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Am J Physiol Heart Circ Physiol 288: H1955-H1964, 2005. First published November 24, 2004; doi:10.1152/ajpheart.00256.2004
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Met5-enkephalin-induced cardioprotection occurs via transactivation of EGFR and activation of PI3K

Zhiping Cao,2 Lijuan Liu,2 and Donna M. Van Winkle1,2,3

1Research and 2Anesthesiology Services, Veterans Affairs Medical Center, and 3Department of Anesthesiology, Oregon Health and Sciences University, Portland, Oregon

Submitted 15 March 2004 ; accepted in final form 21 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our previous studies indicated that opioid-induced cardioprotection occurs via activation of mitochondrial ATP-sensitive K+ (KATP) channels. However, other elements of the Met5-enkephalin (ME) cardioprotection pathway are not fully characterized. In the present study, we investigated the role of tyrosine kinase, MAPK, and phosphatidylinositol 3-kinase (PI3K) signaling in ME-induced protection. Ca2+-tolerant, adult rabbit cardiomyocytes were isolated by collagenase digestion and subjected to simulated ischemia for 180 min. ME was administered 15 min before the 180 min of simulated ischemia; blockers were administered 15 min before ME. Cell death was assessed by trypan blue as a function of time. The epidermal growth factor receptor (EGFR) kinase inhibitor AG-1478 (250 nM) blocked ME-induced protection, but the inactive analog AG-9 (100 µM) did not. Treatment with herbimycin (1 µM) completely eliminated ME-induced protection. To verify that ME activates EGFR and to determine the involvement of Src, Western blotting of EGFR was performed after ME administration with and without herbimycin A. ME resulted in herbimycin-sensitive robust phosphorylation of EGFR at Tyr992 and Tyr1068. Administration of the selective MAPK inhibitor PD-98059 (10 nM) and the specific MEK1/2 inhibitor U-0126 (10 µM) also inhibited ME-induced cardioprotection. ME-induced ERK1/2 phosphorylation was significantly reduced by PD-98059, the EGFR kinase inhibitor PD-153035 (10 µM), and chelerythrine (2 µM). The PI3K inhibitor LY-294002 (20 µM) abrogated ME-induced protection, and ME-induced Akt phosphorylation at Ser473 was suppressed by LY-294002, PD-153035, and chelerythrine. We conclude that ME-induced cardioprotection is mediated via Src-dependent EGFR transactivation and activation of the PI3K and MAPK pathways.

peptides; opioid; opioid receptor; ischemic preconditioning; signaling

PRECONDITIONING DUE TO ACTIVATION of opioid receptors has been reported to be mediated by a kinase cascade involving protein kinase C (PKC) (20, 21, 24, 35) and via opening of the ATP-sensitive K+ (KATP) channels (4, 16, 18, 43, 45). However, the details of the signal pathway involved in opioid peptide-induced cardioprotection have remained unclear. Opioid receptors belong to the superfamily of G protein-coupled receptors (GPCRs), and GPCR-induced epidermal growth factor (EGF) receptor (EGFR) signal transactivation occurs in a variety of cell types (8, 13, 55). In cardiomyocytes, EGFR signal transactivation has previously been shown to be dependent on PKC activity (30, 47, 49).

EGFR is a 1,186-amino acid single-pass transmembrane tyrosine kinase. An amino-terminal 622-amino acid intracellular domain containing two cysteine-rich domains comprises the ligand-binding domain. The intracellular 542 amino acids contain three domains: the juxtamembrane domain, the tyrosine kinase domain, and the autophosphorylation domain (52). Mutation of the autophosphorylation sites of EGFR have indicated that phospho-Tyr992 (pTyr992) of activated EGFR is a direct binding site for the phospholipase C-{gamma} SH2 domain. This binding results in activation of phospholipase C-{gamma}-mediated downstream signaling (14). Phospho-Tyr1068 (pTyr1068) of activated EGFR is a direct binding site for the Gra2/SH2 domain (42). This binding results in Ras/MEK (14) activation through a Grb2/Sos-1 signaling mechanism (54). The EGFR plays a key role in the regulation of essential normal cellular processes as well as pathophysiological states. GPCRs are able to utilize the EGFR as a downstream signaling partner in cellular signal transduction and diversification (15). Transactivation of the EGFR is essential for activation of GPCRs by agonists to produce responses attributed to tyrosine kinase receptors (11, 12).

