Cyclooxygenase-2-derived prostacyclin mediates opioid-induced late phase of preconditioning in...

transcript

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Cyclooxygenase-2-derived prostacyclin mediates opioid-induced late phase of

preconditioning in isolated rat hearts

Ken Shinmura, Maiko Nagai, Kayoko Tamaki,

Masato Tani, and *Roberto Bolli.

Department of Internal Medicine, Keio University School of Medicine, Tokyo, JAPAN

160-8252 and *Division of Cardiology, University of Louisville and Jewish Hospital

Heart and Lung Institute, Louisville, KY USA 40202.

Running title: PGI2 mediates opioid-induced late PC

Address for correspondence: Ken Shinmura, M.D., Ph.D.Department of Internal Medicine,Keio University School of Medicine35 Shinanomachi, Shinjuku-kuTokyo, JAPAN 160-8582Telephone: +81-(3)-3353-1211, Ext. 62915Fax: +81-(3)-5269-2468E-mail: shimmura@sc.itc.keio.ac.jp

AJP-Heart Articles in PresS. Published on August 8, 2002 as DOI 10.1152/ajpheart.00209.2002

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ABSTRACT

Opioids confer biphasic (early and late) cardioprotection against myocardial infarction

by opening mitochondrial KATP channels. It is unknown whether cyclooxygenase-2

(COX-2), which mediates ischemia-induced late preconditioning, also mediates opioid-

induced cardioprotection. Isolated perfused rat hearts were subjected to 20 min of

global ischemia followed by 20 min of reperfusion. BW373U86 (BW), a δ-opioid

receptor agonist, was administered 1, 12, or 24 h before sacrifice. The recovery of left

ventricular developed pressure (LVDP) after ischemia/reperfusion improved when BW

was administered 1 or 24 h before ischemia (control: 57±8, BW 1 h: 75±5, BW 24 h:

85±6%) but not when it was administered 12 h before (60±5%). The levels of 6-keto-

PGF1α (a stable metabolite of PGI2) in coronary effluent after 20 min of reperfusion

were higher with 24-h BW pretreatment than in controls (1053±92 vs. 724±81 pg/mL),

whereas 6-keto- PGF1α levels at baseline did not differ. Administration of a selective

COX-2 inhibitor, NS-398, abolished the late phase of cardioprotection (recovery of

LVDP, 53±8%) and attenuated the increase in PGI2 (706±138 pg/mL) but did not block

the early phase of cardioprotection. The selective COX-1 inhibitor, SC-560, did not

affect either phase of protection. Western immunoblotting revealed upregulation of

PGI2 synthase protein 24 h after BW administration without changes in COX-1 and

COX-2 protein levels. In conclusion, the late (but not the early) phase of δ-opioid

receptor-induced preconditioning is mediated by COX-2. A functional coupling

between COX-2 and upregulated PGI2 synthase appears to underlie this

cardioprotective phenomenon in the rat.

Keywords: cyclooxygenase, myocardial ischemia, opioid, prostacyclin, reperfusion

injury.

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Opioids have been shown to confer biphasic (early phase and late phase)

cardioprotection against myocardial infarction [12-16,28,31,32] similar to ischemic

preconditioning (PC) [4,41]. Opioid-induced cardioprotection was first described by

Schultz et al. [31], who demonstrated that opioids mimic the early phase of ischemic

PC via the activation of the δ1-opioid receptors and a Gi/o protein-mediated mechanism.

Fryer et al. [12] reported that stimulation of δ1-opioid receptors 24 to 48 h before an

ischemic insult also induces a late phase of cardioprotection against myocardial

infarction. Their recent studies have revealed that activation of PKC-δ and subsequent

activation of the p44-isoform of extracelluar signal-regulated kinase and tyrosine

kinases are essential in the development of opioid-induced cardioprotection [13-

15,21,28]. In contrast to the intense research related to the signaling pathways that

lead to δ-opioid-dependent cardioprotection, little is known regarding the effectors

(mediators) of this phenomenon. Gross and his colleagues demonstrated that both

phases of opioid-induced cardioprotection are abolished by the administration of 5-

hydroxydecanoic acid [12,21,26,32], indicating that opening of mitochondrial KATP

channels is involved. However, the cardioprotective protein(s) that mediate the

beneficial effects of opioids remain to be identified.

Ischemic PC is a biphasic phenomenon [4,41]. The rapid nature of the early

phase suggests that it involves the modification of proteins that are already present. In

contrast, the late phase of ischemic PC requires the synthesis of cardioprotective

proteins that are the effectors (mediators) of protection 12 to 72 h after ischemic PC

[4]. Pharmacologic and genetic evidence indicates that upregulation of inducible NO

synthase (iNOS) is essential for late PC [3,5,18]. In addition to iNOS, we recently found

that cyclooxygenase-2 (COX-2) mediates the protective effects of ischemia-induced

late PC in rabbits and mice [17,33,34]. Analysis of COX byproduct levels suggests that

COX-2 mediates the late phase of cardioprotection via increased production of

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cytoprotective prostanoids, mainly PGI2 and PGE2 [33,34]. In contrast, COX-2 does not

mediate late PC induced pharmacologically by activation of adenosine A1 or A3

receptors [23]. The discrepancy between these findings suggests that differences in the

signaling pathways exist between ischemic and pharmacological PC. Recent findings

suggest that COX-2 mediates opioid receptor-induced late PC in rabbits [24].

