic PC [31.8 ± 3.2% (n = 9)]. The selective COX-2 inhibitor N-(2-[cyclohexyloxy]4-nitrophenyl)methanesulfonamide (NS-398, 5 mg/kg), which abolished the protective effect of ischemic late PC, failed to block the protection of either CCPA or IB-MECA, indicating that COX-2 does not mediate the delayed protection of either CCPA or IB-MECA [CCPA + NS-398, 29.1 ± 3.4% (n = 7); IB-MECA + NS-398, 34.9 ± 2.9% (n = 8)]. NS-398 in itself did not affect infarct size [54.9 ± 3.7% (n = 9)]. Taken together, these results demonstrate that, in contrast to ischemia-induced late PC, the mechanisms of adenosine A₁ or A₃ receptor-induced late PC is independent of COX-2.

myocardial ischemia; myocardial reperfusion; 2-chloro- N⁶-cyclopentyl-adenosine; N⁶-(3-iodobenzyl)adenosine-5'-N-methyluronamide; NS-398

	INTRODUCTION

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RECENT STUDIES HAVE DEMONSTRATED that a delayed cardioprotective effect against myocardial infarction, late preconditioning (PC), can be elicited not only by exposure to a sublethal ischemic stress (2, 3, 17, 19, 22, 27, 30, 33, 37, 38), but also by pretreatment in the absence of ischemia with ligands that activate either the A₁adenosine receptor, 2-chloro-N⁶-cyclopentyl-adenosine (CCPA) (3, 4, 10, 12, 13, 32, 39), or the A₃ adenosine receptor, N⁶-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA)(32). These findings, coupled with the demonstration that adenosine receptor antagonists block the development of ischemia-induced late PC against infarction (3), point to an important role of A₁ and A₃ adenosine receptors as triggers of late PC (7).

However, the mechanism(s) whereby activation of A₁ or A₃ adenosine receptors results in the subsequent development of a cardioprotected phenotype remain(s) unclear. Two studies have demonstrated that nitric oxide synthase (NOS) inhibitors abrogate the delayed infarct-sparing effects of CCPA, implicating NOS as a mediator of this type of late PC (32, 39); in contrast, other investigations have concluded that NOS does not play a necessary role in CCPA-induced late PC (5, 11). Even less is known regarding the mechanism(s) underlying A₃ adenosine receptor-induced late PC; the only piece of information available is that this phenomenon cannot be blocked by the NOS inhibitor N-nitro-L-arginine (L-NNA) (32).

Another enzyme that has recently emerged as an obligatory mediator of the protective effects of the late phase of ischemia-induced late PC is cyclooxygenase-2 (COX-2) (7). Shinmura et al. (30) have demonstrated that ischemic PC results in rapid upregulation of COX-2 expression and activity, and that the administration of COX-2 selective inhibitors blocks the infarct-sparing effects of late PC in conscious rabbits. Similar findings have been obtained in mice (15). However, whether COX-2 also mediates late PC induced pharmacologically by activation of A₁ or A₃ adenosine receptors remains unknown.

Thus the goal of the present study was to determine the role of COX-2 in the delayed infarct-sparing effects elicited by adenosine A₁ or A₃ receptor agonists. To this end, we utilized a well-established rabbit model of late PC (2, 3, 17, 19, 22, 27, 30, 33, 37, 38) to investigate whether the administration of CCPA or IB-MECA results, 24 h later, in upregulation of COX-2 protein expression and whether CCPA- and IB-MECA-induced delayed cardioprotection is abrogated by the COX-2 selective inhibitor NS-398. All of the studies were performed in conscious animals in an effort to rigorously test the role of COX-2 under conditions that are as physiological as possible. Open-chest animal preparations are associated with a number of factors, such as anesthesia, surgical trauma, fluctuations in temperature, elevated catecholamine and cytokine levels, abnormal hemodynamics, exaggerated formation of reactive oxygen species (6, 20, 35), etc., which may interfere not only with the regulation of COX-2 (36) and the production of arachidonic acid metabolites (25), but also with myocardial infarction (14, 18, 29) and/or ischemic PC (16, 28). In addition, we felt it was important to eliminate experimental conditions, such as surgical trauma, that lead to inflammatory reactions since COX-2 has been implicated as a mediator of inflammation (31, 36). Accordingly, all rabbits were allowed to recover for a minimum of 14 days after surgery.

