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Am J Physiol Heart Circ Physiol 283: H1943-H1957, 2002. First published July 11, 2002; doi:10.1152/ajpheart.00150.2002
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Vol. 283, Issue 5, H1943-H1957, November 2002

delta -Opioid receptor-induced late preconditioning is mediated by cyclooxygenase-2 in conscious rabbits

Eitaro Kodani, Yu-Ting Xuan, Ken Shinmura, Hitoshi Takano, Xian-Liang Tang, and Roberto Bolli

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although activation of delta -opioid receptors is known to induce both early and late preconditioning (PC) against myocardial infarction, the mechanisms for this salubrious effect are unclear. Furthermore, it is unknown whether delta -opioid receptors can also induce late PC against myocardial stunning. By using conscious rabbits (n = 120) in this study, we found that the delta -opioid receptor agonist (±)-4-{(alpha -R*)-alpha -[(2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl}-N,N-diethylbenzamide (BW-373U86) induced late PC against myocardial stunning 24 h after treatment and that this effect was abolished by the selective cyclooxygenase-2 (COX-2) inhibitors N-[2-(cyclohexyloxy)4-nitrophenyl]methanesulfonamide (NS-398) and celecoxib. This protective effect was also abrogated by the selective delta 1-opioid receptor antagonist 7-benzylidenenaltrexone, indicating that the delta 1-opioid receptor is necessary for BW-373U86-induced late PC. BW-373U86 did not induce early PC against stunning. In addition, BW-373U86 induced late PC against infarction, which was blocked by NS-398. At 24 h after BW-373U86 administration, myocardial COX-2 protein expression and PGE2 and 6-keto-PGF1alpha levels were significantly increased. These results demonstrate that activation of delta -opioid receptors induces late PC against both stunning and infarction via a COX-2-dependent mechanism.

ischemic-reperfused; BW-373U86; 7-benzylidenenaltrexone


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE LATE PHASE OF ISCHEMIC preconditioning (PC) is a cardioprotective phenotypic shift, whereby exposure to a brief ischemic stress increases the tolerance of the heart to stunning and infarction 24-72 h later (4-6, 8-11, 28, 32, 37, 38, 72). Besides ischemia, a delayed cardioprotective effect can be elicited by pretreatment with a variety of pharmacological agents, including nitric oxide (NO) donors (3, 26, 63), adenosine A1 or A3 receptor agonists (2, 6, 30, 60), endotoxin or endotoxin derivatives (17, 68, 69, 73), and bradykinin (31). Recently, activation of delta -opioid receptors has also been shown to induce late PC against myocardial infarction (18, 19). However, whether stimulation of these receptors can also induce late PC against myocardial stunning remains unknown.

Recent evidence indicates that the mechanism responsible for the various forms of late PC is not necessarily identical. Ischemia-induced late PC has been found to be comediated by increased expression and activity of inducible NO synthase (iNOS) (11, 61) and cyclooxygenase-2 (COX-2) (57). NOS has been shown to mediate adenosine A1 receptor-induced late PC against infarction (23, 30, 60, 74). However, NOS does not mediate adenosine A3 receptor-induced late PC (60) and COX-2 does not mediate either adenosine A1 or A3 receptor-induced late PC (30), indicating that adenosine receptor- and ischemia-induced late PC are mechanistically different. Although iNOS is known to mediate delta 1-opioid receptor-induced late PC (23), the role of COX-2 in delta -opioid receptor-induced late PC is unknown.

The present study was undertaken to elucidate these issues. The specific goals were to determine 1) whether pretreatment with the delta -opioid receptor agonist (±)-4-{(alpha -R*)-alpha -[(2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl}-N,N-diethylbenzamide (BW-373U86) induces late PC against myocardial stunning; 2) if so, whether this protection is mediated by COX-2 activity; 3) whether it is mediated via the delta 1-opioid receptor subtype; and 4) whether delta -opioid-induced late PC against infarction is mediated by COX-2. To address these issues, in phase I we tested whether the administration of BW-373U86 affects the severity of myocardial stunning 24 h later and whether this effect is abolished by the administration of the selective COX-2 inhibitors N-[2-(cyclohexyloxy)4-nitrophenyl]methanesulfonamide (NS-398) and celecoxib. Furthermore, we tested whether the selective delta 1-opioid receptor antagonist 7-benzylidenenaltrexone (BNTX) abrogates the ability of BW-373U86 to induce late PC against myocardial stunning. In phase II, we tested whether NS-398 blocks BW-373U86-induced late PC against infarction. In phase III, 24 h after the administration of BW-373U86, we measured the myocardial protein expression of COX-2 and the myocardial content of PGE2 and 6-keto-PGF1alpha , the two major metabolites of arachidonic acid that have previously been shown to increase 24 h after ischemic PC (57).