GPCR-evoked signal transduction pathways leading to the activation of extracellular signal-regulated kinases (ERK) are quite different among cell types. In cardiomyocytes, much attention has been focused on the activation of PKC and MEK. The synthetic {delta}-opioid peptide [D-Ala2,D-Leu5]enkephalin has been reported to activate the p42 and p44 isoforms of ERK in rat fibroblasts stably transfected with the cloned {delta}-opioid receptor (3) and to promote cell survival via the MEK/ERK pathway in PC12 cells (29). Moreover, in anesthetized rats subjected to acute myocardial ischemia-reperfusion, blockade of MEK1/2 with PD-98059 eliminated the infarct limitation conferred by the synthetic {delta}-agonist TAN-67 (19), as did the tyrosine kinase inhibitor genistein (25). In addition, the phosphatidylinositol 3-kinase (PI3K)-Akt (protein kinase B) signaling pathway has been shown to contribute to cardioprotection induced by ischemic and pharmacological preconditioning (10, 37, 39) in a manner that is sensitive to tyrosine kinase and c-Src blockade (32). Recently, Gross and colleagues (26) reported that opioid agonists result in infarct limitation and phosphorylation of the Akt target glycogen synthase kinase-{beta} in rats. However, the contributions of EGFR and the downstream signaling pathways utilized by endogenous opioid peptides are incompletely described.

Therefore, we tested the hypothesis that Met5-enkephalin (ME)-induced protection is mediated via transactivation of EGFR, which leads to the activation of the PKC, MEK/ERK, and/or PI3K pathways.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animals were allowed access to food and water ad libitum until induction of anesthesia. With local Institutional Animal Care and Use Committee approval, all animals received humane treatment in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Research, National Research Council, National Academy Press, 1996).

Cell isolation. Ca2+-tolerant, adult rabbit cardiomyocytes were isolated by collagenase digestion as previously reported (51). Male New Zealand White rabbits (2.4–3.1 kg) were anesthetized with pentobarbital sodium (40 mg/kg) via a marginal ear vein. A tracheotomy was performed, and positive-pressure ventilation with 100% oxygen was established at a rate of 35 breaths/min. After exposure of the myocardium via a left thoracotomy, the heart was rapidly excised and mounted on a nonrecirculating Langendorff apparatus. The heart was perfused at 37°C with oxygenated Krebs-Henseleit buffer (in mM: 118.5 NaCl, 24.8 NaHCO3, 10.0 glucose, 4.7 KCl, 2.0 CaCl2, 1.2 KH2PO4, and 1.2 MgSO4, pH 7.4) to wash out intravascular blood (5 min). Hearts were then perfused with Ca2+-free buffer (in mM: 118.5 NaCl, 24.8 NaHCO3, 10.0 glucose, 4.7 KCl, 1.2 KH2PO4, and 1.2 MgSO4, pH 7.4) for 5 min or until they ceased to contract. On cessation of contractile activity, the hearts were switched to a recirculating perfusion mode at ~100 cmH2O. Collagenase (type II, Worthington Biochemical) was added to a final concentration of ~1 mg/ml, and perfusion continued until the hearts became dilated and started to soften, ~20 min. Hearts were then removed from the perfusion apparatus, trimmed of atria and great vessels, placed in a beaker with a small volume of oxygenated collagenase solution, and gently agitated in a reciprocating shaker bath to disperse the cells. In an iterative fashion, supernatant containing dispersed cells was removed from the beaker and replaced with fresh oxygenated collagenase solution. The collected digest was washed, filtered through a nylon mesh, and resuspended in warm oxygenated incubation buffer [118.5 mM NaCl, 24.8 mM NaHCO3, 10.0 mM glucose, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 30.0 mM HEPES, 60.0 mM taurine, 20.0 mM creatine, 0.68 mM glutamine, 1% Eagle’s basal medium (BME) amino acids, 1% MEM nonessential amino acids, and 1% BME vitamin solution, pH 7.4]. After a 30-min equilibration period, Ca2+ was gradually reintroduced to a final concentration of 1.25 mM. Before the experimental protocol was begun, cells were washed twice and resuspended in fresh incubation buffer, and 1-ml aliquots were gently pipetted into 1.8-ml microcentrifuge tubes. Isolate yield was sufficient for five experimental groups plus an oxygenated time control. Isolates containing <80% rods were not used.