However, whether COX-2 or any prostanoid is involved in opioid-induced

cardioprotection in other species has not been examined. Furthermore, it remains

unknown whether opioid-induced late PC is mediated by an increase in the expression

of COX-2 itself or in one of the PG synthases that operate downstream of COX-2 [36].

The aims of this study were (1) to determine whether COX-1 or COX-2 mediates

opioid-induced cardioprotection in rats, and (2) to determine the mechanism(s)

whereby COX-2 is involved in cardioprotection. Using a potent nonpeptide δ-opioid

receptor agonist, BW373U86, we demonstrate that the opioid-induced late phase of

cardioprotection is mediated by COX-2. In addition, we found evidence for a novel,

heretofore unrecognized functional coupling between COX-2 and upregulated PGI2

synthase during opioid-induced late PC.

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METHODS

Materials

(±)-[1(S*),2α,5β]-4-{[2,5-Dimethyl-4-(2-propenyl)-1-piperazinyl](3-hydroxyphenyl)

methyl}-N,N-diethylbenzamide hydrochloride (BW373U86) was purchased from Sigma-

RBI (St. Louis, MO). NS-398, valeryl salicylate (VSA) and monoclonal antibodies

against PGI2 synthase were purchased from Cayman Chemical (Ann Arbor, MI). PGE2,

6-keto-PGF1α and TXB2 EIA kits and vistra ECL Western blotting kit were purchased

from Amersham Pharmacia biotech (Buckinghamshire, England). Monoclonal

antibodies against COX-2 were purchased from BD (Franklin Lakes, NJ). Monoclonal

antibodies against COX-1 were purchased from Alexis (San Diego, CA).

Langendorff perfusion of the hearts

All procedures in the present study conformed to the principles outlined in the Guide for

the Care and Use of Laboratory Animals published by the USA National Institutes of

Health (NIH Publication No. 85-23, revised 1996).

One-hundred-eight 12-week-old male Fischer 344 rats weighing 210 to 250 g were

anesthetized by an intraperitoneal injection of sodium pentobarbital (40 mg/kg). Hearts

were excised quickly and perfused with modified Krebs-Henseleit buffer (118 mmol/L

NaCl, 25 mmol/L NaHCO3, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4,

1.75 mmol/L CaCl2, 0.5 mmol/L EDTA, 11 mmol/L glucose, and 5 mmol/L pyruvate)

gassed with 95% O2/5% CO2 at 37°C according to the Langendorff procedure.

Coronary perfusion pressure was maintained at 70 mm Hg.

Measurement of left ventricular function

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A plastic catheter with a latex balloon was inserted into the left ventricle through the left

atrium. Before the induction of ischemia, hearts were paced at 5 Hz, and the left

ventricular (LV) end-diastolic pressure (LVEDP) was adjusted to 10 mm Hg by filling

the balloon with water. Pacing was turned off during global ischemia and turned on 10

and 20 min after reperfusion to measure the recovery of LV function. To measure LV

pressure, at 10 min of reperfusion the hearts that were in ventricular fibrillation (VF)

were converted to sinus rhythm by tapping. The balloon was also deflated during global

ischemia and during the first 10 min of reperfusion. Indices of LV function [LV systolic

pressure, LVSP; LV developed pressure (LVDP=LVSP-LVEDP); and LV peak positive

and negative dP/dt] were recorded as described previously [37].

Experimental protocols

Rats were assigned to twelve groups (Fig. 1). All groups received 10 min of initial

perfusion in a recirculating mode and then the isolated perfused hearts were subjected

to 20min of global ischemia followed by 20 min of reperfusion.

(A) Dose response and time-course of BW373U86-induced cardioprotection

An initial dose-response for BW337U86 was established to determine the optimal dose

for inducing late phase of cardioprotection [Fig.1 (A-1)]. Group I (control) received

vehicle (sterile water, 500 µl/kg) injected subcutaneously and underwent 20 min of

global ischemia followed by 20 min of reperfusion 24 h later. In groups II to IV [BW 0.1,

BW 0.33, BW 1.0 (BW 24h)], different doses of BW373U86 were administered

subcutaneously (0.1, 0.33, or 1.0 mg/kg) 24 h before sacrifice. BW373U86 was

dissolved in sterile water just before injection. We used the dose of BW373U86 (1.0

mg/kg) that produced the greatest recovery of LV function after ischemia/reperfusion

for subsequent groups. The second series of rats were used to define the time-course

of opioid-induced cardioprotection [Fig. 1 (A-2)]. Groups V and VI (BW 1h, BW 12h)

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received 1.0 mg/kg of BW373U86 injected 1 h or 12 h before sacrifice and underwent

the 20 min of global ischemia followed by 20 min of reperfusion.