We also sought to compare the effects of BW-373U86 with those of ischemic PC. Accordingly, we utilized a well-characterized rabbit model in which brief bouts of ischemia have been shown to induce robust protection against myocardial stunning (9, 10, 51). In an effort to investigate these issues under conditions that are as physiological as possible, all studies were performed in conscious, chronically instrumented rabbits. The use of a conscious animal model obviates the potential confounding factors associated with open-chest preparations, such as anesthesia, surgical trauma, fluctuations in temperature, elevated catecholamine and cytokine levels, abnormal hemodynamics, exaggerated formation of reactive oxygen species (7, 33, 64) etc., which may interfere with the severity of myocardial stunning (7, 33) and/or ischemic PC (25, 56). Because COX-2 has been implicated as a mediator of inflammation (59, 65), we felt it was important to perform all studies in the absence of inflammatory reactions to recent trauma. Accordingly, rabbits were allowed to recover for a minimum of 14 days after surgery.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental Preparation and Protocol

The experimental preparation has been described in detail previously (1, 9, 11, 29, 30, 36, 46, 51, 52, 61-63, 71). Briefly, New Zealand White male rabbits (weight, 2.5 ± 0.1 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 ECG leads on the chest wall. The chest wound was closed in layers, and a small tube was left in the thorax for 3 days to aspirate air and fluid postoperatively. Gentamicin was administered before surgery and on the first and second postoperative days (5 mg/kg im each day). Animals were allowed to recover for a minimum of 14 days after surgery. Throughout experiments, rabbits were kept in a cage in a quiet, dimly lit room. Left ventricular systolic wall thickening (WTh), range gate depth, and the ECG were recorded throughout the experiments on a thermal array chart recorder (model TA6000, Gould, Valley View, OH).

Phase I. Studies of Myocardial Stunning

The experimental protocol consisted of three consecutive days of coronary artery occlusions (days 1-3). On each day, the rabbits were subjected to a sequence of six 4-min coronary occlusion/4-min reperfusion cycles (Fig. 1). Performance of successful coronary occlusions was verified by observing development of ST-segment elevation and changes in the QRS complex on the ECG and the appearance of paradoxical systolic wall thinning on the ultrasonic crystal recordings. No sedative or antiarrhythmic agent was given at any time.


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  		Fig. 1.   Experimental protocol for the studies of myocardial stunning (phase I). Seven groups of rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles followed by a 5-h observation period for 3 consecutive days (days 1-3). Rabbits in group I (control) received no pretreatment; rabbits in group II (BW-373U86 on day 0) received BW-373U86 (10 µg/kg iv) 24 h before the first sequence of occlusion-reperfusion cycles; rabbits in group III (BW-373U86 on day 1) received BW-373U86 (10 µg/kg iv) 30 min before the first sequence of occlusion-reperfusion cycles on day 1 without pretreatment; rabbits in group IV (BW-373U86 + NS-398) received BW-373U86 on day 0 and NS-398 (5 mg/kg ip) 30 min before the first sequence of occlusion-reperfusion cycles on day 1; rabbits in group V (BW-373U86 + celecoxib) received BW-373U86 on day 0 and celecoxib (3 mg/kg ip) 30 min before the first sequence of occlusion-reperfusion cycles on day 1; rabbits in group VI [7-benzylidenenaltrexone(BNTX) + BW-373U86] received BNTX (3 mg/kg iv) and BW-373U86 (10 µg/kg iv) 10 min later on day 0; rabbits in group VII (BNTX on day 0) received BNTX alone (3 mg/kg iv) 24 h before the first sequence of occlusion-reperfusion cycles.

Rabbits were assigned to seven groups (Fig. 1). Group I (control) underwent the coronary artery occlusion-reperfusion protocol on days 1-3 without any treatment. To determine whether activation of delta -opioid receptors induces late PC against stunning, in group II (BW-373U86 on day 0), rabbits received an intravenous bolus injection of BW-373U86 (10 µg/kg) 24 h before the first sequence of coronary occlusion-reperfusion cycles (day 0). To determine whether activation of delta -opioid receptors induces early PC against stunning, in group III (BW-373U86 on day 1), rabbits received the same dose of BW-373U86 (10 µg/kg iv) 30 min before the first sequence of coronary occlusion-reperfusion cycles on day 1. This dose of BW-373U86 did not cause any hemodynamic changes in pilot studies (Table 1). To determine whether BW-373U86-induced late PC is mediated by COX-2, in groups IV (BW-373U86 + NS-398) and V (BW-373U86 + celecoxib), rabbits were preconditioned with BW-373U86 on day 0 and then received NS-398 (5 mg/kg ip) or celecoxib (3 mg/kg ip) 30 min before the first sequence of occlusion-reperfusion cycles on day 1. These doses of NS-398 and celecoxib were chosen because they were previously shown to inhibit COX-2 activity during ischemia-induced late PC in this rabbit model and to abolish the protective effect of late PC against myocardial stunning and infarction without causing any hemodynamic changes (57). Furthermore, these doses of NS-398 and celecoxib, in themselves, did not affect the severity of myocardial stunning or infarct size (57). To determine whether BW-373U86 induces late PC via activation of delta 1-opioid receptors, in group VI (BNTX + BW-373U86), rabbits received BNTX (3 mg/kg iv) 10 min before the injection of BW-373U86 on day 0. To determine whether BNTX, in itself, affects the severity of myocardial stunning on day 1, in group VII (BNTX on day 0), rabbits received BNTX (3 mg/kg iv) 24 h before the first sequence of occlusion-reperfusion cycles on day 0. This dose of BNTX has been reported to abolish the infarct-sparing effect of late PC in rats (18, 53, 55). In addition, this dose of BNTX did not change hemodynamic variables in pilot studies (Table 1). BW-373U86 (Research Biochemical International, Natick, MA) was dissolved under sterile conditions in normal saline and diluted to a concentration of 25 µg/ml. NS-398 (Cayman Chemicals; Ann Arbor, MI) and celecoxib (Searle) were dissolved in DMSO (20 mg/ml) and then diluted with normal saline (final concentration, 20% DMSO in saline). BNTX (Tocris Cookson; Mallwin, MO) was dissolved in DMSO (30 mg/ml) and then diluted with normal saline (final concentration, 10% DMSO in saline). All solutions were filtered through a 0.2-µm Millipore filter to ensure sterility.