Simulated ischemia. Cells were pelleted by brief centrifugation (35 g for 20 s), and the supernatant was discarded. The volume of each cell pellet was ~0.2 ml. Mineral oil (0.5 ml) was then layered on top of the cell pellet to exclude oxygen delivery, and the cells were incubated without agitation at 37°C for 180 min.

Drugs. AG-1478, AG-9, herbimycin A, LY-294002, PD-98059, U-0126, U-0124, and chelerythrine were obtained from Calbiochem (La Jolla, CA); Met5-enkephalin was obtained from Sigma (St. Louis, MO). All drugs were dissolved in distilled water (chelerythrine, ME, and naloxone) or DMSO (AG-1478, AG-9, herbimycin A, LY-294002, PD-98059, U-0126, and U-0124), aliquoted, and frozen until use. On the day of the experiment, 1 µl of stock solution was diluted directly into 1 ml of the cell suspension. Doses were chosen on the basis of our previous studies and from the literature. Agonists were administered to the cell suspension for 15 min before the 180-min pelleting; antagonists were administered to the cell suspension for 15 min before agonist treatment.

Determination of cell viability. Cell viability was determined before any experimental maneuvers (baseline), immediately before the 180-min simulated ischemia (time 0), and every 30 min thereafter. For each of the groups, a 15-µl aliquot of cells was withdrawn from the pellet by pipette, resuspended in 150 µl of hypotonic buffer (85 mosM) containing 3 mM amytal sodium as a mitochondrial inhibitor, and allowed to equilibrate for 3–4 min. On a microscope slide, a 15-µl sample of this solution was then mixed with an equal volume of trypan blue solution (0.5% glutaraldehyde in 85 mosM NaCl-deficient Tyrode solution containing 1% trypan blue). Three widely separated fields at x100 magnification were then examined to determine cell morphology (rod, round, or square) and permeability (blue vs. not blue), and the results were averaged for each group (1). More than 300 cells were examined in each sample. Cells that were not able to exclude trypan blue were considered to have membrane failure and, therefore, to be nonviable. All viability experiments were accompanied by an untreated oxygenated time control group.

Western blotting. Rabbit cardiomyocytes isolated as described above were cultured for 24 h in serum-free OPTI-MEM (GIBCO) supplemented 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µM cytosine arabinoside (to inhibit fibroblast proliferation). To exclude nonmuscle cells, isolated cells were first plated at 37°C for 1 h under a water-saturated atmosphere of 5% CO2-95% O2, and the suspended cells were then collected and plated at a density of 1.0 x 105 cells/cm2. Cells were treated with ME for 15 min and then sampled. Inhibitors were added 15 min before ME treatment. Myocyte proteins (~20 µg/lane) were separated on 4–20% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad) and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% dry milk and incubated with primary antibodies [anti-Akt, anti-pAkt (Ser473), anti-pAkt (Thr308), anti-ERK1/2, anti-pERK1/2, anti-EGFR, anti-pEGFR (Tyr992), or anti-pEGFR (Tyr1068), 1:1,000 in 5% dry milk]. Antigens were detected by the luminescence method (ECL-plus Western blotting detection kit, Amersham) with peroxidase-linked anti-rabbit (1:1,000 in 5% dry milk). All antibodies were obtained from Cell Signaling (Beverly, MA), except pTyr992 antibody, which was obtained from Biosource (Camarillo, CA). After immunoblotting was completed, the band intensity was assessed with a Kodak 1D Image Analysis System.