(B) Effect of COX selective inhibitors on BW373U86-induced cardioprotection

The third series of the experiments used either selective COX-1 or COX-2 inhibitor [Fig.

1 (B-1) (B-2)]. In group VII (NS+BW 1h), rats were pretreated with an intraperitoneal

injection of the selective COX-2 inhibitor NS-398 (5 mg/kg) 30 min before BW373U86

injection and were sacrificed 1 h after injection. Isolated hearts were subjected to the

20-min global ischemia followed by 20-min reperfusion protocol. In group VIII (SC+BW

1h), rats were pretreated with an intraperitoneal injection of the selective COX-1

inhibitor SC-560 (10 mg/kg) instead of NS-398, received the BW373U86 injection, and

were sacrificed 1 h later. In groups IX and X, rats received 1.0 mg/kg of BW373U86

injection 24 h before sacrifice. Rats were treated with NS-398 (5 mg/kg, group IX: BW

24h+NS) or SC-560 (10 mg/kg, group X: BW 24h+SC) intraperitoneally 30 min before

sacrifice. The hearts were then subjected to the ischemia/reperfusion protocol. Groups

XI (NS) and XII (SC) were the drug control groups. Rats received an intraperitoneal

injection of NS-398 (5 mg/kg) or SC-560 (10 mg/kg) without BW373U86 pretreatment

and were sacrificed 30 min later. NS-398 and SC-560 were dissolved in DMSO (30 and

10 mg/ml, respectively) and diluted twice with sterile water (final volume 1.0 ml/kg,

DMSO 50%). This dose of NS-398 has previously been shown to block COX-2 activity

24 h after ischemic PC in rabbits [17,33]. This dose of SC-560 has previously been

reported to reduce serum TXB2 levels, which reflect COX-1 activity, by more than 90%

in doxorubicin-treated rats [9].

Measurement of CK and LDH activities

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Perfusate was collected at the end of reperfusion to measure the activity of creatine

kinase (CK) and lactate dehydrogenase (LDH) released during the 20 min of

reperfusion. The volume of recirculating coronary perfusate was 50 ml per heart, and

CK and LDH activity was measured by standard enzymatic methods. Total amount of

CK and LDH released in the perfusate are expressed as IU/g wet weight of ventricle.

Measurement of PGE2 and 6-keto-PGF1α

Perfusate was collected after 10 min of preischemic perfusion and after 20 min of

reperfusion to measure prostaglandin (PG) levels in the coronary effluent. The PGE2,

6-keto-PGF1α (a stable metabolite of PGI2) and TXB2 (a stable metabolite of TXA2)

levels were determined using EIA kits.

Western Immunoblotting

Eight rats from groups I (control), V (BW 1h), VI (BW 12h) and IV (BW 24 h) were

euthanized without an ischemic insult 24 h after injection. The heart was excised

quickly, and the left ventricle was stored at –140°C until use. Tissue samples were

homogenized in buffer A (25 mM Tris-HCl [pH 7.4], 0.5 mM EDTA, 0.5 mM EGTA, 1

mM PMSF, 25 µg/mL leupeptin, 1 mM DTT, 25 mM NaF, and 1 mM Na3VO4) and

centrifuged at 1000 g for 10 min. The supernatant (the cytosolic fraction) was carefully

taken off and recentrifuged at 16000 g for 15 min to eliminate any contaminating pellet.

The initial pellet was resuspended in a lysis buffer (buffer A + 1% Triton X-100) and

incubated at 4˚C for 2 h. Samples were centrifuged at 16000 g for 15 min. The

resulting supernatants were collected as membranous fractions [30,33,40]. Standard

SDS-PAGE Western immunoblotting techniques assessed the expressions of COX-2,

COX-1, and PGI2 synthase protein. Briefly, proteins (100 µg) were electrophoresed on

a 10% denaturing gel and then electrophoretically transferred onto nitrocellulose

membranes overnight at 4°C. Gel transfer efficiency was determined by making

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photocopies of membranes dyed with reversible Ponceau staining; gel retention was

determined by Coomassie blue staining [30]. The membranes were incubated in 5%

nonfat dry milk in a washing buffer (10 mM Tris-HCl, [pH 7.2], 0.15 M NaCl, and 0.05%

Tween-20), followed by incubation with specific monoclonal antibodies (1:500 dilution)

at 35°C for 2 h. After rinsing with washing buffer, the membranes were incubated with

alkaline phosphatase-conjugated secondary antibodies (1:3000 dilution) at room

temperature for 1.5 h and developed using the vistra ECL Western blotting kit. The

protein signals and the corresponding records of Ponceau stains of nitrocellulose

membranes were quantitated by an image scanning densitometer, and each protein

signal was normalized to the corresponding Ponceau stain signal [30]. The protein

content was expressed as a percentage of the corresponding protein in group I (control

group).

