The aim of this study was to test whether morphine prevents the mitochondrial permeability transition pore (mPTP) opening through Zn2+ and glycogen synthase kinase 3β (GSK-3β). Fluorescence dyes including Newport Green Dichlorofluorescein (DCF), 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), and tetramethylrhodamine ethyl ester (TMRE) were used to image free Zn2+, nitric oxide (NO), and mitochondrial membrane potential (ΔΨm), respectively. Fluorescence images were obtained with confocal microscopy. Cardiomyocytes treated with morphine for 10 min showed a significant increase in Newport Green DCF fluorescence intensity, an effect that was reversed by the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME), indicating that morphine mobilizes Zn2+ via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented Zn2+ release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on Zn2+ release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of ΔΨm, indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the Zn2+ chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). The action of morphine on the mPTP was lost in cells transfected with the constitutively active GSK-3β mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK-3β. In support, morphine significantly enhanced phosphorylation of GSK-3β at Ser9, and this was blocked by TPEN. GSK-3β small interfering RNA prevented the pore opening in the control cardiomyocytes but failed to enhance the effect of morphine on the mPTP opening. In conclusion, morphine mobilizes intracellular Zn2+ through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3β through Zn2+.
- nitric oxide
- protein kinase G
- guanosine 3′,5′-cyclic monophosphate
- glycogen synthase kinase 3β
both endogenous and exogenous opioids can induce acute or delayed preconditioning (15). The first evidence addressing the important role of opioids in early preconditioning was reported by Schultz et al. (36). In the rat heart, they found that naloxone, a nonselective opioid receptor antagonist, completely blocked the anti-infarct effect of preconditioning administered either before or after preconditioning episodes, suggesting that endogenous opioids are crucial in both triggering and mediating preconditioning. The cardioprotective effects of opioids have been attributed to the activation of δ- (18, 35, 37) or κ− (43, 44) opioid receptors. Many studies have documented that protein kinase C (7, 30), mitochondria ATP-sensitive K+ channels (18, 26), tyrosine kinase (10), and mitogen-activated protein kinase (8, 9) contribute to the mechanism of opioid-induced acute cardioprotection. In these studies, the cardioprotective effects of opioids have been obtained when given before the onset of ischemia. Since pretreatments are seldom possible in the clinical settings of acute cardiac infarction, it is desirable that opioids can protect the myocardium when given after onset of ischemia or at reperfusion. Recently, Gross's group (14) has demonstrated that opioids can reduce infarct size when administered just before reperfusion, an effect that was similar to that observed when given before ischemia, suggesting that opioids can prevent reperfusion injury.
The mitochondrial permeability transition pore (mPTP) opening has been proposed to play a critical role in myocardial ischemia-reperfusion injury (40). The mPTP remains closed during ischemia but opens at the onset of reperfusion (13), and suppression of the mPTP opening at early reperfusion leads to cardioprotection against reperfusion injury (16, 17). Postconditioning has been demonstrated to protect the heart from reperfusion injury by targeting the mPTP through activation of δ-opioid receptors in rat hearts (21). However, the precise signaling events that link opioid receptor activation and the inhibition of mPTP opening remain unclear.
Nitric oxide (NO) has been reported to prevent the mPTP opening (42), and morphine was shown to produce NO by activating δ-opioid receptors in rat cardiomyocytes (21). Our laboratory has demonstrated that NO prevents oxidant-induced mPTP opening by mobilizing intracellular Zn2+ (20) and that exogenous Zn2+ prevents reperfusion injury by targeting the mPTP (4) through inactivation of glycogen synthase kinase 3β (GSK-3β). GSK-3β inactivation is involved in the mechanism by which opioids protects the heart at reperfusion (14) and plays a central role in modulation of the mPTP (22). Therefore, it is reasonable to hypothesize that opioids may prevent the mPTP opening by inactivating GSK-3β through mobilization of Zn2+.
In the present study, we first examined whether morphine could mobilize intracellular Zn2+ via a NO-dependent mechanism in isolated rat cardiomyocytes. We then tested whether morphine could inactivate GSK-3β. Finally, we determined whether morphine prevents the mPTP opening via Zn2+ and GSK-3β.
MATERIALS AND METHODS
This study conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996). All protocols for the experiments using animals were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.
Chemicals and antibodies.
Morphine was purchased from Sigma, and inhibitors were obtained from EMD Biosciences (La Jolla, CA). Fluorescence dyes were purchased from Molecular Probes (Eugene, OR). Antibodies were purchased from Cell Signaling Technology (Beverly, MA).
