Inducible Nitric Oxide Synthase Mediates Delayed Cardioprotection Induced by Morphine In Vivo : Evidence from Pharmacologic Inhibition and Gene-knockout Mice

Adult male iNOS gene-knockout (−/−) B6, 129 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Wild-type (WT) mice (Jackson Laboratory) with a genetic background as close as possible to the iNOS gene-knockout mice (B6129PF2/J) were used as controls.11,21The mean body weight of these animals was 31.4 ± 3.0 g. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Hamamatsu University School of Medicine (Hamamatsu, Japan) and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals .22 

Morphine was purchased from Shionogi Co. (Osaka, Japan). S -methylthiourea sulfate (SMT), a selective inhibitor of iNOS, Evans blue, and triphenyltetrazolium chloride were purchased from Sigma Chemical (St. Louis, MO).

Animals were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneal), and anesthesia was maintained via  supplemental doses of pentobarbital sodium (20 mg/kg intraperitoneal) as needed. A tracheotomy was performed, and the animals were mechanically ventilated using a rodent ventilator (SN-480-7; Shinano Manufacturing Co., Tokyo, Japan). The right carotid artery was cannulated with polyethylene-10 tubing to monitor hemodynamics. Core temperature was measured via  a rectal temperature probe and was maintained at 36.5v–37.5°C with a heating pad. After an equilibration period of 10 min, a thoracotomy was performed. Ligation of the left anterior descending coronary artery was performed using a 7-0 silk suture with a small piece of polyethylene tubing to secure the ligature without damaging the artery. Ischemia was confirmed by visual inspection of blanching in the myocardium distal to the site of occlusion. After a period of coronary occlusion of 45 min, the 7-0 silk ligature was removed. Reperfusion was confirmed visually by the return of a red color in the region that was previously pale.

Mice were pretreated with morphine or an equal volume of saline 24 h before a 45-min period of coronary occlusion followed by 120 min of reperfusion. Seven groups were enrolled (n = 8 or 9 mice/group), as shown in figure 1. WT mice were randomly divided into the following five groups: (1) saline: pretreated with 0.1 ml intraperitoneal normal saline; (2) M0.1: pretreated with 0.1 mg/kg intraperitoneal morphine; (3) M0.3: pretreated with 0.3 mg/kg intraperitoneal morphine; (4) saline + SMT: pretreated with 0.1 ml intraperitoneal saline and 3 mg/kg intraperitoneal SMT, which was administered 30 min before coronary occlusion; and (5) M0.3 + SMT: pretreated with 0.3 mg/kg intraperitoneal morphine and 3 mg/kg intraperitoneal SMT, which was administered 30 min before coronary occlusion. iNOS gene-knockout mice were assigned randomly to another two groups: (6) iNOS-KO + saline: pretreated with 0.1 ml intraperitoneal saline; and (7) iNOS-KO + M0.3: pretreated with 0.3 mg/kg intraperitoneal morphine.

At the end of the 120-min reperfusion period, the left anterior descending coronary artery was occluded again, and 1.0% Evans blue (1.5 ml) was infused into the carotid artery catheter in a retrograde manner to delineate the ischemic (area at risk [AAR]) from the nonischemic zones. The heart was then excised and cut into five transverse slices of approximately equal thickness (approximately 1 mm). Incubation of sections in 1% triphenyltetrazolium chloride for 5 min at 37°C allowed differentiation between the viable (brick red) and necrotic (pale) areas of the previously rendered ischemic myocardium. All atrial and right ventricular tissues were excised. After fixation in a 10% formalin solution, each slice was weighed and photographed. The left ventricular area, AAR (absence of blue dye), and area of infarction (pale) for each slice were then determined by computer planimetry using National Institutes of Health Image software (Bethesda, MD). The weight of the myocardial infarct was determined as (A¹× W¹) + (A²× W²) + (A³× W³) + (A⁴× W⁴) + (A⁵× W⁵), where A is the percent area of infarction obtained by planimetry from subscripted numbers 1–5 representing sections, and W is the weight of the corresponding numbered sections. The weight of the AAR was calculated in a similar fashion. The AAR was expressed as a percentage of the weight of left ventricle, and the infarct size (IS) was expressed as a percentage of the AAR.15,23 