Statistical analysis

Data are reported as the mean ± SEM. For intragroup comparisons, hemodynamic

variables were analyzsed by a two-way repeated measures ANOVA (time and group),

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 ANOVA (time and group), as appropriate, followed by Student's t

tests for paired data with the Bonferroni correction. All statistical analyses were

performed using the SAS software system.

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RESULTS

BW373U86-induced cardioprotection against ischemia/reperfusion injury

Pretreatment with BW373U86 24 h before ischemia improved the recovery of LVSP,

LVDP, and peak positive and negative dP/dt at doses of 0.33 and 1.0 mg/kg (groups III

and IV) (Fig. 2, Tables 1 and 2). In contrast, 0.1 mg/kg of BW373U86 failed to induce

appreciable protection (group II) (Fig. 2, Tables 1 and 2). Since the duration of global

ischemia was short, only small amounts of CK and LDH were released and there was

no difference among the control and BW groups (groups I to IV) (Table 3).

While recovery of LV function after ischemia/reperfusion improved when

BW373U86 was administered 1 h or 24 h before the ischemic insult (groups IV and V)

(Fig. 2, Tables 1 and 2), pretreatment with BW373U86 12 h before the ischemic insult

was not effective (group VI) (Fig. 2, Tables 1 and 2), confirming the biphasic nature of

opioid-induced PC. These results are consistent with the report by Fryer et al [12]. in

which infarct size was compared between the control and opioid-pretreated groups. CK

and LDH release during reperfusion was similar in the control and BW groups (groups

I, V, and VI) (Table 3).

Effect of selective COX inhibitor on BW373U86-induced cardioprotection

Pretreatment with the selective COX-2 inhibitor, NS-398, 30 min before the

administration of BW373U86, did not block the BW373U86-induced early phase of

cardioprotection (group VII) (Fig. 3, Tables 1 and 2). In contrast, the administration of

NS-398 30 min before the induction of prolonged ischemia abolished BW373U86-

induced late phase of cardioprotection (group IX) (Fig. 3, Tables 1 and 2). The

selective COX-1 inhibitor SC-560 had no effect on either the early or the late phase of

cardioprotection (groups VIII and X) (Fig. 3, Tables 1 and 2). Neither NS-398 nor SC-

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560 in itself affected the recovery of LV function after ischemia/reperfusion (groups XI

and XII) (Fig. 3, Tables 1 and 2).

There was no difference in CK and LDH release between the control and

selective COX inhibitor treated hearts (groups XI and XII), indicating that NS-398 and

SC-560 in itself did not exacerbate ischemia/reperfusion injury (Table 3). CK and LDH

release during reperfusion was similar in the control and all COX inhibitor-treated

groups (groups VII-XII)(Table 3).

Effect of BW373U86 on myocardial prostanoids levels

Changes in the levels of PGE2, TXB2 and 6-keto-PGF1α in the coronary effluent after

the administration of BW373U86 were evaluated (Figs. 4 and 5). 6-keto-PGF1α levels

after 10 min of preischemic perfusion (an index of 6-keto-PGF1α levels at baseline)

were similar among the control and all BW373U86-treated groups (groups I, V, VI, and

IV) (Fig. 4). However, 6-keto-PGF1α levels after 20 min of reperfusion were higher in

group IV (BW 24h) than in group I (control). The PGE2 and TXB2 levels were similar

after 10 min of preischemic perfusion and after 20 min of reperfusion among the

control and all BW373U86-treated groups (Fig. 5). These results indicate that the

increase in PGI2 observed in group IV was independent of the production of other

prostanoids. The increase in PGI2 production during reperfusion seen in group IV (BW

24h) was completely suppressed by the administration of NS-398 (group IX) (Fig. 4).

NS-398 in itself, however, did not reduce the 6-keto-PGF1α levels either after 10 min of

preischemic perfusion or after 20 min of reperfusion (group XI).

Effect of BW373U86 on protein expression of COX and PGI2 synthase

Expression of COX-1 and COX-2 did not change after the administration of BW373U86

at any time-point (groups V, VI and IV) (Fig. 6). There was no change in PGI2 synthase

protein levels among groups I (control), V (BW 1h) and VI (BW 12h) (Fig. 7). However,

PGI2 synthase protein expression increased (+56% in the membranous fraction, +49%

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in the cytosolic fraction) significantly 24 h after the administration of BW373U86 (group

IV)(P<0.05).

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DISCUSSION

This study provides three major findings: (1) COX-2 does not contribute to BW373U86-

induced early PC, (2) COX-2 mediates BW373U86-induced late PC by increasing PGI2

production, and (3) the increase in PGI2 production results from upregulation of PGI2

synthase rather than COX-2.

BW373U86-induced cardioprotection in isolated perfused rat hearts

Gross et al. have demonstrated the biphasic nature of opioid-induced cardioprotection

against myocardial infarction in rats [12,29,31,32]. Similar cardioprotective effects of

opioids have been observed in mice [16], rabbits [28], and even human myocytes [1].

Recent advances in pharmacologic technology have made it possible to develop drugs

with high selectivity for the δ-opioid receptor, minimizing the risk of side effects, such as

addiction [8]. Therefore, opioids have potential for development as therapeutic

cardioprotective agents.