Isolation of adult rat cardiomyocytes.
Rat cardiomyocytes were isolated enzymatically (45). Male Wistar rats weighing 250–350 g were anesthetized with thiobutabarbital sodium (100 mg/kg ip). A midline thoracotomy was performed, and the heart was removed and rapidly mounted on a Langendorff apparatus. The heart was perfused in a nonrecirculating mode with Krebs-Henseleit buffer (37°C) containing (in mM) 118 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.25 CaCl2, and 10 glucose for 5 min to wash out blood. The buffer was bubbled with 95% O2-5% CO2. The heart was then perfused with a calcium-free buffer that contained all of the above components except CaCl2. After 5 min of perfusion, collagenase (type II) was added to the buffer (0.1%) and the heart was perfused in a recirculating mode for ∼15 min. The heart was removed from the apparatus, and the ventricles were placed into a beaker containing the calcium-free buffer. The ventricles were agitated in a shaking bath (37°C) at a rate of 50 cycles/min until individual cells were released. The released cells were suspended in an incubation buffer containing all the components of the calcium-free buffer, 1% bovine serum albumin, 30 mM HEPES, 60 mM taurine, 20 mM creatine, and amino acid supplements at 37°C. Calcium was gradually added to the buffer containing the cells to a final concentration of 1.2 mM. The cells were filtered through nylon mesh and centrifuged briefly. Finally, the cells were suspended in culture medium M199 for 4 h before experiments.
H9c2 cell culture.
Rat heart tissue-derived H9c2 cardiac myoblast cell line was purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 U penicillin-streptomycin at 37°C in a humidified 5% CO2-95% air atmosphere.
GSK-3β plasmid DNA.
The constitutively active GSK-3β (GSK-3β-S9A-HA) mutant plasmids containing HA-tag was kindly provided by Dr. Morris Birnbaun (University of Pennsylvania School of Medicine). After the plasmid DNA was purified using Endofree Maxi kit (Qiagen), transient transfections were performed on 12-well plates using Fugene6 with 2 μg DNA according to the manufacturer's instructions (Roche). Briefly, H9c2 cells were seeded in a 12-well plate at a 50% confluency. Two hours after cell seeding, cells were transfected with β-gal (pCDNA-His-LacZ) or the GSK-3β mutants using Fugene6 transfection reagents (DNA:reagent ratio, 1:3). Cells were replaced with fresh medium 24 h after the transfection. All experiments were done 48 h after transfection. The transfection efficiency test using β-galactosidase assay kit (Invitrogen) revealed that over 80% of cells expressed β-galactosidase (>80% transfection; data not shown). To test the protein expression levels of the mutant genes, total GSK-3β protein levels were analyzed with Western blot. Since both mutants include HA tags, the expressed constructs could be distinguished from the endogenous GSK-3β (29).
Confocal imaging of intracellular Zn2+.
Intracellular Zn2+ was detected with Newport Green Dichlorofluorescein (DCF) (20). Cardiomyocytes cultured in a specific temperature-controlled culture dish were incubated with 2 μM Newport Green DCF diacetate in standard Tyrode solution containing (in mM) 140 NaCl, 6 KCl, 1 MgCl2, 1 CaCl2, 5 HEPES, and 5.8 glucose (pH 7.4) for 20 min. Cells were then mounted on the stage of an Olympus FV 500 laser scanning confocal microscope. The green fluorescence was excited with the 488-nm line of argon-krypton laser and imaged through a 525-nm long-path filter. Temperature was maintained at 37°C with Delta T Open Dish Systems (Bioptechs, Butler, PA). The images recorded on a computer were quantified using ImageJ.
Confocal imaging of mitochondrial membrane potential.
Mitochondrial membrane potential (ΔΨm) was measured using confocal microscopy as reported previously (45). Briefly, cells were incubated with tetramethylrhodamine ethyl ester (TMRE; 100 nM) in standard Tyrode solution for 20 min. The red fluorescence was excited with a 543-nm line of argon-krypton laser line and imaged through a 560-nm long-path filter.
Confocal imaging of NO.
To measure intracellular NO concentration, cardiomyocytes were loaded with 2 μM 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate (45). The green fluorescence was excited at 488 nm and imaged through a 525-nm long-path filter.
Transfection of small interfering RNA.
GSK-3α/β small interfering RNA (siRNA) was obtained from Cell Signaling. Rat cardiomyocytes were transfected with siRNA (100 nM) according to according to the manufacturer's instruction. Imaging studies were conducted 48 h after transfection.