In a parallel series of experiments, ventricular tissue samples were collected from the saline, M0.1, M0.3, iNOS-KO + saline, and iNOS-KO + M0.3 groups (n = 3–7/group). To determine the time course of iNOS expression, WT mice hearts were removed at 0.5, 1, 2, 6, 12, 18, and 24 h after 0.3 mg/kg morphine administration (n = 3/group). Three additional nontreated hearts were used as controls. Samples were homogenized in 1 ml ice-cold RIPA buffer. The homogenate was centrifuged at 4°C for 30 min at 15,000 rpm, and the supernatant was recovered as the total cellular protein. All protein extracts were analyzed simultaneously for protein concentration to ensure equal protein loading between samples. Protein extracts were separated on 8% sodium dodecyl sulfate–polyacrylamide gels (60 μg protein/lane) and then transferred onto a nitrocellulose membrane. Blots were blocked for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline and 0.1% Tween-20 and were then washed in Tween-20. The membrane was incubated with a polyclonal rabbit antibody against mouse iNOS or eNOS (dilution, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. After a washing with Tween-20, the membrane was incubated with an anti-rabbit horseradish peroxidase–linked antibody (dilution, 1:5000; Nacalai Tesque, Kyoto, Japan) for 1 h. The membranes were developed using enhanced chemiluminescence and exposed to x-ray film for the appropriate duration. The optical density for each band on the Western blot was quantified using the National Institutes of Health Image program and was normalized against the background density for each gel.24Each protein signal was also normalized to the corresponding Ponceau stain signal.

All values are reported as mean ± SD. Differences among the groups were analyzed with multiple analyses of variance for repeated measures followed by Student-Newman-Keuls post hoc  test. Statistical significance was defined as P < 0.05.

A total of 61 mice were enrolled in the current study. Ten mice were excluded because of death or marked hypotension (mean arterial blood pressure below 30 mmHg) during coronary occlusion and reperfusion. Another two mice were omitted as a result of technical difficulties with the experimental preparation. Complete data were obtained in the remaining 49 mice. Final numbers in the groups are as follows: saline, n = 7; M0.1, n = 6; M0.3, n = 7; saline + SMT, n = 6; M0.3 + SMT, n = 8; iNOS-KO + saline, n = 7; iNOS-KO + M0.3, n = 8.

Mean arterial blood pressure and heart rate were recorded in all groups throughout the experimental protocols. The rate–pressure product (mean arterial blood pressure × heart rate/1,000) was calculated as an index of oxygen demand. Table 1indicates the hemodynamic values from all seven groups. No significant differences in baseline hemodynamic values were observed among these groups. Compared with the saline group, the mean arterial blood pressure was higher in the M0.3 group after 20 min of ischemia (P < 0.05) and was decreased in the M0.3 + SMT group after 60 min of reperfusion (P < 0.05). After 60 min reperfusion, the M0.3 group exhibited a lower heart rate than the saline group (P < 0.05). No significant differences in rate–pressure product were found among these groups at any time point. Coronary artery occlusion and reperfusion produced similar increases in heart rate and decreases in mean arterial blood pressure and rate–pressure product in all experimental groups.

Figure 2summarizes the data of the AAR and IS in all groups. No significant differences in AAR/left ventricle were observed between all groups. IS/AAR was 43.1 ± 5.3% in the saline group. Pretreatment with 0.3 mg/kg morphine markedly reduced the IS/AAR to 22.4 ± 4.4% (P < 0.05 vs.  saline group); however, pretreatment with 0.1 mg/kg morphine did not reduce the infarct size (41.1 ± 5.4%; P > 0.05 vs.  saline group). SMT abolished this cardioprotective effect, as indicated by an increase of IS/AAR to 43.3 ± 3.9% (P > 0.05 vs.  saline group). SMT by itself did not affect the infarct size. Morphine (0.3 mg/kg) pretreatment in iNOS knockout mice did not reduce the infarct size (42.3 ± 4.7%; P > 0.05 vs.  iNOS-KO + saline group). No significant differences in IS/AAR were observed between the saline group and iNOS-KO + saline group, as reported previously.12,25 