This study demonstrates that BW373U86 can induce both an early and a late

phase of cardioprotection in isolated perfused rat hearts, suggesting that opioid-

induced cardioprotection is independent of circulating factors and neural modulation.

Although opioids can protect myocytes directly during simulated ischemia/reperfusion

[21,26], opioid-induced late PC has been demonstrated only in in vivo experiments

[12,16], and thus a role of indirect (neural) mechanisms cannot be ruled out. Our

results show that the myocardium itself acquires tolerance against

ischemia/reperfusion injury following the administration of BW373U86. The dosage of

BW373U86 that we used (1 mg/kg) was higher than that used by Patel et al. [29], who

administered BW373U86 intravenously at 0.1 mg/kg. The different route of

administration may account for the differences in the sensitivity of hearts to BW373U86

in that study and ours.

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Role of COX-2 in opioid-induced early PC

Opioids can mimic the early phase of ischemic PC by opening mitochondrial KATP

channels [12,21,26,32], but it has not been determined whether COX-2 is involved in

opioid-induced early PC. Since expression of COX-2 protein is detectable even in the

normal heart [33,34], it is theoretically conceivable that COX-2 may play a role.

Li and Kloner found that preadministration of 10 mg/kg of aspirin before

repetitive cycles of brief ischemia did not abolish the early phase of ischemic PC and

concluded that the cardioprotective effects of ischemic PC are not mediated by

prostanoids [25]. Camitta et al. demonstrated that targeted disruption of the COX-1 or

COX-2 gene did not affect the early phase of ischemic PC, although myocardial

ischemia/reperfusion injury was exacerbated in COX-1-/- or COX-2-/- mice compared

with control wild-type mice [6]. In accordance with these studies, we found that

BW373U86-induced early PC was not abolished by 5 mg/kg of NS-398, which

completely blocked late PC. In addition, there was no difference in the PG levels either

at baseline or during reperfusion between the control and BW373U86-treated hearts

(Figs. 4 and 5). These findings suggest that fundamentally different mechanisms are

responsible for opioid-induced early and late PC, and further corroborate the notion

that prostanoids do not contribute to early PC.

Role of COX-2 in opioid-induced late PC

COX-2 mediates the protective effects of ischemia-induced late PC in rabbits and mice

[17,33], but does not mediate late PC induced by activation of adenosine A1 or A3

receptors in rabbits [23]. In the present study, the selective COX-2 inhibitor NS-398

completely abolished the late PC induced by BW373U86 in rats. These results are

congruent with our recent finding that COX-2 mediates δ-opioid receptor-induced late

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PC in rabbits [24], indicating that the contribution of COX-2 to δ-opioid late PC is not

species specific. Furthermore, our data suggest that COX-2 plays a key role at least in

some types of pharmacological PC.

Involvement of COX-2 in opioid-induced late PC

The next important question was how COX-2 mediates opioid-induced cardioprotection

in isolated perfused hearts. We have found that COX-2 is upregulated after repetitive

episodes of brief ischemia, leading to increased synthesis of PGE2 and PGI2 in the

preconditioned myocardium [33,34]. Both PGI2 and the PGE family have been reported

to have cardioprotective properties [11,20,22,35]. Thus, COX-2-dependent production

of these prostanoids might protect the myocardium from ischemia/reperfusion injury

during the late phase of ischemic PC. In recent studies in rabbits, we found that COX-

2 protein expression was increased 24 h after BW373U86 [24]. Surprisingly, in this

study we found that COX-2 protein was not upregulated during opioid-induced late PC

in rats, even though opioid-induced cardioprotection was COX-2-dependent. Analysis

of prostanoids released into the perfusate demonstrated that 24 h after the

administration of BW373U86 PGI2 production was increased but PGE2 production was

not (Figs. 4 and 5). This is consistent with our finding that PGI2 synthase protein

content was increased by 56% 24 h after the administration of BW373U86 (Fig. 7).

COX-1 and COX–2 are located upstream of respective prostanoid synthases and

regulate the synthesis of prostanoids by supplying PGH2, the common precursor of

bioactive prostanoids [39]. COX-1 is responsible for constitutive PG formation, while

COX-2 is induced in response to stress, but is also constitutively expressed [39].

Increasing evidence indicates that constitutive COX-2 does not contribute to the basal

levels of PG production in the myocardium [6,33,34]. However, it is unknown whether

constitutive COX-2 produces PGH2 that each prostanoid synthase could utilize under

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pathological conditions. Every prostanoid synthase is known to utilize COX-1-derived

PGH2; recently, a functional coupling between COX-2 and specific prostanoid

synthases has also been proposed [7,38]. Ueno et al. have demonstrated that the

perinuclear enzymes thromboxane synthase and PGI2 synthase generate their

respective products via COX-2 rather than COX-1 in HEK293 cells cotransfected with

