Morphine Mimics the Cardioprotective Effect of Ischemic Preconditioning via a Glibenclamide-Sensitive Mechanism in the Rat Heart
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 δ-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.
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 δ-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 δ1-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.
Materials and Methods
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.
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).
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).
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.
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.
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.
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).
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 α1B-adrenoceptors,27 β-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 δ-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.
Selected Abbreviations and Acronyms
|AAR||=||area at risk|
|KATP channel||=||ATP-sensitive K+ channel|
|MBP||=||mean arterial blood pressure|
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.
- © 1996 American Heart Association, Inc.
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