© 1996 American Heart Association, Inc.
Articles |
Morphine Mimics the Cardioprotective Effect of Ischemic Preconditioning via a Glibenclamide-Sensitive Mechanism in the Rat Heart
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.
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Abstract |
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Abstract Previous results from our laboratory have suggested that opioid receptors are involved in ischemic preconditioning (PC) in rat heart. Furthermore, other investigators have suggested that µ- and
-opioid receptors mediate analgesia and hypoxic
cerebral vasodilatation via opening of ATP-sensitive K+
(KATP) channels. Thus, the purpose of the present study
was to test the hypothesis that activation of opioid receptors mimics
the cardioprotective effect of ischemic PC and that this effect
is produced by activation of KATP channels in the rat
heart. Anesthetized open-chest Wistar rats were subjected
to six different protocols. All groups were subjected to 30 minutes of
occlusion and 2 hours of reperfusion. Ischemic PC was elicited
by three 5-minute occlusion periods interspersed with 5 minutes of
reperfusion. Similarly, morphine-induced PC was elicited by three
5-minute drug infusions (100 µg/kg IV) interspersed with 5-minute
drug-free periods before the prolonged 30-minute occlusion. Infarct
size (IS) as a percentage of the area at risk (AAR) was determined by
triphenyltetrazolium staining.
Ischemic PC and morphine infusions resulted in similar
reductions in IS/AAR from 56±5% to 11±3% and 12±5%, respectively
(P<.05). Administration of glibenclamide (0.3 mg/kg IV), a
KATP channel antagonist, or naloxone (3 mg/kg
IV), a nonselective opioid receptor antagonist, both
blocked the cardioprotective effects of morphine. These results
indicate that opioid receptor stimulation results in a reduction in
infarct size similar to that produced by ischemic PC. The
effect of morphine is most likely mediated via an opioid
receptor-KATP channel–linked mechanism in the rat
heart, since glibenclamide abolished its protection.
Key Words: myocardial infarction • ATP-sensitive K+ channels • naloxone • opioid receptors
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Introduction |
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Opioid receptors have been implicated in protecting several organ systems from hypoxic or ischemic events.1 2 In this regard, our laboratory, using an in vivo rat model of myocardial infarction, was the first to show that naloxone, a nonselective opioid receptor antagonist, abolished the protective effect of ischemic PC.3 Recently, Chien et al4 have also shown that opioid receptors are involved in PC in rabbit heart. However, at present, the mechanism by which opioid receptors produce their cardioprotective actions is not clear.
It is known that certain opioid receptors, most notably µ- and
-opioid receptors, are linked to K+ channels via G
proteins.5 6 7 Several investigators have demonstrated the
involvement of KATP channels in morphine-induced
analgesia8 9 10 11 and morphine withdrawal.12
Besides the involvement of opioid receptors and KATP
channels in analgesia, this receptor-effector interaction has also
been implicated in cerebral vascular control. Shankar and
Armstead13 showed that endogenous and
synthetic µ- and 1-opioid receptor agonists acted via
KATP channels to produce hypoxia-induced pial
vasodilation. Taken together, these results suggest that the
interaction of opioid receptors and KATP channels may be
involved in protecting tissue during hypoxic and ischemic
events. Therefore, the following experiments were designed to test the
hypothesis that opioid receptors are involved in myocardial protection
and that this cardioprotection is produced by activating
KATP channels in the rat heart.
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Materials and Methods |
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The present study was performed in accordance with the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association of Laboratory Animal Care.
General Surgical Preparation
Male Wistar rats weighing 350 to 450 g were used. The rats
were anesthetized by intraperitoneal
administration with the long-acting thiobutabarbital, inactin (100
mg/kg IV). A tracheotomy was performed, and the rat was intubated with
a cannula connected to a rodent ventilator (model 683, Harvard
Apparatus). The rats were ventilated with room air at 65 to
70 breaths per minute. Atelectasis was prevented by maintaining a
positive end-expiratory pressure of 5 to 10 mm of H2O.