COX and prostanoid synthase [38]. They also found that the COX selectivity of these

lineage-specific prostanoid synthases was affected by the concentration of arachidonic

acid. Although it is unknown whether the interaction between COX-2 and prostanoid

synthases in rat myocardium is similar to that in transfected cultured cells, we propose

that a functional coupling between COX-2 and upregulated PGI2 synthase is

established during opioid-induced late PC. Our finding that the PGI2 levels at baseline

did not change in opioid-treated hearts suggests that this coupling becomes

functionally active during ischemia/reperfusion, possibly as a result of the release of

arachidonic acid during this condition [10]. Indeed, the fact that opioid-induced

protection was abolished by the administration of a selective COX-2 inhibitor, but not a

selective COX-1 inhibitor, suggests that COX-2-derived PGH2 plays an important role

on the development of opioid-induced late PC. Recent evidence that COX-2 is the

major source of systemic biosynthesis of PGI2 in healthy volunteers [27] and in patients

with atherosclerosis [2] supports this hypothesis. The precise reason why PGI2

synthase couples preferentially to COX-2 as opposed to COX-1 during opioid-induced

late PC is unknown. Nevertheless, our data identify, for the first time, upregulation of

PGI2 synthase as a critical element in late PC, thereby revealing a mechanism of

delayed cardioprotection that was heretofore unknown. These findings warrant further

studies aimed at defining the preferential functional coupling between upstream COX

and specific prostanoid synthesis in the cardiovascular system.

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Our present finding that opioid-induced late PC is associated with upregulation

of PGI2 synthase but not COX-2 itself differs from our recent findings in rabbits, in

which COX-2 expression was increased [24], and reveals possible species-specific

mechanisms underlying the functional involvement of COX-2-dependent prostanoid

synthesis in opioid-induced cardioprotection. The mechanism(s) whereby PGI2

synthase is upregulated after stimulation of opioid receptors is unknown. It has been

reported that tumor necrosis factor-α and interleukin-1 upregulate PGI2 synthase

mRNA [7,19,42]. The promoter of the PGI2 synthase gene contains several response

elements, including NF-κB, NF-IL6, and SP1 [42]. NF-κB has been shown to play an

essential role in ischemia-induced late PC [40] but its role in opioid-induced late PC is

unknown. The study of the signaling pathways that lead to upregulation of PGI2

synthase is potentially a fruitful ore because regulation of PGI2 synthase has important

clinical implications.

Conclusions

This study offers novel insights into the mechanism of opioid-induced late PC and late

PC in general. Our findings show that COX-2 does not mediate opioid-induced early

PC, whereas it does mediate late PC by increasing PGI2 production. Surprisingly,

however, the increased COX-2 dependent biosynthesis of PGI2 cannot be explained by

increased expression of COX-2 protein but appears instead to be the result of

upregulated expression of PGI2 synthase, suggesting that a functional coupling

between COX-2 and PGI2 synthase plays an important role in opioid-induced late PC.

To our knowledge, this is the first time that PGI2 synthase has been shown to be

upregulated during late PC and that an interaction between COX and a specific

prostanoid synthase has been shown in the heart. We propose that COX-2-dependent

synthesis of PGI2 via preferential coupling of COX-2 with upregulated PGI2 synthase is

a previously unrecognized mechanism for cardioprotection and a new pathway

H00209-2002.R2- 18 -

whereby opioid receptors protect the ischemic myocardium during late PC. The

concept that cardiac PGI2 production is affected by stimulation of opioid receptors

suggests novel therapeutic strategies aimed at enhancing the production of

cardioprotective prostanoids in the ischemic myocardium using selective opioid

receptor agonists.

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ACKNOWLEDGEMENTS

This study was supported in part by the Ueda Memorial Trust Fund for Research of

Heart Disease, by the Mitsui-Sumitomo Insurance Health Promotion Foundation, by the

Medical Research Grant Program of Keio Health Consulting Center (Dr. Shinmura) and

by NIH grants HL-43151, HL-55757, and HL-68088 (Dr. Bolli). We thank Dr. Eitaro

Kodani (University of Louisville, KY, USA) for his contribution to this work.

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H00209-2002.R2- 26 -

FIGURE LEGENDS

Figure 1. Schematic diagram illustrating experimental protocols. (See text for details).

Figure 2. Percent recovery of left ventricular developed pressure (LVDP) after

ischemia/reperfusion. (A-1) Dose-response experiment. (A-2) Time-course experiment.

Percent recovery of LVDP was calculated by dividing the LVDP 20 min after

reperfusion by the baseline LVDP. Data are expressed as the mean ± SEM. *: P<0.05

vs. corresponding value in group I (control)

Figure 3. Percent recovery of left ventricular developed pressure (LVDP) after

ischemia/reperfusion (B-1) Early preconditioning experiment. (B-2) Late

preconditioning experiment. Percent recovery of LVDP was calculated by dividing the

LVDP 20 min after reperfusion by the baseline LVDP. Data are expressed as the mean

± SEM. *: P<0.05 vs. corresponding value in group I (control), +: P<0.05 vs.

corresponding value in group IV (BW 24h), #: P<0.05 vs. corresponding value in group

XII (SC)

Figure 4. 6-keto-PGF1Αlevels in the perfusate after 10 min of perfusion (A) and 20 min

after ischemia/reperfusion (B). Data are expressed as the mean ± SEM. *: P<0.05 vs.