	MATERIALS AND METHODS

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Experimental Preparation and General Protocol

The experimental preparation has been described in detail previously (1, 8, 9, 21, 23, 26, 27, 33, 34, 37). Briefly, New Zealand White male rabbits (weight, 2.0-2.5 kg; age, 3-4 mo) were instrumented under sterile conditions with a balloon occluder around a major branch of the left coronary artery, a 10-MHz pulsed Doppler ultrasonic crystal in the center of the region to be rendered ischemic, and bipolar electocardiogram (ECG) leads on the chest wall. The animals were allowed to recover for a minimum of 10 days after surgery. Throughout the experiments, rabbits were kept in a cage in a quiet, dimly lit room. Left ventricular (LV) systolic wall thickening (WTh), range gate depth, and the ECG were recorded throughout the experiments on a thermal array chart recorder (Gould TA6000, Valley View, OH). After the administration of adenosine receptor agonists, arterial pressure was measured by cannulating the ear dorsal artery with a 22-gauge angiocatheter under local anesthesia (benzocaine) as previously described (1, 8, 9, 23, 26, 33, 34).

Measurement of COX-2 Protein

To determine whether COX-2 is induced 24 h after pharmacological activation of adenosine A₁ or A₃ receptors, the expression of COX-2 was assessed by standard SDS-PAGE Western immunoblotting techniques (24, 26, 37). Rabbits were assigned to four groups (Fig. 1). Group I (control) did not undergo coronary occlusion. Myocardial samples were rapidly removed from the anterior and posterior LV wall, frozen in liquid N₂, and stored at 140°C until used. Group II (PC) underwent a sequence of six 4-min coronary occlusions interspersed with 4 min of reperfusion without any treatment and was euthanized 24 h later. Myocardial samples were rapidly removed from the ischemic-reperfused region, frozen in liquid N₂, and stored at 140°C until used. Groups III (CCPA) and IV (IB-MECA) received an intravenous bolus of CCPA (100 µg/kg) and IB-MECA (300 µg/kg), respectively, 24 h earlier, and myocardial samples were obtained and stored as in group I. Tissue samples were homogenized in buffer A [25 mM Tris · HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, 1 mM dithiothreitol, 25 mM NaF, and 1 mM Na₃VO₄] and centrifuged at 14,000 g for 12 min at 4°C, and the resulting supernatants were collected as cytosolic fractions (24). The pellets were incubated in a lysis buffer (buffer A + 1% Triton X-100) for 2 h and centrifuged at 14,000 g for 15 min at 4°C, and the resulting supernatants were collected as membranous fractions (24). Gel transfer efficiency was carefully recorded by making photocopies of membranes dyed with reversible Ponceau staining (24, 26); gel retention was determined by Coomassie blue staining (24, 26). Specific monoclonal anti-COX-2 antibodies were purchased from Transduction Laboratories. The COX-2 signals and the corresponding records of Ponceau stains of nitrocellulose membranes were quantitated by an image scanning densitometer, and each COX-2 signal was normalized to the corresponding Ponceau stain signal (24, 26). In all samples, the content of COX-2 protein was expressed as a percentage of the COX-2 protein content in group I (control).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Experimental protocol for measurement of cyclooxygenase-2 (COX-2) protein. Rabbits were assigned to four groups. On day 1, rabbits in group I did not receive any treatment, rabbits in group II underwent six 4-min occlusion (O)/4-min reperfusion (R) cycles, rabbits in group III received an intravenous bolus of 2-chloro-N6-cyclopentyladenosine (CCPA, 100 µg/kg), and rabbits in group IX received an intravenous bolus of N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA, 300 µg/kg). On day 2, tissue samples were obtained in all groups. PC, preconditioning.