                              
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  		Table 1.   Hemodynamic variables after administration of BW-373U86 or BNTX

Phase II. Studies of Myocardial Infarction

To examine the effect of BW-373U86 pretreatment on myocardial infarction, 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 four groups (Fig. 2). Group VIII (control) underwent the 30-min occlusion without any pretreatment. Group IX (PC) was preconditioned with six 4-min occlusion/4-min reperfusion cycles 24 h before the 30-min occlusion. Group X (BW-373U86) received an intravenous bolus of BW-373U86 (10 µg/kg) 24 h before the 30-min coronary occlusion. Group XI (BW-373U86 + NS-398) received on day 0 the same dose of BW-373U86 as group X; 24 h later (on day 1), the rabbits were given an intravperitoneal injection of NS-398 (5 mg/kg) 30 min before the 30-min occlusion. Previous studies have shown that this dose of NS-398 abolishes the protective effect of late PC against infarction (57), but it, in itself, does not affect infarct size (30, 57).


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  		Fig. 2.   Experimental protocol for the studies of myocardial infarction (phase II; A) and COX-2 expression and activity (phase III; B). In phase II, four groups of rabbits underwent a 30-min coronary occlusion followed by 72 h of reperfusion. Rabbits in group VIII (control) received no pretreatment; rabbits in group IX (PC) underwent a sequence of six 4-min occlusion/4-min reperfusion cycles 24 h before the 30-min occlusion (day 0); rabbits in group X (BW-373U86) received BW-373U86 (10 µg/kg iv) on day 0; rabbits in group XI (BW-373U86 + NS-398) received BW-373U86 on day 0 and NS-398 (5 mg/kg ip) 30 min before the 30-min occlusion on day 1. In phase III, rabbits in group XII (control) were euthanized without any pretreatment, whereas rabbits in groups XIII (BW-373U86) and XIV (BNTX + BW-373U86) received BW-373U86 (10 µg/kg iv) in the absence (group XIII) or the presence of BNTX (3 mg/kg iv, 10 min before BW-373U86; group XIV) and were euthanized 24 h later.

Measurement of regional myocardial function. Regional myocardial function was assessed as systolic thickening fraction using the pulsed Doppler probe as previously described (9). In studies of myocardial stunning, the total deficit of systolic WTh (an integrative assessment of the overall severity of myocardial stunning) was calculated by measuring the area comprised between the systolic WTh-versus-time line and the baseline (100% line) during the 5-h recovery phase after the sixth reperfusion (1, 9, 11, 29, 36, 51, 63). In all animals, measurements from at least 10 beats were averaged at baseline and from at least five beats at all subsequent time points.

Measurement of region at risk. At the conclusion of the study, 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 hearts were 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 (30, 52, 61, 63). Each 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. In the studies of myocardial stunning (phase I), the region at risk (identified by the absence of blue dye) was separated from the rest of the left ventricle, and both components were weighed. In the studies of myocardial infarction (phase II), 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 (30, 46, 52, 57, 61, 63, 71).

Phase III. Studies of COX-2 Expression and Activity

Rabbits were assigned to three groups (Fig. 2). On day 0, group XII (control) did not receive any treatment, whereas group XIII (BW-373U86) received BW-373U86 and group XIV (BNTX + BW-373U86) received BW-373U86 in the presence of BNTX. BNTX was given intravenously 10 min before BW-373U86. Twenty-four hours later (day 1), rabbits were euthanized and myocardial samples (congruent 500 mg) were rapidly removed from the left ventricular walls, frozen in liquid N2, and stored at -140°C until used.