Group I (control), +: P<0.05 vs. Group IV (BW 24h)

Figure 5. PGE2 and TXB2 levels in the perfusate after 10 min of perfusion (A) and 20

min after ischemia/reperfusion (B). Data are expressed as the mean ± SEM.

Figure 6. Densitometric analysis of COX-1 (A) and COX-2 (B) protein signals in the

membranous fraction and the cytosolic fraction. The densitometric measurements of

protein immunoreactivity are expressed as a percentage of the average value

measured in control hearts. Data are expressed as the mean ± SEM.

Figure 7. (Left) Representative Western immunoblots showing the expression of PGI2

synthase protein. (Right, C) Densitometric analysis of PGI2 synthase protein signals in

H00209-2002.R2- 27 -

the membranous fraction and the cytosolic fraction. The densitometric measurements

of protein immunoreactivity are expressed as a percentage of the average value

measured in control hearts. Data are expressed as the mean ± SEM. *: P<0.05 vs.

Group I (control)

Table 1. Left ventricular pressure

At the Baseline 10 min after Reperfusion 20 min after Reperfusion

LVSP LVEDP DP LVSP LVEDP DP LVSP LVEDP DP(mmHg) (mmHg) (mmHg) (mmHg) (mmHg) (mmHg) (mmHg) (mmHg) (mmHg)

Group I (control) n=10 79 6 10.3 0.4 69 6 50 9 10.3 0.4 40 7 48 5 10.4 0.5 37 5

Group II (BW 0.1) n=9 79 9 10.1 0.3 68 9 54 7 10.0 0.2 44 7 57 8 10.3 0.3 46 9

Group III (BW 0.33) n=9 69 6 10.6 0.3 58 6 52 7 9.9 0.2 43 7 62 8 * 10.9 0.2 51 8 *

Group IV (BW 1.0=BW 24h) n=9 75 8 10.5 0.2 65 8 58 6 * 10.2 0.3 47 6 * 67 8 * 10.0 0.2 57 8 *

Group V (BW 1h) n=9 79 4 10.1 0.3 69 4 55 5 10.6 0.3 44 5 62 4 * 10.6 0.3 52 4 *

Group VI (BW 12h) n=9 82 2 10.3 0.2 72 3 52 5 9.9 0.3 42 5 53 3 10.2 0.4 43 3

Group VII (NS+BW 1h) n=10 77 4 10.1 0.3 66 4 57 4 10.6 0.3 46 3 60 5 * 10.2 0.1 50 5 *

Group VIII (SC+BW 1h) n=9 86 7 10.4 0.2 75 6 60 6 * 10.6 0.3 49 6 * 63 6 * 10.3 0.2 52 6 *

Group IX (BW 24h+NS) n=8 80 9 10.5 0.3 69 9 44 8 + 10.4 0.3 33 9 + 50 11 + 10.5 0.2 40 11 +

Group X (BW 24h+SC) n=9 85 6 10.0 0.3 75 6 70 9 # 10.1 0.2 60 9 # 69 8 # 10.3 0.2 58 8 #

Group XI (NS) n=8 75 5 10.5 0.3 64 5 45 3 10.4 0.3 35 2 48 4 10.3 0.2 38 4

Group XII (SC) n=9 89 7 10.3 0.2 79 7 51 6 10.2 0.2 41 6 51 4 10.2 0.2 41 4

LVSP: Left ventricular systolic pressure, LVEDP: LV end-diastolic pressure, LVDP: LV developed pressure=LVSP-LVEDP BW: BW373U86, NS: NS-398, SC: SC-560

*: P <0.05 vs. Group I (control), +: P <0.05 vs. Group IV (BW 24h), #: P <0.05 vs. Group XII (SC)

Table 2. LV peak positive and negative dP /dt

At the Baseline 20 min after Reperfusion

peak positive dP /dt peak negative dP /dt % recovery of p dP /dt % recovery of n dP /dt(mmHg/s) (mmHg/s) (%) (%)

Group I (control) n=10 1850 140 960 100 58 9 66 10

Group II (BW 0.1) n=9 2000 230 980 130 68 8 71 8

Group III (BW 0.33) n=9 1710 150 870 80 79 6 * 86 6 *

Group IV (BW 1.0=BW 24h) n=9 1810 200 870 100 81 7 * 85 7 *

Group V (BW 1h) n=9 2000 120 920 70 75 5 * 87 6 *

Group VI (BW 12h) n=9 2040 110 1030 70 60 4 67 5

Group VII (NS+BW 1h) n=10 1920 120 890 60 77 8 * 88 6 *

Group VIII (SC+BW 1h) n=9 2110 230 1060 120 75 7 * 85 7 *

Group IX (BW 24h+NS) n=8 1810 250 1000 140 63 11 + 60 11 +

Group X (BW 24h+SC) n=9 2160 210 1020 100 77 10 # 78 9 #

Group XI (NS) n=8 1780 120 880 90 64 8 66 11

Group XII (SC) n=9 2170 190 1070 130 56 7 62 8

BW: BW373U86, NS: NS-398, SC: SC-560 *: P <0.05 vs. Group I (control), +: P <0.05 vs. Group IV (BW 24h), #: P <0.05 vs. Group XII (SC)