Experimental Protocol for the Studies of Infarct Size

To determine whether constitutively expressed COX-2 activity contributes to late PC, rabbits were subjected to a 30-min coronary artery occlusion followed by 3 days of reperfusion. Diazepam was administered 20 min before the onset of ischemia (6 mg/kg ip) to relieve the stress caused by the coronary occlusion. No antiarrhythmic agent was given at any time. Rabbits were assigned to seven groups (Fig. 2). Group V (control) underwent the 30-min occlusion without any pretreatment. Group VI (PC) was preconditioned with six 4-min occlusion/4-min reperfusion cycles 24 h before the 30-min occlusion. Groups VII (CCPA) and VIII (IB-MECA) received an intravenous bolus of CCPA (100 µg/kg) and IB-MECA (300 µg/kg), respectively, 24 h before the 30-min coronary occlusion. These doses of CCPA and IB-MECA have previously been shown to elicit late PC against infarction in this model (32). Groups IX (CCPA + NS-398) and X (IB-MECA + NS-398) received on day 1 the same doses of CCPA and IB-MECA as groups VII and VIII, respectively; 24 h later (on day 2), the rabbits were given an intraperitoneal injection of NS-398 (5 mg/kg) 30 min before the 30-min occlusion. To exclude the possibility that NS-398 itself affects infarct size, group XI (NS-398) was given NS-398 30 min before the 30-min occlusion at the same dose given in groups IX and X. NS-398 has been shown to be 168 times more selective for COX-2 versus COX-1 (36). We (30) have previously found that this dose of NS-398 abolishes the increase in COX-2 activity associated with ischemia-induced late PC and abrogates the concomitant protective effects against myocardial stunning and infarction without any hemodynamic changes. CCPA (Sigma; St. Louis, MO) was dissolved in 0.5 ml of dimethyl sulfoxide (DMSO) and 0.5 ml of normal saline (total volume injected, 1 ml). IB-MECA (Research Biochemicals International; Natick, MA) was dissolved in 0.5 ml of DMSO and 0.5 ml of saline (total volume infused, 1 ml). NS-398 (Cayman Chemicals; Ann Arbor, MI) was dissolved in DMSO (20 mg/ml) and diluted in normal saline (final concentration, 20% DMSO in saline). All solutions were filtered through a 0.2-µm Millipore filter to ensure sterility.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Experimental protocol for the studies of myocardial infarction. Seven groups of rabbits were studied. On day 2, all groups underwent a 30-min coronary occlusion followed by 72 h of reperfusion. On day 1, rabbits in group VI underwent six 4-min occlusion/4-min reperfusion cycles. Rabbits in groups VII and IX received an intravenous bolus of CCPA (100 µg/kg) and rabbits in groups VIII and X received an intravenous bolus of IB-MECA (300 µg/kg). Rabbits in groups IX, X, and XI received an intraperitoneal injection of N-(2-[cyclohexyloxy]4-nitrophenyl)methanesulfonamide (NS-398, 5 mg/kg) 30 min before the 30-min occlusion on day 2.

Measurement of Regional Myocardial Function

Regional myocardial function was assessed as systolic thickening fraction using the pulsed Doppler probe, as previously described (1, 8, 9, 21, 23, 26, 27, 33, 34, 37). The total deficit of systolic WTh over the 3-day reperfusion period (an integrative assessment of the overall severity of contractile dysfunction during this time interval) was calculated by measuring the area between the systolic WTh versus time line and the baseline (100% line) during the 3-day recovery phase after the 30-min coronary occlusion (23, 27, 33, 34). In all animals, measurements from at least 10 beats were averaged at baseline, and at least five beats were averaged at all subsequent time points.

Measurement of Region at Risk and Infarct Size

At the conclusion of the study, the rabbits were given heparin (1,000 units iv), after which they were anesthetized with pentobarbital sodium (50 mg/kg iv) and euthanized with KCl. The heart was excised and the size of the ischemic-reperfused region (region at risk) was determined by tying the coronary artery at the site of the previous occlusion and by perfusing the aortic root for 2 min with a 5% solution of Phthalo blue dye in normal saline at a pressure of 70 mmHg using a Langendorff apparatus (27, 33, 34). The heart was then cut into 6-7 transverse slices, which were incubated for 10 min at 37°C in a 1% solution of triphenyltetrazolium chloride in phosphate buffer (pH = 7.4). All atrial and right ventricular tissues were excised. The slices were weighed, fixed in a 10% neutral buffered formaldehyde solution, and photographed (Nikon AF N6006). Transparencies were projected onto a paper screen at a 10-fold magnification, and the borders of the infarcted, ischemic-reperfused, and nonischemic regions were traced. The corresponding areas were measured by computerized planimetry (Adobe Photoshop, version 4.0), and from these measurements the weight of the infarct was calculated as a percentage of the weight of the region at risk (23, 27, 30, 33, 34, 37).