Western immunoblotting analysis. Tissue samples were homogenized in buffer A, which contained (in mM) 25 Tris · HCl (pH 7.4), 0.5 EDTA, 0.5 EGTA, 1 PMSF, 1 DTT, 25 NaF, and 1 Na3VO4 and 25 µg/ml leupeptin, and centrifuged at 14,000 g for 12 min at 4°C, and the resulting supernatants were collected as cytosolic fractions (47). The pellets were incubated in a lysis buffer (buffer A + 1% Triton X-100) for 2 h and centrifuged 14,000 g for 15 min at 4°C, and the resulting supernatants were collected as membranous fractions (47). Expression of COX-2 was assessed by standard SDS-PAGE immunoblotting techniques (47, 51, 71). Gel transfer efficiency was recorded carefully by making photocopies of membranes dyed with reversible Ponceau staining (47, 51); gel retention was determined by Coomassie blue staining (47, 51). Specific monoclonal anti-COX-2 antibodies were purchased from Transduction Laboratories (Lexington, KY). COX-2 signals and 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 (47, 51). In all samples, the content of COX-2 protein was expressed as a percentage of the COX-2 protein in group XII (control).

PG enzyme immunoassay. PGs were extracted from tissue samples using octadecylsilyl-silica reverse-phase columns (Sep-Pak C18, Waters Associates) as described by Powell (49). By using [3H]PGE2 as an internal standard, percent recovery was estimated to be 81 ± 1 (n = 10). Myocardial content of PGE2 and 6-keto-PGF1alpha was determined using enzyme immunoassay (EIA) kits (PGE2 kit from Cayman Chemical; 6-keto-PGF1alpha kit from Amersham Life Science) as described (43, 50, 57, 58) and expressed as picograms per milligram of protein.

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 one-way or 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 analysis of covariance (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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A total of 120 rabbits were used in this study (13 for the pilot studies, 52 for the studies of myocardial stunning, 38 for the studies of myocardial infarction, and 17 for the studies of COX-2 expression and activity).

Pilot Studies

Pilot studies were conducted in 10 rabbits to identify a dose of BW-373U86 that has no effect on heart rate, arterial blood pressure, and systolic WTh. The concern was that hemodynamic perturbations caused by this agent (e.g., a fall in blood pressure or an increase in heart rate) could contribute nonspecifically to induce a late PC effect unrelated to delta -opioid receptor stimulation. Arterial pressure was measured by cannulating the ear dorsal artery with a 22-gauge angiocatheter under local anesthesia (benzocaine), as previously described (9). Rabbits were given BW-373U86 as an intravenous bolus injection. In one rabbit, a dose of 300 µg/kg of BW-373U86 caused a sustained (120 min) increase in heart rate (+47%) and a decrease in mean arterial pressure (-13%). In three rabbits, 30 µg/kg of BW-373U86 also caused a significant increase in heart rate (+31 ± 8%) and decrease in mean arterial pressure (-11 ± 2%). Therefore, we reduced the dose of BW-373U86 to 10 µg/kg, which did not cause any appreciable changes in heart rate, mean arterial pressure, or WTh in six rabbits (Table 1). In addition, we measured hemodynamic variables after escalating doses of BNTX in three rabbits. Even at the highest dose (3 mg/kg), which was previously used in rats (18, 53, 55), BNTX did not cause any change in heart rate or mean arterial pressure (Table 1). On the basis of these pilot studies, we selected a dose of 10 µg/kg of BW-373U86 and 3 mg/kg of BNTX.

Phase I. Studies of Myocardial Stunning

Exclusions and postmortem analysis. Of the 52 rabbits instrumented for the studies of myocardial stunning, seven were assigned to group I (control), nine to group II (BW-373U86 on day 0), seven to group III (BW-373U86 on day 1), nine to group IV (BW-373U86 + NS-398), six to group V (BW-373U86 + celecoxib), seven to group VI (BW-373U86 + BNTX), and seven to group VII (BNTX on day 0). A total of six rabbits were excluded. Two rabbits (in group II) developed myocardial infarction after the six coronary occlusion-reperfusion cycles on day 1. Four rabbits died of ventricular fibrillation during coronary occlusion on day 2 (one rabbit in group IV) or day 3 (one rabbit each in groups IV, VI, and VII). Therefore, a total of seven rabbits completed the experimental protocol in groups I-IV and six in groups V-VII. Postmortem analysis showed that the size of the occluded-reperfused vascular bed did not differ significantly in the nine groups (data not shown). Tissue staining with triphenyltetrazolium confirmed the absence of infarction in all animals. In all rabbits, the ultrasonic crystal was found to be at least 3 mm from the boundaries of the ischemic-reperfused region.

Hemodynamic variables. There were no significant differences in heart rate 30 min after the administration of BW-373U86 or BNTX on day 0 in groups II and IV-VII (Table 2). On days 1-3 there were no appreciable differences in heart rate among the seven groups either during the sequence of coronary occlusion-reperfusion cycles or during the 5-h reperfusion period (Table 2).

                              
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  		Table 2.   Heart rate during coronary occlusion and reperfusion in phase I

Regional myocardial function. There were no significant differences in baseline systolic thickening fraction among all groups on the same day, or among different days within the same group (Table 4). Furthermore, within the same group there were no significant differences among days 1-3 with respect to the extent of paradoxical systolic thinning during the six occlusions (Figs. 3-6).