Western blotting analysis.
Cardiomyocytes were homogenized in ice-cold lysis buffer. Equal amounts of protein were loaded and eletrophoresed on SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Membranes were probed with the primary antibody that recognizes phosphorylation of GSK-3β (Ser9). The primary antibody bindings were detected with a secondary anti-rabbit antibody and visualized by the enhanced chemiluminescence method. Equal loading of samples was confirmed by reprobing membranes with anti-GSK-3β (total) antibody.
In the experiments monitoring changes in the intracellular Zn2+ and NO levels, morphine was given immediately after baseline (0′) measurements, whereas the inhibitors were applied 10 min before the application of morphine. To determine the effects of morphine on ΔΨm, rat cardiomyocytes were exposed to 100 μM H2O2 for 20 min. Morphine was given 10 min before exposure to H2O2. Inhibitors were given 10 min before the exposure to morphine. To test the effect of morphine (and Zn2+) on GSK-3β phosphorylation at Ser9, cells were exposed to 1 μM morphine for 10 min. In experiments investigating the role of GSK-3β in the action of morphine, cardiac H9c2 cells were exposed 600 μM H2O2 for 20 min and morphine was applied 10 min before exposure to H2O2.
Data are expressed as means ± SE and were obtained from 5 to 10 separate experiments. Statistical significance was determined using Student's t-test or one-way ANOVA followed by Tukey's test. A value of P < 0.05 was considered as statistically significant.
Morphine (1 μM) significantly enhanced Newport Green DCF fluorescence intensity (155.9 ± 7.2% of baseline) compared with the control (109.8 ± 2.4% of baseline), an effect that was abolished by the NO synthase (NOS) inhibitor N G-nitro-l-arginine methyl ester (l-NAME), suggesting that morphine mobilizes intracellular Zn2+ by producing NO in isolated rat cardiomyocytes (Fig. 1). H2O2 and l-NAME did not alter the fluorescence intensity. To confirm the above observation that morphine releases Zn2+ via NO, we then tested whether morphine could produce NO in cardiomyocytes. Figure 2 shows that morphine markedly enhanced DAF-FM fluorescence intensity (154.9 ± 19.7% of baseline) 10 min after the treatment compared with the control (102.2 ± 2.4% of baseline), suggesting that morphine indeed produces NO in cardiomyocytes. The effect of morphine on NO generation was reversed by the NOS inhibitor l-NAME (99.6 ± 4.3% of baseline), indicating that morphine produces NO via NOS. Figure 3 shows that the effect of morphine (155.9 ± 7.2% of baseline) on Newport Green DCF fluorescence was blocked by ODQ (119.3 ± 6.3% of baseline), a potent and selective inhibitor of NO-sensitive guanylyl cyclase, implying that cGMP is responsible for the Zn2+-releasing effect of morphine. The involvement of cGMP in the action of morphine was confirmed by further experiments in which the action of morphine was reversed by another specific and irreversible inhibitor of guanylyl cyclase: NS2028 (105.8 ± 4.1% of baseline; Fig. 3). Figure 3 further shows that KT5823, a highly specific cell-permeable inhibitor of protein kinase G (PKG), nullified the action of morphine (106.0 ± 3.3% of baseline), suggesting that PKG contributes to the action of morphine.
To examine whether morphine prevents the mPTP opening by the cGMP/PKG/Zn2+ pathway, we tested the effect of morphine on the oxidant-induced loss of ΔΨm in rat cardiomyocytes. Treatment of cardiomyocytes with 100 μM H2O2 dramatically decreased TMRE fluorescence (45.1 ± 8.2 of baseline in the control), indicating that oxidant stress caused loss of ΔΨm (Fig. 4). Because the loss of ΔΨm is caused by the mPTP opening (6), this result may indicate that oxidant stress caused the mPTP opening. Treatment of cells with 1 μM morphine prevented the loss of TMRE fluorescence (90.0 ± 2.0% of baseline), indicating that morphine can modulate the mPTP opening. This effect of morphine was abolished by the PKG inhibitor KT5823 (51.1 ± 4.8% of baseline). Further experiments showed that morphine was not able to preserve TMRE fluorescence in cells pretreated with the Zn2+ chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) (57.5 ± 2.0% of baseline), indicating that morphine prevent the mPTP opening by mobilizing intracellular Zn2+. ZnCl2 (1 μM) in the presence of the zinc ionophore pyrithione mimicked the action of morphine to preserve TMRE fluorescence (Fig. 4). The recognized closer of cyclosporine A (0.2 μM) also prevented the loss of TMRE fluorescence, confirming that morphine and zinc moderate the pore opening in cardiomyocytes. KT5823, ODQ, and TPEN did not alter TMRE fluorescence.