Statistical Analysis

Data are reported as means ± SE. For intragroup comparisons, hemodynamic variables and WTh were analyzed by a one-way repeated-measures ANOVA followed by Student's t-tests for paired data with the Bonferroni correction. For intergroup comparisons, data were analyzed by either a one-way or a two-way repeated-measures (time and group) ANOVA, as appropriate, followed by unpaired Student's t-tests with the Bonferroni correction. The relationship between infarct size and risk region size was compared among groups with an ANCOVA using the size of the risk region as the covariate. The correlation between infarct size and risk region size was assessed by linear regression analysis using the least-squares method. All statistical analyses were performed using SPSS for Windows version 8.0 and SigmaStat for Windows version 2.0.

	RESULTS

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Exclusions

A total of 82 rabbits were used in this study. All of the 12 rabbits used for the measurement of COX-2 protein completed the protocol (groups I, II, III, and IV). Of the 70 rabbits instrumented for the studies of infarct size, 10 were assigned to group V (control), 9 to group VI (PC), 10 to group VII (CCPA), 12 to group VIII (IB-MECA), 9 to group IX (CCPA + NS-398), 9 to group X (IB-MECA + NS-398), and 11 to group XI (NS-398). Seven rabbits died of ventricular fibrillation during coronary occlusion (1 in group V, 3 in group VIII, 1 in group IX, 1 in group X, and 2 in group XI). One rabbit (group IX) was excluded because of occluder malfunction. One rabbit (group VII) was excluded because of technical problems during the postmortem analysis. Therefore, 9 rabbits completed the experimental protocol in groups V, VI, VII, VIII, and XI, 7 in group IX, and 8 in group X. No rabbit included in the final analysis was subjected to defibrillation.

Studies of COX-2 Expression

In control rabbits (group I), over 99% of total COX-2 protein was found in the membranous fraction, which is consistent with previous reports (30, 31). A representative Western immunoblotting analysis of COX-2 is illustrated in Fig. 3A. A weak COX-2 signal was detected in control hearts (group I; Fig. 3A). When rabbits were given CCPA or IB-MECA 24 h earlier (groups III and IV), the expression of COX-2 did not increase. However, when rabbits were preconditioned with ischemia 24 h earlier (group II), the expression of COX-2 increased markedly (+572 ± 320%; Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of CCPA and IB-MECA on the expression of COX-2 protein in rabbit myocardium. Tissue samples were obtained from the anterior left ventricular (LV) wall of control rabbits (group I), of rabbits given CCPA (group III) or IB-MECA (group IV) 24 h earlier, or from the ischemic-reperfused region of rabbits that underwent ischemic PC with six 4-min coronary occlusion/4-min reperfusion cycles 24 h earlier (group II). A: COX-2 immunoreactivity in the membranous fraction did not increase in the rabbits preconditioned with either CCPA or IB-MECA. Robust COX-2 expression was observed in the ischemic-reperfused region 24 h after ischemic PC and in rabbit kidney and in murine macrophages stimulated with interferon- and lipopolysaccharide (positive controls). B: densitometic analysis of COX-2 signals in the membranous fraction. In all samples, the densitometric measurements of COX-2 immunoreactivity were expressed as a percentage of the average value measured in the anterior LV wall of control rabbits. Data are means ± SE; n, number of rabbits.

Studies of Infarct Size

Hemodynamic variables. Heart rate and WTh fraction on day 1 were comparable in groups VII-X at baseline (Table 1). In group VII, the administration of CCPA reduced heart rate by 24% and mean arterial pressure by 13% at 30 min after injection of the ligand (P < 0.05; Table 1). In group VIII, the administration of IB-MECA reduced heart rate by 12% at 30 min after IB-MECA (P < 0.05) but did not cause any appreciable changes in mean arterial pressure (Table 1). In group IX, the administration of CCPA caused a decrease in heart rate at 30 and 60 min similar to that in group VII (Table 1). In group X, the administration of IB-MECA caused a decrease in heart rate at 60 min similar to that in group VIII (Table 1). However, all of these changes resolved by 2 h after treatment (except heart rate in group IX); on the next day (day 2), baseline hemodynamics were similar among all groups (Table 2).