Experimental protocols. To test whether EGFR transactivation is involved in ME-induced protection, we assessed the role of EGFR in ME-induced preservation of cellular viability, measured ME-induced EGFR phosphorylation, and determined the opioid-receptor dependence of EGFR phosphorylation. To determine whether blockade of EGFR alters ME-induced cardioprotection, four groups were studied: control, 100 µM ME, 250 nM AG-1478, and 100 µM ME + 250 nM AG-1478. Four additional groups were examined as a negative control: control, 100 µM ME, 100 µM AG-9, and 100 µM ME + 100 µM AG-9. To determine whether ME results in phosphorylation of EGFR, Western blots of total and phosphorylated EGFR were performed after ME treatment; treatment with EGF was included as a positive control in control, 100 µM ME, and 16 nM EGF groups. To test whether ME-induced phosphorylation of EGFR is an opioid receptor-dependent event, four groups were examined: control, 100 µM ME, 100 µM naloxone, and 100 µM ME + 100 µM naloxone. Finally, we tested whether EGFR transactivation involves activation of Src. To assess the role of Src, we examined ME-induced protection and ME-induced phosphorylation in the presence and absence of the Src kinase inhibitor herbimycin A in control, 100 µM ME, 1 µM herbimycin A, and 100 µM ME + 1 µM herbimycin A groups.

Second, we examined potential downstream signaling components of EGFR. To test whether MEK is involved in opioid-induced cardioprotection, we assessed ME-induced cardioprotection and phosphorylation of ERK1/2 in the absence and presence of the selective inhibitors of MEK1/2, PD-98059, and U-0126 in control, 100 µM ME, 10 nM PD-98059, and 100 µM ME + 10 nM PD-98059 groups and in control, 100 µM ME, 100 µM U-0126, and 100 µM ME + 100 µM U-0126 groups. Four additional groups were examined as a negative control: control, 100 µM ME, 100 µM U-0124, and 100 µM ME + 100 µM U-0124. To test the involvement of PI3K in ME-induced protection, we examined ME-induced cardioprotection as well as phosphorylation of the downstream target of PI3K, Akt, in the absence and presence of selective PI3K inhibition with LY-294002 in control, 100 µM ME, 20 µM LY-294002, and 100 µM ME + 20 µM LY-294002 groups.

We also examined interrelations between the signaling components. To assess whether ME-induced activation of PI3K and MEK occurs via EGFR signaling, we examined ME-induced phosphorylation of Akt and ERK1/2 in the absence and presence of the EGFR inhibitors AG-1478 and PD-153035 in control, 100 µM ME, 250 nM AG-1478, and 100 µM ME + 250 nM AG-1478 groups and in control, 100 µM ME, 10 µM PD-153035, and 100 µM ME + 10 µM PD-153035 groups. To investigate whether ERK activation occurs via PI3K signaling, we examined ME-induced phosphorylation of ERK1/2 in the absence and presence of the PI3K blocker LY-294002 in control, 100 µM ME, 20 µM LY-294002, and 100 µM ME + 20 µM LY-294002 groups. Finally, to determine the role of PKC in ME-induced protection, we assessed ME-induced cardioprotection and phosphorylation of Akt and ERK1/2 in the absence and presence of the PKC inhibitor chelerythrine in control, 100 µM ME, 2 µM chelerythrine, and 100 µM ME + 2 µM chelerythrine groups. A summary of the experiments is given in Table 1.