Arterial pH, Pco2, and Po2
were monitored at baseline, 15 minutes of occlusion, 15 minutes of
reperfusion, 60 minutes of reperfusion, and 120 minutes of reperfusion
by a blood gas system (AVL 995, Automatic Blood Gas System) and
maintained within a normal physiological range (pH
7.35 to 7.45; Pco2, 25 to 40 mm Hg; and
Po2, 80 to 110 mm Hg) by adjusting the respiratory
rate and/or tidal volume. Bicarbonate was administered if the pH was
lowered because of metabolic acidosis. Body temperature was
monitored (Yellow Springs Instruments Tele-Thermometer) and maintained
at 37±1°C (mean±SEM) by using a heating pad. Blood glucose for the
glibenclamide protocol was measured at the same time points as the
blood gas measurements, including a postdrug measurement using a blood
glucose monitor (model 780, Tracer II, Boehringer Mannheim
Diagnostics) and glucose reagent strips (Tracer bG strips,
Boehringer Mannheim).
The right carotid artery was cannulated to measure MBP and HR via a
Gould PE-50 or Gould PE-23 pressure transducer, which was connected to
a Grass (model 7) polygraph. The right jugular vein was cannulated to
infuse saline or drugs. A left thoracotomy was performed 
20 mm from
the sternum to expose the heart at the fifth intercostal space. The
pericardium was removed, and the left atrial appendage was moved to
reveal the location of the left coronary artery. The vein
descending along the septum of the heart was used as the marker for the
left coronary artery. A ligature (6-0 prolene), along with a
snare occluder, was placed around the vein and left coronary
artery close to the place of origin. After surgical preparation, the
rat was allowed to stabilize for 15 minutes.
Drugs
Inactin and naloxone were purchased from Research Biochemicals
International. TTC and glibenclamide, a sulfonylurea KATP
channel antagonist, were purchased from Sigma Chemical Co.
Morphine was purchased from Mallinckrodt Chemical Works. Inactin,
naloxone, and morphine were dissolved in saline. Glibenclamide was
dissolved in a 1:1:1:2 cocktail mixture of polyethylene glycol, 95%
ethanol, 0.1N sodium hydroxide, and 0.09% saline, respectively. TTC
was dissolved in 100 mmol/L phosphate buffer (pH 7.4).
Study Groups and Experimental Protocols
The present study consisted of rats randomly assigned to one
of six experimental groups (Fig 1
). Group I (the control
group) was subjected to 30 minutes of occlusion and 2 hours of
reperfusion. In group II (ischemic PC group), ischemic
PC was elicited by three 5-minute occlusion periods interspersed with 5
minutes of reperfusion. Group III (GLY+PC group) consisted of rats
administered glibenclamide (0.3 mg/kg IV) 30 minutes before
ischemic PC. In group IV (MOR PC group), to determine if opioid
receptor stimulation mimicked ischemic PC, morphine, a
µ-opioid receptor agonist, was given as three 5-minute infusions
interspersed with a 5-minute drug-free period (100 µg/kg per
infusion; total dose, 300 µg/kg). In group V (NL+MOR PC group),
naloxone (3 mg/kg IV), a nonselective opioid receptor
antagonist, was administered 10 minutes before
morphine-induced PC. Finally, in group VI (GLY+MOR PC group),
to test the interaction of the µ-opioid receptor and
KATP channels in myocardial protection, glibenclamide (0.3
mg/kg IV) was given 30 minutes before morphine-induced PC. Saline
or glibenclamide vehicle was used in 50% of the control and
ischemic PC groups, and no influence of vehicle was noted (data
not shown).
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Determination of Infarct Size
After each experiment, the left coronary artery was
reoccluded, and patent blue dye was injected into the venous catheter
to stain the normally perfused region of the heart. The rat was
euthanized with 15% KCl through the arterial catheter. The
heart was excised, and the left ventricle was removed and sliced into
five cross-sectional pieces. This procedure allowed for
visualization of the normal, nonischemic region and the
AAR. The AAR was separated from the normal area using a dissecting
microscope (Cambridge Instruments). Both tissue regions
(nonischemic and AAR) were incubated at 37°C for 15
minutes in a 1% TTC stain in 100 mmol/L phosphate buffer (pH 7.4). TTC
is used as an indicator to separate out viable and nonviable
tissue.14 The tissue was stored overnight in a 10%
formaldehyde solution. The following day, the infarcted tissue was
separated from the AAR by using the dissecting scope. The different
regions (nonischemic, AAR, and infarct) were determined by
gravimetry, and IS was calculated as a percentage of the AAR
(IS/AAR).