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  		Fig. 3.   Systolic thickening fraction in the ischemic-reperfused region in group I (control) 5 min before the 1st occlusion (baseline), 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval after the 6th occlusion. Thickening fraction is expressed as a percentage of baseline values. Data are means ± SE.



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  		Fig. 4.   Systolic thickening fraction in the ischemic-reperfused region in group II (BW-373U86 on day 0) and group III (BW-373U86 on day 1) 5 min before the first occlusion or before the administration of BW-373U86 (baseline), just before the first occlusion (pre-O), 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval after the 6th occlusion. To facilitate comparisons, the data pertaining to day 1 of group I (control) are also shown (dashed and dotted line without symbols). Thickening fraction is expressed as a percentage of baseline values. Data are means ± SE.



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  		Fig. 5.   Systolic thickening fraction in the ischemic-reperfused region in group IV (BW-373U86 + NS-398) and group V (BW-373U86 + celecoxib) before the administration of NS-398 or celecoxib (baseline), just before the first occlusion, 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval after the 6th occlusion. To facilitate comparisons, the data pertaining to day 1 of group I (control) are also shown (dashed and dotted line without symbols). Thickening fraction is expressed as a percentage of baseline values. Data are means ± SE.



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  		Fig. 6.   Systolic thickening fraction in the ischemic-reperfused region in group VI (BNTX + BW-373U86) and group VII (BNTX on day 0) 5 min before the 1st occlusion (baseline), 3 min into each coronary occlusion, 3 min into each reperfusion, and at selected times during the 5-h reperfusion interval after the 6th occlusion. To facilitate comparisons, the data pertaining to day 1 of group I (control) are also shown (dashed and dotted line without symbols). Thickening fraction is expressed as a percentage of baseline values. Data are means ± SE.

Group I (control). On day 1, the thickening fraction remained significantly (P < 0.05) depressed for 5 h after the sixth reperfusion (Fig. 3), indicating that the sequence of six 4-min occlusion/4-min reperfusion cycles resulted in severe myocardial stunning. On days 2 and 3, however, the recovery of WTh after the six occlusion-reperfusion cycles was markedly improved compared with day 1 (Fig. 3). The total deficit of WTh after the sixth reperfusion was 40% less on day 2 and 42% less on day 3 compared with day 1 (P < 0.05) (Fig. 7). Thus, as expected (9, 11, 51), myocardial stunning was attenuated markedly, and to a similar extent, on days 2 and 3 compared with day 1.


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  		Fig. 7.   Total deficit of wall thickening after the 6th reperfusion on days 1-3 in groups I-VII. Data are means ± SE.

Effect of the delta -opioid receptor agonist on myocardial stunning (groups II and III). Although the extent of paradoxical wall thinning on day 1 was similar in group II compared with that noted in control rabbits, the recovery of WTh after the sixth reperfusion was markedly faster than in the control group, and this improvement was sustained throughout the entire reperfusion interval (Fig. 4A). The total deficit of WTh in group II was 56% less than that observed in control rabbits on day 1 (P < 0.05) and similar to that observed in control rabbits on days 2 and 3 (Fig. 7). On days 2 and 3, there was no further improvement in either the recovery of WTh (Fig. 4A) or the total deficit of WTh (Fig. 7) compared with day 1. Thus pretreatment with BW-373U86 24 h before the sequence of six coronary occlusion-reperfusion cycles resulted in an attenuation of myocardial stunning on day 1 that was essentially indistinguishable from that effected by ischemic PC, indicating the development of delta -opioid receptor-induced late PC against myocardial stunning; the superimposition of ischemic PC had no additive effect on myocardial stunning on day 2. In contrast, when rabbits received BW-373U86 30 min before the first sequence of six occlusion-reperfusion cycles on day 1 (group III), the recovery and total deficit of WTh were similar to those noted in control rabbits (Figs. 4B and 7). Thus BW-373U86 given on day 1 did not affect either the severity of myocardial stunning on day 1 (indicating that delta -opioid receptor activation does not induce early PC against stunning) or the development of late PC against stunning on day 2.

Effect of COX-2 inhibitors on myocardial stunning (groups IV and V). To determine the role of COX-2 in BW-373U86-induced late PC, rabbits in groups IV and V were given NS-398 or celecoxib on day 1. In contrast to group II, in groups IV and V the recovery and total deficit of WTh on day 1 were not improved compared with control rabbits (Fig. 5). Specifically, in groups IV and V, the thickening fraction remained significantly (P < 0.05) depressed for 4 h after the sixth reperfusion and was essentially superimposable to that noted in control rabbits on day 1 (Fig. 5); furthermore, the total deficit of WTh was indistinguishable from that measured in control rabbits on day 1 (Fig. 7), indicating that both NS-398 and celecoxib abolished BW-373U86-induced late PC. On days 2 and 3, the recovery of WTh (Fig. 5) and the total deficit of WTh (Fig. 7) were significantly improved compared with day 1 in both groups IV and V, indicating that the ischemic stimulus associated with the six occlusion-reperfusion cycles on day 1 induced a late PC effect against myocardial stunning on days 2 and 3.