Densitometric analysis of the Western blot images revealed that pretreatment with 0.3 mg/kg but not 0.1 mg/kg morphine resulted in significantly enhanced expression of myocardial iNOS compared with the saline-treated hearts. Consistent with the knockout status of the mice, no iNOS protein was expressed in either of the iNOS-KO + saline or iNOS-KO + M0.3 groups (fig. 3). A representative photograph of a Western blot from hearts harvested at different time points after 0.3 mg/kg morphine pretreatment and controls is shown in figure 4. Densitometric analysis indicated that the induction of iNOS reached significance 18 and 24 h after morphine administration, compared with controls.

Endothelial nitric oxide synthase was constitutively expressed in both WT and iNOS gene-knockout mouse hearts. The higher dose (0.3 mg/kg) of morphine caused a slight increase, whereas the lower dose (0.1 mg/kg) caused a slight decrease in eNOS expression; however, these differences were not significant (P > 0.05). No significant differences in eNOS expression was found between the iNOS-KO + saline and iNOS-KO + M0.3 groups (P > 0.05). A representative Western blot image and the results of the densitometric analysis are depicted in figure 5.

The most important findings of this study are summarized as follows: (1) Higher doses of morphine (0.3 mg/kg), when administered 24 h before ischemia and reperfusion, resulted in a significant reduction of myocardial infarct size. (2) This cardioprotective effect was abolished by previous pharmacologic inhibition of iNOS and was absent in mice with targeted ablation of the iNOS gene. (3) The cardioprotective dose of morphine pretreatment significantly enhanced the expression of iNOS in myocardium. In summary, these data demonstrate that high-dose morphine induces a significant delayed cardioprotection in mice and that iNOS mediates this effect.

Using selective δ-opioid receptor agonists, the biphasic nature of the opioid-induced cardioprotection against myocardial infarction has been demonstrated in several mammalian species4–6and even human myocytes.26δ-Opioid agonist mediated delayed cardioprotection has been also found to be only partially dependent on δ-opioid receptor stimulation and via  a free radical mechanism.27Morphine, a μ-opioid receptor agonist with δ-opioid receptor properties,28has been shown to induce acute cardioprotection by a large number of in vivo  and in vitro  studies.3,7–9However, it remains unknown whether morphine could also induce delayed cardioprotection. The current investigation demonstrates that morphine induced late cardioprotective effects in a dose-dependent manner. A higher dose (0.3 mg/kg) of morphine was necessary to achieve significant reduction of the infarct size, whereas the lower dose (0.1 mg/kg) was ineffective. These data are consistent with the findings of a recent study in which a higher dose of morphine, but not a lower dose, induced acute cardioprotection in the rat heart.9 

In recent investigations, iNOS has received considerable attention as the trigger and mediator of delayed cardioprotection induced by divergent pathophysiologic stimuli or pharmacologic agents. iNOS induction has been shown to be necessary for the development of delayed protection conferred by ischemic preconditioning in rabbit models of myocardial infarction29and stunning.30The late phase of ischemic preconditioning is abrogated by targeted disruption of the iNOS gene.11Furthermore, a direct cause-and-effect relation of iNOS in adenosine-induced late cardioprotection was demonstrated by increased iNOS expression, which occurred concomitant with the lack of protective effect of adenosine A(1) receptor activation in iNOS gene-knockout mice.12iNOS has also been found to mediate the delayed cardioprotective effects induced by systemic hypoxia,24endotoxin derivatives,13and whole body hyperthermia.31In the isolated perfused myocardium, Rebrova et al.  32reported that through activation of NOS, stimulation of the δ-opioid receptor increases the tolerance of the heart to oxidative stress. However, no studies are available that demonstrate a link between morphine and activation of a NOS-dependent signaling cascade. In the current study, the morphine-induced late cardioprotection was abolished by previous administration of SMT, a selective iNOS antagonist. SMT is a non–amino acid analog of l-arginine, which has been reported to be 10- to 30-fold more potent than aminoguanidine and the l-arginine analogs at inhibiting iNOS activity.33Targeted disruption of iNOS in the gene-knockout mice also eliminated the delayed protection induced by morphine, whereas morphine was successful in preconditioning hearts from WT mice. Morphine has been found to stimulate iNOS expression in human macrophages.20In the current study, morphine pretreatment significantly up-regulated the iNOS protein content in the myocardium 18–24 h after administration. Taken together, these data provide the unique observation that iNOS is a mediator of the morphine-induced late cardioprotection.

