This Article Abstract Full Text (PDF) All Versions of this Article: 109/10/1219    most recent 01.CIR.0000121422.85989.BDv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peart, J. N. Articles by Gross, G. J. Search for Related Content PubMed PubMed Citation Articles by Peart, J. N. Articles by Gross, G. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB MORPHINE Related Collections Ischemic biology - basic studies Chronic ischemic heart disease

(Circulation. 2004;109:1219-1222.)
© 2004 American Heart Association, Inc.

Brief Rapid Communications

Morphine-Tolerant Mice Exhibit a Profound and Persistent Cardioprotective Phenotype

Jason N. Peart, PhD; Garrett J. Gross, PhD

From the Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee.

Correspondence to Garrett J. Gross, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail ggross{at}mcw.edu

Received December 10, 2003; revision received January 9, 2004; accepted January 21, 2004.

	Abstract

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Background— Morphine and other opioids continue to be used as the major treatment for acute pain both before and after surgery. In this regard, much research has focused on the mechanisms of morphine tolerance and dependence in the central nervous system; however, few studies have examined the effect of morphine on peripheral organs, such as the heart, in morphine-tolerant animals. Here, we examine the effect of tolerance to the analgesic effect of morphine on ischemic tolerance in mice after prolonged morphine exposure and withdrawal.

Methods and Results— Male C57/BL6 mice were implanted subcutaneously with either placebo or morphine pellets (25 or 75 mg). After prolonged exposure to and/or withdrawal from morphine or placebo, the hearts were excised and subjected to 25 minutes of ischemia and 45 minutes of reperfusion. Morphine-tolerant mice exhibited a markedly improved functional recovery compared with placebo and mice subjected to acute morphine. Lactate dehydrogenase release was also significantly reduced. The protection observed was equieffective 48 hours after withdrawal of pellet, whereas the onset of protection preceded analgesic tolerance.

Conclusions— These data demonstrate that chronic exposure to morphine unexpectedly results in a profound and persistent cardioprotective phenotype.

Key Words: ischemia • reperfusion • myocardial stunning • myocardial infarction • occlusion

	Introduction

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Myocardial binding studies demonstrate that - and -opioid receptors are present on adult ventricular myocytes of rats and humans.^1,2 Opioid receptors ( and ) have also been linked to a second messenger system involving phosphoinositol turnover,² and opioid receptor stimulation directly regulates ion channels via an interaction with inhibitory G proteins.^3,4

Exogenous opioids have previously been shown to elicit cardioprotection against both myocardial stunning and infarction^5,6 via activation of G_i/o proteins, protein kinase C, and the mitochondrial K_ATP channel.² Opioid receptor antagonists and selective ₁-opioid receptor antagonists have been shown to inhibit the cardioprotective effects of ischemic preconditioning.^7,8

Although much research has focused on the acute cardioprotective effects of opioid receptor activation, there are no reports regarding chronic morphine exposure. Here, we show that chronic exposure to morphine affords a marked increase in ischemic tolerance that is significantly greater than acute morphine treatment. Furthermore, the protection persists for at least 48 hours after drug withdrawal.

	Methods

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Perfused Heart Preparation
The following investigations conformed to the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association of Laboratory Animal Care. A total of 133 male C57/Bl6 mice (25 to 30 g; Charles River, Wilmington, Mass) were anesthetized with sodium pentobarbital (60 mg/kg). Hearts were excised and perfused in Langendorff mode as described previously.⁹ After equilibration, hearts were subjected to 25 minutes of global ischemia and 45 minutes of reperfusion.

Analysis of Enzyme Efflux
Coronary venous effluent was sampled and frozen at -80°C until analyzed for lactate dehydrogenase (LDH) with a commercially available enzymatic assay kit (Diagnostic Chemicals Limited). Total efflux during the 45-minute postischemic period is reported as IU/g.

Pellet Implantation and Withdrawal
Mice were briefly anesthetized with halothane. Using aseptic techniques, a small incision was made at the base of the neck. Placebo or morphine (25 or 75 mg) pellets (National Institute of Drug Abuse) were inserted in the dorsal subcutaneous space before the site was closed with 9-mm wound clips. Pellets were left in place for the specified length of time. For withdrawal studies, after 5 days of pellet implantation, the mice were again anesthetized with halothane, and the wound clip and pellet were removed. The wound was again closed with a clip, and the mice were given a nonselective opioid antagonist, naloxone (7 mg/kg IP) to rapidly precipitate withdrawal.