View this table: [in this window] [in a new window]   Table 1.   Hemodynamic variables and systolic thickening fraction on day 1 in groups VII, VIII, IX, and X

View this table: [in this window] [in a new window]   Table 2.   Heart rate on day 2 at baseline, during occlusion, and during the 72-h reperfusion period

On day 2, there were no appreciable differences in heart rate among groups V, VI, VII, VIII, IX, X, and XI during the 30-min coronary occlusion or during the ensuing 72 h of reperfusion (Table 2). This is consistent with previous studies in conscious rabbits showing that the dose of NS-398 used in this study does not alter heart rate or systemic arterial pressure (30).

Baseline systolic thickening fraction on day 2 was also similar among the seven groups (35.0 ± 4.0%, 31.7 ± 2.6%, 29.1 ± 2.8%, 31.4 ± 3.0%, 28.3 ± 2.0%, 29.4 ± 3.1%, and 34.5 ± 1.9% in groups V, VI, VII, VIII, IX, X, and XI, respectively). Neither CCPA nor IB-MECA caused any appreciable changes in systolic thickening fraction in groups VII, VIII, IX, and X (Table 1).

Region at risk and infarct size. There were no significant differences among the seven groups with respect to the weight of the region at risk [0.92 ± 0.15 g (19.1 ± 2.3% of LV wt), 0.74 ± 0.09 g (17.1 ± 2.1%), 0.73 ± 0.09 g (17.0 ± 1.9%), 0.76 ± 0.09 g (17.0 ± 1.6%), 0.68 ± 0.10 g (19.4 ± 2.5%), 0.58 ± 0.10 g (17.8 ± 3.0%), and 0.84 ± 0.08 g (23.9 ± 2.2%) in groups V-XI, respectively]. The average infarct size was 39% smaller in group VII (CCPA) and 48% smaller in group VIII (IB-MECA) compared with group V (control) (36.2 ± 4.0 and 31.2 ± 4.7% vs. 59.5 ± 3.8% of the risk region, respectively; P < 0.05; Fig. 4), indicating that both CCPA and IB-MECA elicited delayed protection 24 h later. The infarct size in these two groups was similar to that observed in group VI (PC, 31.8 ± 3.2% of the risk region), indicating that the protection induced by CCPA or IB-MECA was equivalent to that induced by ischemic PC. The cardioprotective effects of CCPA and IB-MECA cannot be ascribed to the vehicle used (DMSO) because the same doses of DMSO have been shown previously to have no effect on infarct size 24 h later in this model (32). Infarct size in group IX (CCPA + NS-398, 29.1 ± 3.4% of risk region) and group X (IB-MECA + NS-398, 34.9 ± 2.9% of risk region) did not differ from that in groups VII and VIII, respectively, indicating that the selective COX-2 inhibitor NS-398 failed to abolish the delayed protective effects of either CCPA or IB-MECA. In group XI (NS-398), infarct size (54.9 ± 3.7% of risk region) was indistinguishable from that measured in the control group, indicating that NS-398 does not affect cell death per se (Fig. 4). We (30) have previously demonstrated that the dose of DMSO used as a vehicle for NS-398 on day 2 has no effect on infarct size in this model.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Myocardial infarct size in groups V (control), VI (PC), VII (CCPA), VIII (IB-MECA), IX (CCPA + NS-398), X (IB-MECA + NS-398), and XI (NS-398). Infarct size is expressed as a percentage of the region at risk of infarction. Open circles represent individual rabbits, whereas solid circles represent mean ± SE. *P < 0.05 vs. group V (controls).