Table 3. CK and LDH release in the perfusate

20 min after Reperfusion

CK LDH(IU/g wt) (IU/g wt)

Group I (control) n=10 15 3 10 2

Group II (BW 0.1) n=9 13 2 8 2

Group III (BW 0.33) n=9 15 3 11 2

Group IV (BW 1.0=BW 24h) n=9 14 2 10 2

Group V (BW 1h) n=9 20 6 13 3

Group VI (BW 12h) n=9 17 2 10 1

Group VII (NS+BW 1h) n=10 18 6 12 4

Group VIII (SC+BW 1h) n=9 18 4 14 2

Group IX (BW 24h+NS) n=8 15 5 10 3

Group X (BW 24h+SC) n=9 17 5 11 4

Group XI (NS) n=8 14 3 9 2

Group XII (SC) n=9 21 7 14 6

BW: BW373U86, NS: NS-398, SC: SC-560 *: P <0.05 vs. Group I (control), +: P <0.05 vs. Group IV (BW 24h)

GROUP I(control)

GROUP II (BW 0.1)

GROUP III (BW 0.33)

GROUP IV (BW 1.0=BW 24h)

GROUP V (BW 1h)

GROUP VI (BW 12h)

GROUP VII (NS+BW 1h)

GROUP VIII (SC+BW 1h)

GROUP IX (BW 24h+NS)

GROUP X (BW 24h+SC)

GROUP XI (NS)

GROUP XII (SC)

10 min

20 min Ischemia

20 minReperfusion

10 min

20 min Ischemia

20 minReperfusion

10 min

20 min Ischemia

20 minReperfusion

10 min

20 min Ischemia

20 minReperfusion

10 min

20 min Ischemia

20 minReperfusion

10 min

20 min Ischemia

20 minReperfusion

1 h or 12 h

1 h30 min

30 min24 h

30 min

Vehicle (sterile water) sc

BW373U86 0.1, 0.33, or 1.0 mg/kg sc

BW373U86 1.0 mg/kg sc

NS-398 5 mg/kg ip or SC-560 10 mg/kg ip

BW373U861.0 mg/kg sc

NS-398 5 mg/kg ip or SC-560 10 mg/kg ip

Figure 1

(A-1) Dose-Response%

GROUP I(control)

GROUP III(BW 0.33)

GROUP II(BW 0.1)

GROUP IV(BW 1.0)

10 min after reperfusion

* P<0.05 vs Group I (control)

(A-2) Time-Course

GROUP IV(BW 24 h)

GROUP I(control)

GROUP V(BW 1 h)

GROUP VI(BW 12 h)

Figure 2

(B-1) Early Preconditioning%

GROUP I(control)

GROUP VII(NS+

BW 1h)

GROUP V(BW 1h)

GROUP VIII(SC+

BW 1h)

* P<0.05 vs Group I (control)+ P<0.05 vs Group IV (BW 24h)

(B-2) Late Preconditioning

GROUP X

(BW 24h

GROUP I

(control)

GROUP IV

(BW 24h)

GROUP IX

(BW 24h

GROUP XI

GROUP XII

Figure 3

# P<0.05 vs Group XII (SC)

(pg/ml)

A) After 10 min of Pre-ischemic perfusion

B) After 20 min of Reperfusion

(pg/ml)

Figure 4

GF 1α

GROUP I (control)

GROUP IV (BW 24h)

GROUP IX (BW 24h+NS)

GROUP XI (NS)

* P<0.05 vs Group I (control)

GROUP V (BW 1h)

GROUP VI (BW 12h)

+ P<0.05 vs Group IV (BW 24h)

(pg/ml)0

(pg/ml)

A) After 10 min of Pre-ischemic perfusion

B) After 20 min of Reperfusion

(pg/ml)

Figure 5

GROUP I (control)

GROUP IV (BW 24h)

GROUP V (BW 1h)

GROUP VI (BW 12h)

(A) COX-1 PROTEIN C

Membranous fraction

Cytosolic fraction

(B) COX-2 PROTEIN

Membranous fraction

Cytosolic fraction

GROUP I (control) GROUP IV (BW 24h) GROUP V (BW 1h) GROUP VI (BW 12h)

Figure 6

(C) PGI2 SYNTHASE PROTEIN

PGI 2 S

Membranous fraction

Cytosolic fraction

GROUP I (control)

GROUP IV (BW 24h)

GROUP V (BW 1h)

GROUP VI (BW 12h)

Figure 7

P<0.05

GROUP I(control)

GROUP IV(BW 24h)

Membranousfraction

Cytosolicfraction

Cyclooxygenase-2-derived prostacyclin mediates opioid-induced late phase of preconditioning in...

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