Tail-Flick Test and Analgesic Tolerance
The tail-flick test used to assess antinociception was described by Dewey et al.¹⁰ Briefly, a high-intensity light beam was applied to the tail, and the tail-flick latency was quantified. A 10-second maximum (test) cutoff was applied to prevent tail damage. Morphine tolerance was indicated when tail-flick latency returned to baseline values. To verify tolerance, mice were injected with morphine (20 mg/kg IP), and latencies were repeated. Mice were determined to be tolerant if latency did not increase from baseline after administration of morphine.

Statistical Analysis
All values are expressed as mean±SEM. One-way ANOVA with Newman-Keuls post hoc test was used to determine significant differences between groups. Tail-flick latencies were compared directly by unpaired t tests. Significant differences were determined at a value of P<0.05.

	Results

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Tail-Flick Latency and Tolerance
Measurement of tail-flick latency was used to determine the presence of tolerance. Tolerance was apparent after 5 days of implantation (Figure 1). Furthermore, a challenge dose of morphine (20 mg/kg) failed to alter the tail-flick latency when measured 30 minutes after administration on day 5 (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 1. Development of morphine tolerance and onset of cardioprotection after chronic exposure to morphine. A, Onset of morphine tolerance as determined by tail-flick latency, with chronic placebo (P, [open bars]) or morphine (M, [solid bars]) treatment. B, Functional recovery of rate-pressure product (left ventricular developed pressure times heart rate) expressed as percentage of preischemic function in untreated hearts, hearts treated with 30 µmol/L morphine 10 minutes before ischemia and throughout reperfusion, and hearts with either placebo or morphine pellets implanted for 5 days (data from Figure 2A). C and D, Recovery of end-diastolic pressure and rate-pressure product (percent of preischemic function) after 25 minutes of ischemia and 45 minutes of reperfusion in a separate subset of hearts from mice implanted with either placebo or morphine (75 mg) for 3, 4, or 5 days. *P<0.01, morphine vs placebo; P<0.05, acute morphine exposure vs chronic exposure; n5 for each group.

View larger version (35K): [in this window] [in a new window]   Figure 2. Protection afforded by chronic exposure to morphine persists after withdrawal. A, B, C, and D, Recovery of end-diastolic pressure ([open bars]) and rate-pressure product ([solid bars]) in placebo- and morphine (25 and 75 mg)–treated hearts. Mice had implants for 5 days before heart isolation or precipitated withdrawal for 1, 24, or 48 hours before isolation and perfusion. LDH efflux as collected throughout reperfusion in placebo- and morphine (25 and 75 mg)–treated hearts after 5 days of exposure (E) or after 5 days of exposure and 24 hours of withdrawal (F). *P<0.01, morphine vs placebo; P<0.05, morphine vs placebo; n6 for each group.

Baseline Hemodynamics and Ischemic Tolerance
After implantation for the allotted time, hearts were rapidly excised and perfused. After 20 minutes of equilibration, baseline measurements were taken. No differences were noted between placebo- and morphine-treated mice. Global ischemia was induced for 25 minutes, followed by 45 minutes of reperfusion. On termination of reperfusion, placebo-treated hearts exhibited a poor recovery of left ventricular contractile function and an elevated end-diastolic pressure (Figure 1).

Morphine Treatment
Mice implanted with morphine pellets for 5 days demonstrated a marked improvement in ischemic tolerance that was significantly greater than that afforded by acute morphine treatment (30 µmol/L, infused for 10 minutes before ischemia and throughout reperfusion). End-diastolic pressure returned to baseline, whereas left ventricular contractile function returned to 75% to 80% of baseline after 45 minutes of reperfusion (Figure 1). Interestingly, protection was apparent after 3 days of implantation, preceding the development of analgesic tolerance. LDH release was also dramatically reduced after chronic morphine (Figure 2).