In all seven groups, the size of the infarction was positively and linearly related to the size of the region at risk (r = 0.93, 0.66, 0.82, 0.78, 0.91, 0.90, and 0.89, respectively). The regression line was shifted downward in the CCPA and IB-MECA groups compared with the control group (P < 0.05 by ANCOVA; Fig. 5, A and B), whereas the regression line in the NS-398 group did not differ from that observed in the control group (Fig. 5, A and B). In the CCPA + NS-398 and IB-MECA + NS-398 groups, the regression lines were similar to those observed in the CCPA (Fig. 5A) and IB-MECA (Fig. 5B) groups, respectively. These data indicate that for any given size of the region at risk, the resulting infarct size was reduced by pretreatment with CCPA and IB-MECA and that this change was not affected by NS-398.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Relationship between size of the region at risk and size of myocardial infarction. Illustrated are individual values and regression lines obtained by linear regression analysis for control group (group V), PC group (group VI), CCPA group (group VII), and CCPA + NS-398 group (group IX) (A) and control group (group V), PC group (group VI), IB-MECA group (group VIII), and IB-MECA + NS-398 group (group X) (B). In all groups, infarct size was positively and linearly related to risk region size. The linear regression equations were as follows: control group, y = 0.65x  0.04 (r = 0.93); PC group, y = 0.27x + 0.03 (r = 0.66); CCPA group, y = 0.41x  0.03 (r = 0.82); IB-MECA group, y = 0.52x  0.15 (r = 0.78); CCPA + NS-398 group, y = 0.41x  0.07 (r = 0.91); IB-MECA + NS-398 group, y = 0.41x  0.03 (r = 0.90) and NS-398 group, y = 0.75x  0.16 (r = 0.89). ANCOVA demonstrated that the slopes of the regression lines for the PC, CCPA, IB-MECA, CCPA + NS-398, and IB-MECA + NS-398 groups (groups VI, VII, VIII, IX, and X) were significantly less than that for the control group (P < 0.05 for each comparison), indicating that for any given risk region size, infarct size was smaller in rabbits preconditioned with ischemia, CCPA, or IB-MECA even when NS-398 was given on day 2.

In keeping with the infarct size data, the recovery of systolic WTh after the 30-min occlusion was significantly improved in the CCPA and IB-MECA groups compared with that measured in the control group (4.4 ± 4.8 and 6.8 ± 9.7% of baseline vs. 15.0 ± 3.4%, respectively, at 72 h, P < 0.05; Fig. 6, A and B). In the CCPA + NS-398 and IB-MECA + NS-398 groups, the recovery of systolic WTh was also improved compared with the control group (0.0 ± 6.4 and 1.1 ± 6.2% of baseline vs. 15.0 ± 3.4%, respectively, at 72 h, P < 0.05; Fig. 6, A and B). These data provide an independent confirmation of the results obtained with the measurement of infarct size.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Systolic thickening fraction in the ischemic-reperfused region. Measurements were obtained at baseline, 5 min before the 30-min occlusion (Pre-Occl), 15 min into the 30-min occlusion (Occl), and at selected times during the 72-h reperfusion interval. Because of Doppler probe malfunction, complete measurements of thickening fraction could be obtained only in 8 rabbits in group VI, 5 in group VII, 7 in group VIII, and 6 in group IX. Thickening fraction is expressed as a percentage of baseline values. The total deficit of wall thickening (WTh) after infarction is depicted in the inset. The total deficit of WTh was calculated by measuring the area between the systolic WTh versus time line and the baseline (100% line) during the 3-day reperfusion period after the 30-min occlusion (see text). Data are means ± SE. *P < 0.05 between group VII and group V in A and between group VIII and group V in B. In the inset, *P < 0.05 vs. the control group (group V).

	DISCUSSION

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The mechanism of adenosine A₁ or A₃ receptor-induced late PC remains unclear and is the focus of much current investigation. The present study demonstrates that pharmacological activation of either A₁ or A₃ receptors 24 h before myocardial infarction confers significant cardioprotection, confirming our previous findings in this model (32). In contrast to ischemic PC, which elicited a robust upregulation of COX-2 protein expression, pharmacological PC with either CCPA or IB-MECA failed to increase the protein expression of COX-2 24 h later despite the presence of a cardioprotected state. Furthermore, the administration of the COX-2 selective inhibitor NS-398, in doses previously shown to block both COX-2 activity and ischemic PC in this model (30), failed to abolish either CCPA- or IB-MECA-induced late PC. Taken together, these results demonstrate that, in contrast to ischemia-induced late PC, adenosine A₁ or A₃ receptor-induced late PC does not require upregulation of COX-2. The differential role of COX-2 in delayed cardioprotection induced by ischemia versus adenosine receptor activation reveals heretofore unrecognized differences in the mechanisms of these types of cardiac adaptation and suggests that the phenotype of late PC may be underlain by different molecular mechanisms in a stimulus-specific fashion.