Effect of the delta 1-opioid receptor antagonist on myocardial stunning (groups VI and VII). To determine whether BW-373U86 induces late PC via activation of delta 1-opioid receptors, in group VI the selective delta 1-opioid receptor antagonist BNTX (48) was coadministered with BW-373U86 on day 0. In contrast to group II, in group VI the recovery and total deficit of WTh were similar to those noted in control rabbits (Figs. 6A and 7), indicating that BNTX prevented the development of BW-373U86-induced late PC. When BNTX was given on day 0 without BW-373U86 (group VII), the recovery and total deficit of WTh were also similar to those noted in control rabbits (Figs. 6B and 7), indicating that BNTX in itself did not affect myocardial stunning on day 1.

Phase II. Studies of Myocardial Infarction

Exclusions. Of the 38 rabbits instrumented for the studies of myocardial infarction, nine were assigned to group VIII (control), nine to group IX (PC), eight to group X (BW-373U86), and 12 to group XI (BW-373U86 + NS-398). Six rabbits (one rabbit each in groups VIII and IX, and four in group XI) died of ventricular fibrillation during coronary occlusion. Therefore, a total of eight rabbits completed the experimental protocol in groups VIII-XI.

Hemodynamic variables. There were no significant differences in heart rate 30 min after the administration of BW-373U86 on day 0 in groups X and XI (Table 3). On day 1, there were no appreciable differences in heart rate among the four groups during the 30-min coronary occlusion or during the ensuing 72 h of reperfusion (Table 3). Baseline systolic thickening fraction did not differ among all groups on the same day or among different days within the same group (Table 4).

                              
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  		Table 3.   Heart rate during coronary occlusion and reperfusion in phase II


                              
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  		Table 4.   Systolic thickening fraction on each experimental day

Region at risk and infarct size. There were no significant differences among the four groups with respect to the weight of the region at risk (data not shown). The average infarct size was 42% smaller in group X (BW-373U86) compared with group VIII (control) (32.1 ± 3.3 vs. 54.9 ± 3.1% of the risk region, P < 0.05; Fig. 8), indicating that BW-373U86 elicited delayed protection 24 h later. The infarct size in group X was similar to that observed in group IX (PC, 34.2 ± 3.4% of the risk region), indicating that the protection induced by BW-373U86 was equivalent to that induced by ischemic PC. Infarct size in group XI (BW-373U86 + NS-398, 52.2 ± 4.2% of risk region), however, did not differ from that in group VIII, indicating that NS-398 abolished the delayed protective effects of BW-373U86 (Fig. 8).


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  		Fig. 8.   Myocardial infarct size in groups VIII-XI. Infarct size is expressed as a percentage of the region at risk of infarction. Open circles represent individual rabbits, whereas solid circles represent means ± SE. *P < 0.05 vs. group VIII (control); dagger P < 0.05 vs. group X (BW-373U86).

In all four groups, the size of the infarction was positively and linearly related to the size of the region at risk (r = 0.92, 0.78, 0.95, and 0.92, respectively). The regression line was shifted downward in the PC and BW-373U86 groups compared with the control group (P < 0.05 by ANCOVA) (Fig. 9), whereas the regression line in the BW-373U86 + NS-398 group did not differ from that observed in the control group (Fig. 9). These data indicate that for any given size of the region at risk, the resulting infarct size was reduced by pretreatment with BW-373U86 and that this change was abolished by NS-398.


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  		Fig. 9.   Relationship between size of the region at risk and size of myocardial infarction. Both individual values and regression lines obtained by linear regression analysis for the control group (group VIII), the PC group (group IX), the BW-373U86 group (group X), and the BW-373U86 + NS-398 group (group XI) are shown. In all groups, infarct size was positively and linearly related to risk region size. The linear regression equations were as follows: control group, y = 0.48x+0.05 (r = 0.92); PC group, y = 0.40x -0.03 (r = 0.78); BW-373U86 group, y = 0.50x -0.13 (r = 0.95); BW-373U86 + NS-398 group, y = 0.44x +0.08 (r = 0.92). The regression line was shifted downward in the PC and BW-373U86 groups compared with the control group (P < 0.05 by analysis of covariance, respectively), indicating that for any given risk region size, infarct size was smaller in rabbits preconditioned with ischemia or BW-373U86 compared with control rabbits. In contrast, in the BW-373U86 + NS-398 group, the regression line did not differ from that observed in the control group, indicating that the infarct-sparing effect of BW-373U86 was abolished by treatment with NS-398 on day 1.