Dose and Withdrawal
To examine potential differences between 2 doses of morphine and the effects of morphine withdrawal, mice were implanted with either placebo or 25- or 75-mg morphine pellets for 5 days. After 5 days, hearts were excised or withdrawal was precipitated via pellet removal and administration of naloxone (7 mg/kg). All withdrawn mice displayed classic withdrawal behavior. Mice were withdrawn for 1 hour, 24 hours, or 48 hours before isolation and heart perfusion. After 5 days of implantation, no difference was observed between mice treated with 25- or 75-mg pellets (Figure 2). Furthermore, no reduction in protection afforded by chronic preconditioning was observed for either dose up to 48 hours after withdrawal (Figure 2).

	Discussion

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The results of our present study show that chronic preconditioning with morphine, most likely via nonselective activation of - and -opioid receptors, leads to a pronounced cardioprotective phenotype, affording greater protection than acute treatment, and this protection does not seem to be associated with morphine tolerance. Furthermore, the degree of protection persists for at least 48 hours without waning after drug withdrawal.

The protection observed was 20% greater than that seen with acute morphine treatment, even though the blood concentration of morphine was calculated to be only 650 nmol/L after 5 days of exposure.¹¹ This was surprising, because acute treatments generally show greater degrees of cardioprotection than that seen in delayed preconditioning.¹² However, some studies report the lack of an acute protective effect from an agonist, whereas that same agonist affords delayed cardioprotection.¹³ Moreover, the protection seen during withdrawal (1, 24, and 48 hours) failed to show a reduction of protection at 1 or 24 hours, suggesting that this effect may not follow the typical time course of preconditioning, whereby protection wanes after 1 to 2 hours and reappears after 24 hours. Another interesting observation is that the protection persisted in the presence of morphine tolerance. This conflicts with the findings of Tsuchida et al,¹⁴ who reported that the preconditioning effect of an adenosine A₁ receptor agonist (CCPA) diminished after tachyphylaxis. However, Dana and colleagues¹⁵ reported that chronic, intermittent administration of CCPA, without tachyphylaxis, maintained a preconditioned state. Interestingly, the protection seen after chronic, intermittent administration of CCPA was unchanged from single-dose treatment. In addition, Cohen et al¹⁶ demonstrated that conscious rabbits may become tolerant to repetitive cycles of ischemic preconditioning; however, this may be in part a result of the depletion of adenosine.¹⁷ The diminished protection seen 1 to 2 hours after the preconditioning stimuli may also be a result of receptor internalization and desensitization. Interestingly, chronic morphine exposure has been shown to prevent internalization and desensitization of opioid receptors.¹⁸

The mechanism leading to protection observed after chronic morphine exposure is unknown. However, chronic opioid exposure has led to considerable research, and the knowledge afforded by central nervous system studies may provide an insight into the mechanism of protection seen in the heart. Chronic opioid treatment leads to an increase in the release of endogenous substances such as substance P, calcitonin gene–related peptide¹⁹ and adenosine.²⁰ Indeed, chronic morphine exposure increases adenosine receptor sensitivity.²¹ After chronic activation of opioid receptors, the opioid receptors may convert from inhibitory (G_i-coupled) to excitatory (G_s-coupled).²² Furthermore, chronic exposure to opioids confers a superactivation of adenylyl cyclase.²³ Moreover, chronic exposure may afford activation of multiple redundant signaling pathways.²³

In summary, we have demonstrated that chronic preconditioning with morphine provides a markedly ischemia-tolerant phenotype in murine hearts. This protection occurs independently of morphine tolerance and persists for at least 48 hours after withdrawal without any change in the degree of protection. As such, the protection observed does not follow the traditional time course of preconditioning-mediated cardioprotection. Obviously, further studies are needed to determine the potential mechanisms responsible for this marked cardioprotection produced by chronic exposure to morphine.

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-08311 (Dr Gross) and an American Heart Association Postdoctoral Fellowship (Dr Peart).

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 109/10/1219    most recent 01.CIR.0000121422.85989.BDv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peart, J. N. Articles by Gross, G. J. Search for Related Content PubMed PubMed Citation Articles by Peart, J. N. Articles by Gross, G. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB MORPHINE Related Collections Ischemic biology - basic studies Chronic ischemic heart disease