It has also been reported that eNOS mediates delayed cardioprotection.17In the current study, no significant increase of eNOS expression was detected 24 h after morphine pretreatment compared with the control groups. Furthermore, the morphine-induced cardioprotection was completely blocked by administration of an iNOS antagonist and was absent in the iNOS gene-knockout mice. These data refute the role of eNOS as a mediator of the morphine-induced delayed protection. However, the role of eNOS during the trigger phase remains unclear. Nitric oxide derived from eNOS at the time of morphine treatment may initiate the signaling cascade leading to the resulting increase of iNOS expression.34,35 

The opening of a mitochondrial adenosine triphosphate–sensitive potassium (mitoK^ATP) channel or a mitochondrial site of action seems to be an important trigger for the mediation of cardioprotection. Both ischemic and pharmacologic preconditioning may be dependent on the activation of mitoK^ATPchannels. Numerous studies have shown that morphine-induced and opioid-induced cardioprotection were mediated by opening of K^ATPchannels.3,4,7–9,36However, the mediator of the opening of the K^ATPchannel after preconditioning is not fully known. In the current investigation, we observed that iNOS expression was enhanced by morphine pretreatment, which would consequently lead to increased nitric oxide production. Morphine causes the release of nitric oxide in human neutrophils, monocytes, immunocytes,19,37and macrophages.20Opioids have also been found to increase nitric oxide synthesis from vascular endothelial cells and monocytes.38Furthermore, in isolated myocytes, δ-opioid receptor stimulation has been reported to produce nitric oxide, which mediates the cardioprotection.18Nitric oxide may modulate K^ATPchannels via  the second messenger cyclic guanosine monophosphate. Nitric oxide produced by iNOS could potentially activate guanylate cyclase and stimulate cyclic guanosine monophosphate formation. Protein kinase G may be activated by the enhanced cyclic guanosine monophosphate, which can subsequently open mitoK^ATPchannels, resulting in the cardioprotection.39Sasaki et al.  40have reported that nitric oxide directly activates mitoK^ATPchannels in rabbit ventricular myocytes. Functioning as an endogenous mitoK^ATPchannel opener, nitric oxide may titrate the coupling level of the mitochondria to an optimum that blunts mitochondrial calcium overload, without significantly undermining adenosine triphosphate synthetic capacity. Although enhanced iNOS expression could lead to greater nitric oxide production, increasing evidence suggests that iNOS could also generate superoxide rather than nitric oxide under pathophysiologic conditions, such as ischemia, in which l-arginine and tetrahydrobiopterin may be depleted.41,42Therefore, the exact mechanism by which iNOS exerts its effect in morphine-induced protection necessitates further examination.

In conclusion, the current study has demonstrated that morphine induced a delayed cardioprotection against myocardial infarction in mice in a dose-dependent manner. This cardioprotective effect was associated with enhanced expression of iNOS protein in myocardium and was completely abrogated by either selective pharmacologic inhibition of iNOS or targeted disruption the iNOS gene. To the best of our knowledge, this is the first study showing delayed cardioprotection induced by morphine and providing a direct link between morphine preconditioning and the induction of iNOS that subsequently results in delayed cardioprotection. Further investigations are necessary to elucidate the upstream and downstream signaling cascades that lead to the activation of iNOS and whether additional opioid agents are also capable of inducing late preconditioning via  signal transduction pathways analogous to those of morphine.