Phase III. Studies of COX-2 Expression and Activity

Expression of COX-2 protein. In control rabbits (group XII), over 99% of total COX-2 protein was found in the membranous fraction, which is consistent with previous reports (57, 59). A representative Western immunoblotting analysis of both COX-1 and COX-2 is illustrated in Fig. 10. A weak COX-2 signal was detected in control hearts (group XII). When rabbits were given BW-373U86 (10 µg/kg iv) 24 h earlier (group XIII), the expression of COX-2 increased significantly (+81 ± 32% vs. control, P < 0.05) (Fig. 10). BW-373U86-induced increase in COX-2 expression was completely abolished by the coadministration of the selective delta 1-opioid receptor antagonist BNTX (group XIV) (Fig. 10), indicating that activation of delta 1-opioid receptors is necessary for the increase in BW-373U86-induced COX-2 expression. No expression of COX-2 protein was detectable in the cytosolic fractions (data not shown). In contrast to COX-2, COX-1 protein expression in the membranous fraction was not affected by pretreatment with either BW-373U86 alone (group XIII) or BW-373U86 in the presence of BNTX (group XIV) (Fig. 10).


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  		Fig. 10.   Effect of BW-373U86 on the expression of cyclooxygenase (COX)-2 and COX-1 protein in rabbit myocardium. Tissue samples were obtained from the left ventricular wall of control rabbits that were euthanized without any pretreatment (group XII) or 24 h after the administration of BW-373U86 (BW) alone (group XIII) or in the presence of BNTX (group XIV). A, left: COX-2 immunoreactivity in the membranous fraction increased 24 h after the administration of BW-373U86 and the increase was prevented by the coadministration of the delta 1-opioid receptor antagonist BNTX; A, right: in contrast, COX-1 immunoreactivity was not affected by the pretreatment of BW-373U86 24 h earlier. B: densitometic analysis of COX-2 and COX-1 signals in the membranous fraction. In all samples, the densitometric measurements of COX immunoreactivity were expressed as a percentage of the average value measured in the control rabbits. Data are means ± SE.

Myocardial PG content. To determine whether the increase in COX-2 protein expression was associated with increased COX-2 enzymatic activity, the myocardial content of PGE2 and 6-keto-PGF1alpha (the stable metabolite of PGI2) was measured using EIA. We focused on PGE2 and 6-keto-PGF1alpha because these are the two metabolites of arachidonic acid that were previously shown to be increased during ischemia-induced late PC in conscious rabbits (57). The administration of BW-373U86 resulted in a significant increase in both PGE2 and 6-keto-PGF1alpha levels 24 h later compared with control rabbits [1,025 ± 206 vs. 413 ± 58 pg/mg of protein, +148 ± 50% (P < 0.05) and 1,191 ± 82 vs. 841 ± 75 pg/mg of protein, +42 ± 10% (P < 0.05), respectively (Fig. 11)].


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  		Fig. 11.   Effect of BW-373U86 on the myocardial content of PGE2 (A) and 6-keto-PGF1alpha (B; measured by enzyme immunoassay). In rabbits pretreated with BW-373U86 24 h earlier (group XIII), the levels of PGE2 and 6-keto-PGF1alpha were increased vs. control rabbits (group XII). Data are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that activation of delta -opioid receptors induces late (but not early) PC against myocardial stunning in conscious, chronically instrumented rabbits, and that this protective phenomenon is mediated by upregulation of COX-2. The fact that the PC effect induced by BW-373U86 was completely abolished by the coadministration of the selective delta 1-opioid antagonist BNTX demonstrates that it was dependent on activation of delta 1-opioid receptors. Furthermore, this study demonstrates that delta -opioid receptor-induced late PC against infarction is also mediated by COX-2. Previous reports have indicated that activation of delta -opioid receptors induces both early and late PC against myocardial infarction (18-22, 27, 55) and that this protective phenomenon involves free radicals (15, 39, 40, 45), protein kinase C (PKC) (22, 27, 35, 41, 67), Gio proteins (53, 55), ATP-sensitive K+ channels (15, 18, 20, 27, 34, 39, 40, 54, 55), and MAPKs (19, 21, 24). However, it remained unknown whether stimulation of delta -opioid receptors can also induce late PC against myocardial stunning; furthermore, virtually nothing was known regarding the identity of the protein(s) responsible for mediating the delayed cardioprotection induced by delta -opioid receptors. To our knowledge, this is the first study to demonstrate that activation of delta -opioid receptors (and, specifically, of the delta 1-subtype) elicits a robust late PC effect against myocardial stunning and that this effect is equivalent to that elicited by ischemia. This is also the first study to identify COX-2 as an essential mediator of delta -opioid receptor-induced late PC against both reversible (stunning) and irreversible (infarction) injury. The discovery that activation of delta 1-opioid receptors upregulates the expression and activity of COX-2 in the myocardium has potentially important implications for our understanding of the functional significance of these poorly understood receptors as well as for the regulation of COX-2 expression in the heart.