To define the role of GSK-3β in the protective action of morphine, we determined the effect of morphine on GSK-3β activity by detecting its phosphorylation at Ser9 10 min after exposure to morphine. As shown in Fig. 5, morphine significantly enhanced GSK-3β phosphorylation (174.3 ± 16.2% of control), an effect that was reversed by TPEN (114.2 ± 13.6% of control), implying that morphine inactivates GSK-3β via Zn2+ in cardiomyocytes. The effect of morphine on GSK-3β phosphorylation was further reversed by l-NAME (118.2 ± 9.4% of control), ODQ (110.3 ± 7.8% of control), and KT5823 (105.3 ± 9.9% of control). H2O2 (100 μM) reduced GSK-3β phosphorylation (76.2 ± 5.8% of control).
To determine whether morphine prevents the mPTP opening by inactivating GSK-3β, cardiac H9c2 cells were transfected with the constitutively active GSK-3β mutant (GSK-3β-S9A) plasmid and then treated with morphine. As shown in Fig. 6 (left), 600 μM H2O2 caused a dramatic decrease in TMRE fluorescence (38.8 ± 2.0% of baseline), indicating the mPTP opening by oxidant stress. The treatment of cells with 1 μM morphine prevented the loss of TMRE fluorescence (73.5 ± 4.9% of baseline), suggesting that morphine can modulate the mPTP opening in H9c2 cells. In contrast, morphine failed to preserve TMRE fluorescence in cells transfected with GSK-3β-S9A (37.9 ± 2.9% of baseline), suggesting that GSK-3β is critical for the action of morphine on the mPTP opening. To confirm the role of GSK-3β in the action of morphine in rat cardiomyocyte, we suppressed GSK-3β protein expression silencing GSK-3β RNA. GSK-3β silencing prevented the loss of TMRE fluorescence in control hearts, indicating that the suppression of GSK-3β protein levels leads to modulation of the mPTP opening (Fig. 6, right). In contrast, siRNA failed to further increase TMRE fluorescence in the hearts treated with morphine, implying that the preventive effect of morphine on the pore opening was attributable to inactivation of GSK-3β.
In this study, we have demonstrated that morphine mobilizes intracellular Zn2+ via the NO/cGMP/PKG signaling pathway in cardiomyocytes. Zn2+ mediates the inhibitory effect of morphine on the mPTP opening by inactivating GSK-3β.
Postconditioning protects the heart from reperfusion injury by targeting the mPTP through activation of δ-opioid receptors and that the opioid receptor agonist morphine mimicked the effect of postconditioning by modulating oxidant-induced mPTP opening (21). Similarly, a recent study by Obame et al. (32) has also shown that morphine delayed the mPTP opening induced by anoxia/reoxygenation in rat cardiomyocytes. These observations indicate that opioid receptor activation protects the heart from ischemia-reperfusion injury by targeting the mPTP. Although inactivation of GSK-3β was proposed to be responsible for the preventive effect of morphine on the mPTP opening (32), the exact mechanism for the action of morphine on the mPTP remains unclear. We hypothesized that morphine mobilizes intracellular Zn2+ through the NO/cGMP/PKG signaling pathway and Zn2+ modulates the mPTP opening by inactivating GSK-3β. This hypothesis was based on the following observations. First, exogenous NO (11) mobilizes intracellular Zn2+ through the cGMP/PKG pathway and morphine can produce NO in cardiomyocytes (20). Second, Zn2+ inhibits the mPTP opening by inactivating GSK-3β in cardiac cells (4).
It is well known that the major intracellular Zn2+ binding protein is metallothionein (46). Nitrosylation of metallothionein has been proposed to be the mechanism by which NO at high doses (2 mM SNOC or 0.2 and 2 mM DETA/NO) releases Zn2+ (3, 25, 38, 39). In a recent study, we have demonstrated that exogenous NO at a low dose mobilizes intracellular Zn2+ via the cGMP/PKG signaling pathway in cardiomyocytes (20). In the present study, we found that morphine mobilizes intracellular Zn2+ through the same signaling pathway. This was evidenced by the observation that the effect of morphine on Newport Green DCF fluorescence was blocked by the NOS inhibitor l-NAME, the guanylyl cyclase inhibitor ODQ, and the PKG inhibitor KT5823. In addition, morphine was also able to produce NO in cardiomyocytes. It is, therefore, reasonable to propose that morphine produces NO, which in turn mobilizes intracellular Zn2+ through the cGMP/PKG pathway. Since the cGMP/PKG pathway plays an important role in cardioprotection (5), the release of Zn2+ by morphine through this pathway may serve as an important mechanism for cardioprotection against ischemia-reperfusion injury.