Both adenosine A₁ and A₃ receptor agonists have been demonstrated to elicit a delayed cardioprotective effect similar to that elicited by ischemic PC (32). However, the mechanisms responsible for A₁ versus A₃-induced late PC appears to differ. In a recent study (32), we have found that the infarct size limitation observed 24 h after the administration of the A₁-selective agonist CCPA was completely abrogated by the administration of the NOS inhibitor L-NNA given on day 2 before the sustained ischemic episode, implicating NOS (most likely, inducible NOS) as an essential mediator of adenosine A₁-induced late PC. In contrast, the same dose of L-NNA failed to abrogate IB-MECA-induced late PC in the same model, indicating that the delayed cardioprotection triggered by activation of adenosine A₃ receptors does not require enhanced NOS activity (32). Because COX-2 has recently been identified as an obligatory mediator of ischemia-induced late PC (30), we tested the hypothesis that this enzyme could also be a mediator of adenosine A₁ or A₃ receptor-induced late PC. Our results demonstrate that neither CCPA nor IB-MECA produced an appreciable increase in COX-2 protein expression 24 h after their administration, despite the fact that both of these agonists were given in doses known to elicit delayed cardioprotection (32). However, because COX-2 is constitutively expressed in the rabbit heart (Ref. 30 and Fig. 3), it is possible that this enzyme might be activated 24 h after CCPA or IB-MECA treatment and thus contribute to cardioprotection even though its protein expression is unchanged. The studies of infarct size were designed to rule out this possibility. We used the same dose of NS-398 (5 mg/kg) that was previously shown (30) to effectively abolish the enhanced COX-2 activity elicited by ischemia and the concomitant cardioprotection in this same conscious rabbit model. The finding that NS-398 did not inhibit either CCPA- or IB-MECA-induced late PC, coupled with the absence of COX-2 upregulation, demonstrates that COX-2 is not a necessary mediator of these two types of delayed adaptation.

The present study has important corollaries. First, the mechanism that underlies IB-MECA-induced late PC remains unknown, as neither NOS (32) nor COX-2 (present study) is required for this phenomenon to occur. Second, our data imply that the mechanism responsible for adenosine A₁-induced late PC differs from that of ischemia-induced late PC, because COX-2 is necessary for the latter (30) but not for the former (present study). Thus even though ischemia-induced and adenosine A₁-induced late PC share NOS as a common mediator, there are differences with respect to the other protein(s) involved. These differences between adenosine A₁-induced and ischemia-induced late PC were not previously appreciated. An increasingly complex paradigm emerges from the present study taken in conjunction with previous investigations (27, 30, 32, 33), namely, that late PC represents a polygenic adaptation of the heart to stress and that the precise identity of the genes involved in this response varies from one type of stimulus (e.g., ischemia) to other types (e.g., adenosine receptor agonists) and even within the same type of stimuli (e.g., adenosine A₁ versus adenosine A₃ receptor agonists). The diversity of molecular pathways that lead to seemingly identical cardioprotected phenotypes has important implications for future investigations of the manner in which the heart protects itself from ischemia.

	ACKNOWLEDGEMENTS

We gratefully acknowledge Gregg Shirk and Larisa Hodge for expert technical assistance.

	FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Grants R01 HL-43151 and HL-55757 (to R. Bolli), by National Heart, Lung, and Blood Institute Grant R01 HL-60051 and American Heart Association National Center Grant 9630083N (to J. A. Auchampach), by Kentucky American Heart Association Ohio Valley Affiliate Grant 9951533V (to X.-L. Tang), by American Heart Association Kentucky Affiliate Fellowship Award 9804558 (to H. Takano), by the Medical Research Grant Program of the Jewish Hospital Foundation, Louisville, KY, and by the Commonwealth of Kentucky Research Challenge Trust Fund. Eitaro Kodani is an International Research Fellow from Nippon Medical School, Tokyo, Japan.

Address for reprint requests and other correspondence: R. Bolli, Division of Cardiology, Univ. of Louisville, Louisville, KY 40292 (E-mail: rbolli{at}louisville.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 March 2001; accepted in final form 9 April 2001.

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