Shinmura et al. (57) have shown that COX-2 activity is required for the manifestation of ischemia-induced late PC against both myocardial stunning and infarction in conscious rabbits (57). Because of this and because in the present investigation, Western immunoblotting and biochemical analyses demonstrated that activation of delta -opioid receptors upregulates COX-2 protein expression and activity 24 h later (Figs. 10 and 11), we tested the potential role of COX-2 as a mediator of delta -opioid receptor-induced late PC. To this end, we utilized two structurally unrelated COX-2 selective inhibitors, NS-398 and celecoxib, which have been reported to be 168 and 375 times more selective, respectively, for COX-2 vs. COX-1 (65). The doses of NS-398 and celecoxib used in this study have been previously shown to completely inhibit the increased COX-2 activity associated with ischemia-induced late PC in conscious rabbits (57). Our present finding that both NS-398 and celecoxib given on day 1 abrogated delta -opioid receptor-induced late PC demonstrates that COX-2 activity is required for the manifestation of this protective phenomenon. These results further support the concept that COX-2 upregulation is a crucial mechanism of late PC and suggest that this enzyme plays an important role in pharmacologically induced delayed cardioprotection.

Previous studies have demonstrated that the cardioprotective actions of opioid receptor agonists can be ascribed specifically to the delta 1-opioid receptor (18, 53, 55). Although BW-373U86 is known to be a selective delta -opioid receptor agonist (13, 14, 16), it is not selective for the delta 1-subtype (66, 70). Therefore, it is unclear whether BW-373U86 induces late PC specifically via stimulation of delta 1-opioid receptors. To elucidate this issue, we used the selective delta 1-opioid receptor antagonist BNTX, which has been reported to bind with 100-fold more avidity to delta 1 than delta 2 sites in guinea pig brain membranes (48). Our finding that the coadministration of BNTX with BW-373U86 completely abrogated BW-373U86-induced late PC against myocardial stunning indicates that this agent acts, at least in part, via activation of delta 1 receptors, consistent with previous reports (18, 53, 55). Nevertheless, our data do not conclusively demonstrate that the protection against infarction afforded by BW-373U86 was specifically dependent on delta -opioid receptors, because we did not study the effect of BNTX on late PC against infarction.

Results obtained with the coadministration of BNTX and BW-373U86 in the present study differ from those obtained by Patel et al. (45). These authors showed that the delayed cardioprotective effects elicited by BW-373U86 in rats are only partially dependent on activation of delta -opioid receptors, because they could not be completely blocked by BNTX and involve a BW-373U86-initiated free radical mechanism, because they could be blocked by the antioxidant 2-mercaptopropionyl glycine. Possible reasons for this apparent discrepancy are the differences in species (rabbits vs. rats) and doses (0.1 mg/kg vs. 10 µg/kg of BW-373U86 in the present study). Furthermore, because we did not study the effects of BNTX on BW-373U86-induced late PC against infarction, we cannot rule out the possibility that a delta -opioid-independent mechanism of action of this agent could also contribute to the protection against infarction in our rabbit model. The importance of the differences in doses is underscored by the fact that Patel et al. (45) found no changes in heart rate or arterial pressure with 0.1 mg/kg BW-373U86 in rats, whereas in our pilot studies even 30 µg/kg BW-373U86 was sufficient to affect mean arterial pressure and heart rate. It appears, therefore, that the threshold at which BW-373U86 elicits hemodynamic changes is lower in rabbits compared with rats. By using TAN-67, a delta 1-opioid receptor agonist, Fryer et al. (18) found a late PC effect at doses of 10 or 30 mg/kg.

In conclusion, our understanding of the function of delta -opioid receptors in the heart continues to evolve. The present data significantly expand present knowledge regarding these receptors by demonstrating that 1) delta -opioid receptor activation induces a delayed PC effect against myocardial stunning; 2) at the same time, stimulation of these receptors upregulates the protein expression and activity of COX-2 in the heart; and 3) delta -opioid receptor-induced late PC against both stunning and infarction is dependent on COX-2 activity. The fact that the administration of BW-373U86 on day 1 had no effect on the severity of myocardial stunning on the same day demonstrates that stimulation of delta -opioid receptors does not induce an early phase of protection against myocardial stunning, which is consistent with the notion that ischemia does not induce early PC against stunning (8, 12, 42, 44). From a practical standpoint, the demonstration that activation of delta 1-opioid receptors induces late PC against myocardial stunning could have therapeutic implications for the use of opioids in patients with coronary artery disease as well as for the development of new approaches to the protection of ischemic myocardium.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Gregg Shirk and Larisa Hodge for expert technical assistance and Marcia Joines and Carla Hilse for expert secretarial assistance.


    FOOTNOTES

E. Kodani is an International Research Fellow from Nippon Medical School, Tokyo, Japan. This study was supported, in part, by National Heart, Lung, and Blood Institute Grants R01-HL-43151, HL-55757, and HL-68088 (to R. Bolli) and HL-65660 (to Y.-T. Xuan), by National American Heart Association Grant 0150074 (to Y.-T. Xuan), by Kentucky American Heart Association Ohio Valley Affiliate Grant 9951533V (to X.-L. Tang), by the Medical Research Grant Program of the Jewish Hospital Foundation, Louisville, KY, and by the Commonwealth of Kentucky Research Challenge Trust Fund.

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

July 26, 2002;10.1152/ajpheart.00150.2002

Received 25 February 2002; accepted in final form 8 July 2002.


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Am J Physiol Heart Circ Physiol 283(5):H1943-H1957
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