Zn2+ has been demonstrated to prevent apoptosis both in vitro and in vivo models (28, 41). Recently, it has been reported that the treatment of isolated rat hearts at reperfusion with Zn2+ ionophore pyrithione induces cardioprotection against ischemia-reperfusion injury (23). Recent studies from our laboratory have shown that Zn2+ modulates oxidant-induced mPTP opening in isolated rat cardiomyocytes (20) and prevents reperfusion injury by targeting mPTP in cardiac H9c2 cells (4). In this study, the inhibitory effect of morphine on the mPTP was partially but significantly blocked by both the PKG inhibitor KT5823 and the Zn2+ chelator TPEN, suggesting that morphine protects mitochondria by mobilizing Zn2+ via the cGMP/PKG pathway. Although we report the role of zinc in the preventive effect of morphine on the mPTP opening in the setting of oxidant stress in this study, the release of zinc by morphine may play an important role in cardioprotection against reperfusion injury. We have recently demonstrated that intracellular free zinc levels were markedly decreased upon reperfusion. The adenosine receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reversed the decrease in free zinc levels by mobilizing intracellular zinc, and this contributed to the anti-infarct effect of NECA (manuscript under review). Similarly, Karagulova et al. (23) have also demonstrated decreased levels of intracellular free zinc in isolated rat hearts subjected to ischemia-reperfusion. Thus the zinc release by morphine may also prevent the decrease in free zinc levels in the setting of ischemia-reperfusion, which may account in part for the cardioprotective effect of morphine at reperfusion. Mobilization of intracellular zinc at reperfusion may serve as a useful approach to reduce reperfusion injury.
Zn2+ can regulate activities of many intracellular signaling elements including Akt (2), p70S6 kinase (24), mTOR (27), ERK (2), and GSK-3β (19). Among these signaling molecules, GSK-3β has recently been proposed to play a critical role in the modulation of the mPTP opening in cardiomyocytes. Many cardioprotective interventions protect the heart from ischemia-reperfusion injury by targeting the mPTP through inactivation of GSK-3β (12, 22, 31, 33, 34). GSK-3β activity is regulated by the phosphorylation of Ser9 and Tyr216 residues. Phosphorylation of Ser9 decreases, but Tyr216 phosphorylation increases GSK-3β activity. Zn2+ can inactivate GSK-3β (1, 19), and we have demonstrated that Zn2+ prevents reperfusion injury by inactivating GSK-3β in cardiac cells (4). Gross et al. (14) have documented that GSK-3β inactivation accounts for the cardioprotective effect of morphine at reperfusion. In the present study, morphine markedly increased GSK-3β phosphorylation at Ser9 and this was inhibited by the Zn2+ chelator TPEN, suggesting that morphine may inactivate GSK-3β through Zn2+. In addition, the inhibitory effect of morphine on the mPTP opening was blocked by the Zn2+ chelator TPEN and the action of morphine on the mPTP was lost in cells transfected with the constitutively active GSK-3β mutant (GSK-3β-S9A). Moreover, suppression of GSK-3β protein levels with siRNA could prevent the pore opening in the control but failed to enhance the protective effect of morphine. Therefore, it is reasonable to conclude that morphine modulates the mPTP opening by inactivating GSK-3β through Zn2+. As to the mechanism by which mobilized Zn2+ inactivates GSK-3β, we assume that Zn2+ may inhibit GSK-3β through the Akt/phosphatidylinositol 3-kinase signaling pathway, as we documented previously (4).
In summary (Fig. 7), morphine mobilizes intracellular Zn2+ via the NO/cGMP/PKG signaling pathway and modulates the mPTP opening by inactivating GSK-3β through Zn2+. These findings provide new insights into the mechanism by which opioids protect the heart from ischemia-reperfusion injury.
This work was supported by the American Heart Association Grant 0555430U; Grant 2007136 from Bureau of Education, Hebei Province, China; and Grant 20072010 from Bureau of Human Resource, Hebei Province, China.
No conflicts of interest are declared by the author(s).
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