Exclusion Criteria
A total of 55 animals were enrolled in the present
study. Animals were omitted from further data analysis if
intractable ventricular fibrillation occurred or if marked
hypotension (arterial mean pressure below 30 mm Hg) was
observed to the extent that the experiment could not be continued
successfully for the duration of the protocol. In the control group,
three animals were excluded because of intractable
ventricular fibrillation. One animal in the
ischemic PC group, one animal in the GLY+PC group, and four
animals in the MOR PC group were excluded because of severe
hypotension. A total of 46 animals completed the study.
Statistical Analysis
All values are expressed as mean±SEM. ANOVA was used to
determine differences among groups for IS and AAR. Differences between
groups in hemodynamics at various time points were
compared by using a two-way ANOVA for time and treatment with
repeated measures and Fisher's least significant difference test if
significant F ratios were obtained. Statistical differences were
considered significant at a value of P<.05.
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Results |
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Hemodynamics
Table 1
illustrates the
hemodynamic values obtained for HR, MBP, and RPP at
baseline, after 30 minutes of occlusion, and at 2 hours of reperfusion.
There were no differences in baseline hemodynamic
values between groups. At 30 minutes of occlusion, the HRs were
slightly but significantly greater in the three PC (ischemia
and morphine) groups treated with glibenclamide and naloxone than in
the control group; however, the HRs were not different at 2 hours of
reperfusion in these three groups. MBP and RPP were increased at 30
minutes of occlusion in the GLY+PC group. Heart rate was lower in the
ischemic PC group at 2 hours of reperfusion, whereas all other
groups showed no significant differences in the
hemodynamic parameters at this time.
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Infarct Size
Fig 2 and Table 2 depict the effect
of myocardial PC on infarct size. Table 2 summarizes the weights of the
left ventricle, AAR, IS, and IS/AAR in all groups. There were no
significant differences in left ventricular and AAR weights
among groups. IS and IS/AAR for ischemic PC and MOR PC groups
were significantly different than for the control group
(P<.05). IS values of individual hearts and the mean±SEM
for each protocol are shown in Fig 2. The average IS/AAR in the control
group (group I) was 56.4±4.9%. Ischemic PC markedly reduced
IS/AAR to 10.6±2.6% (P<.05). Glibenclamide administered
30 minutes before ischemic PC abolished the
cardioprotective effect of ischemic PC
(45.7±10.6%). Fig 2 also indicates that morphine
administration mimicked the cardioprotective effect of ischemic
PC (12.2±5.2%, P<.05). Naloxone completely eliminated
the protective effect produced by morphine
(67.4±7.2%). Similarly, glibenclamide also abolished
the cardioprotective effect of morphine-induced PC
(51.9±5.7%). Previous results from our laboratory3 have
shown that the doses of naloxone and glibenclamide (authors'
unpublished data, 1996) used in the present study do not change IS
when either agent is administered to non-PC hearts (data not
shown).
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indicates infarct sizes from individual hearts; 
, average
infarct size in each group. Values are mean±SEM. *P<.05 vs
CON.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The endogenous opioid system has been shown to be involved in many pathophysiological states, including hypotension,15 arrhythmogenesis,16 and congestive heart failure.17 This opioid receptor system has also been implicated in the protection of organs during hypoxic and ischemic events.1 2 Our laboratory was the first to show the involvement of opioid receptors in ischemic PC in the rat heart.3
Ischemic PC was first observed and described by Murry et
al18 in studies of the canine heart. Since this initial
observation, a plethora of studies has been performed to determine the
mechanism(s) responsible for this remarkable cardioprotective effect in
the eventual hope of finding a clinically useful PC-like drug.
Unfortunately, no single receptor or effector has been identified that
seems to be involved across all species that might be used clinically.
Gross and Auchampach19 were the first investigators to
demonstrate the involvement of the KATP channel in
ischemic PC in dogs. Since then, other
investigators20 21 22 23 have shown the involvement of the
KATP channel in ischemic PC in other species;
however, Liu and Downey24 and others25 26
were unable to show a role for the KATP
channel–mediated myocardial protection in the intact or isolated
rat heart. Subsequently, studies of the isolated rat heart have
demonstrated the involvement of other receptor signal transduction
pathways, such as 
1B-adrenoceptors,27
ß-receptors,28 nitric oxide,29 and
protein kinase C27 in ischemic PC. In the intact
rat heart, no receptor system has been clearly shown to mediate
ischemic PC, and adenosine, a common trigger for
ischemic PC in other species, does not appear to be
involved.24 Recently, our laboratory found that
ischemic PC is mediated via the KATP channel in the
intact rat heart and that glibenclamide antagonizes ischemic PC
in a time-dependent manner (authors' unpublished data, 1996). On
the basis of our previous work showing an involvement of opioids during
ischemic PC3 and our recent evidence suggesting
that the KATP channel mediates ischemic PC in the
rat, it was of interest to determine the potential role and interaction
of opioid receptors and KATP channels in eliciting
ischemic PC.
In agreement with several laboratories,20 21 22 23 24 our results demonstrate that ischemic PC can produce a marked reduction in infarct size. This cardioprotection could be abolished by the administration of glibenclamide, thereby indicating an involvement of the KATP channel in the protective effect of ischemic PC in rat hearts. Thus, it appears that the KATP channel is a common effector of ischemic PC in all species studied thus far.
Previously, our laboratory demonstrated the involvement of opioid receptors in ischemic PC in the rat by using the nonselective opioid receptor antagonist naloxone.3 More recently, Chien et al4 showed that this protection is mediated by an opioid receptor(s), since the stereoselective (-) isomer of naloxone abolished ischemic PC in the rabbit. The results of the present study are the first to demonstrate that an opioid receptor agonist, morphine, produces a reduction in infarct size similar to that of ischemic PC.
The present results also suggest that morphine-induced cardioprotection is mediated via opioid receptors, since naloxone could abolish this protection. In addition, morphine not only stimulates µ-opioid receptors but can stimulate other opioid receptors as well.30 The dose that was used in the present study is thought to be relatively selective for µ-opioid receptors; however, studies with more selective opioid receptor agonists and antagonists are essential to further determine which opioid receptor subtype is involved in producing the myocardial protection observed.
Finally, the mechanism involved in opioid receptor–induced
cardioprotection appears to be mediated via the KATP
channel, since the protective effect of morphine was abolished by
glibenclamide. Previously, investigators have shown an interaction of
opioid receptors and KATP channels in regulating
antinociception8 9 10 11 and pial arterial
vasodilation.13 These findings are the first to suggest
that opioid receptor stimulation results in activation of the
myocardial KATP channel. At this time, the complete pathway
between opioid receptor stimulation and KATP channel
opening is uncertain, and the mechanism by which opening this channel
produces cardioprotection is unclear. However, it has been recently
demonstrated by Perchenet and Kreher31 that shortening of
the action potential duration is not necessary for ischemic PC
to occur in the rat. Furthermore, it is not known whether any second
messengers are involved in mediating opioid receptor–induced
cardioprotection; however, Chen and Yu7 demonstrated that
µ-opioid receptors are linked to inwardly rectifying
K+ channels via a G protein and are differentially
regulated by protein kinase A and protein kinase C. In addition, North
et al6 revealed the involvement of a G-protein link
between µ- or -opioid receptors and K+ channels,
with no evidence to support the activation of cAMP-dependent protein
kinase and protein kinase C.
In summary, the present results indicate that ischemic PC markedly reduces myocardial infarct size in the rat. This cardioprotection can be completely abolished by the KATP channel antagonist glibenclamide. The opioid receptor agonist morphine mimicked the cardioprotection induced by ischemic PC, and this effect was completely blocked by naloxone, a nonselective opioid receptor antagonist, thereby indicating an opioid receptor–mediated mechanism. That glibenclamide abolished the morphine-induced cardioprotection suggests an involvement of the myocardial KATP channel as an important component of this potent cardioprotective effect. These findings have important clinical ramifications, since morphine is used preoperatively and postoperatively in coronary artery bypass surgery and in the emergency management of pain in patients suffering an acute myocardial infarction. These data also suggest that opioid agonists may possess a previously unrecognized beneficial cardioprotective effect in some of these patients.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grant HL-08311 and an Advanced Predoctoral fellowship from the Pharmaceutical Research and Manufacturers of America Foundation. The investigators would like to acknowledge James M. Fujimoto, PhD, for his helpful suggestions in designing the present study and Blythe Holmes for her assistance with the morphine studies.
Received March 1, 1996; accepted April 1, 1996.
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