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  		Table 1. Experiments

 
Data analysis. Data were analyzed with a personal computer-based statistical software package (Prism 4.0, GraphPad Software, San Diego, CA). For the data of cell viability experiments, the primary measured end point for all studies was cell death, defined as uptake of trypan blue. For each group, the percentage of dead cells was plotted vs. duration of pelleted incubation. The area under these injury curves (AUC) was calculated for each individual experiment. For all the data, differences between groups were assessed by one-way ANOVA with repeated measures, with a Student-Newman-Keuls post hoc test. Statistical significance was assumed for P ≤ 0.05. Values are means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We first assessed the role of EGFR transactivation in ME-induced protection in isolated rabbit cardiomyocytes from simulated ischemia. The EGFR kinase inhibitor AG-1478 abolished ME-induced cardioprotection, whereas the inactive analog AG-9 had no effect (Fig. 1). Western blotting demonstrated that ME induced EGFR phosphorylation at Tyr992 and Tyr1068 (but not Tyr845 or Tyr1173) comparable to that seen with EGF: for ME, 234 ± 29 and 190 ± 2% at pTyr992 and pTyr1068, respectively; for EGF, 238 ± 17 and 282 ± 25% at pTyr992 and pTyr1068, respectively (means ± SE, expressed as percentage of control). Moreover, ME-induced phosphorylation of EGFR was abolished by opioid receptor blockade with naloxone (Fig. 2). When we tested whether EGFR transactivation involves activation of Src, we found that the Src kinase inhibitor herbimycin A totally abolished ME-induced protection as well as EGFR phosphorylation (Fig. 3).



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  		Fig. 1. Epidermal growth factor (EGF) receptor (EGFR) participates in Met5-enkephalin (ME)-induced cardioprotection. A and C: percentage of cell death (%Dead) at 0, 30, 60, 90, 120, 150, and 180 min of simulated ischemia. B and D: area under the curve (AUC) data presented as means ± SE. Selective EGFR antagonist AG-1478 blocks ME-induced protection of isolated cardiomyocytes subjected to simulated ischemia, but its inactive congener AG-9 does not. Differences between slopes were highly significant for control vs. ME (P < 0.0001) and ME vs. AG-1478 + ME (P < 0.0001). AG-1478 and AG-1478 + ME slopes were not significantly different from each other (P > 0.05).

 


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  		Fig. 2. A and B: ME induces EGFR phosphorylation at Tyr992 and Tyr1068, but not Tyr845 and Tyr1173, as assessed by Western blotting. EGF was used as a positive control. C and D: ME-induced EGFR phosphorylation is abolished by opioid receptor blockade with naloxone. Values are means ± SE. phospho, Phosphorylated form. NS, not significant.

 


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  		Fig. 3. Src inhibitor herbimycin A abolishes ME-induced protection and eliminates ME-induced phosphorylation of EGFR. A and B: viability data after simulated ischemia. Differences between slopes of lines were highly significant for control vs. ME (P < 0.0001) and ME vs. herbimycin A + ME (P < 0.0001). Herbimycin A and herbimycin A + ME slopes were not significantly different from each other (P > 0.05). C: densitometric representation of the immuno results shown in D. Values are means ± SE.

 
We next addressed potential downstream signaling components of ME-induced protection. Figure 4 indicates that ME induced ERK1/2 phosphorylation and that the selective MEK1/2 inhibitor PD-98059 completely blocked ME-induced protection and ME-induced ERK1/2 phosphorylation. ME-induced protection was also completely blocked by the specific MEK1/2 inhibitor U-0126 but not by the inactive analog U-0124 (Fig. 5). ME induced Akt phosphorylation at Ser473 (but not Thr308), and the PI3K inhibitor LY-294002 completely abolished ME-induced protection against simulated ischemia and Akt phosphorylation (Fig. 6).



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  		Fig. 4. ME induces ERK1/2 phosphorylation, and inhibition of MAPK completely blocks ME-induced protection and ME-induced ERK1/2 phosphorylation. A and B: PD-98059 blocks ME-induced protection of isolated cardiomyocytes subjected to simulated ischemia. Differences between slopes of lines in A were highly significant for control vs. ME (P < 0.001) and ME vs. PD-98059 + ME (P < 0.001); PD-98059 and PD-98059 + ME slopes were not significantly different from each other (P > 0.05). C: densitometric representation of the Western blot results shown in D. Values are means ± SE.

 


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  		Fig. 5. ME-induced protection is completely blocked by the specific MEK1/2 inhibitor U-0126, but not by the inactive analog U-0124. A and C: percentage of cell death at 0, 30, 60, 90, 120, 150, and 180 min of simulated ischemia. B and D: AUC data presented as means ± SE.

 


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  		Fig. 6. ME induces Akt phosphorylation at Ser473, and inhibition of phosphatidylinositol 3-kinase (PI3K) completely abolishes ME-induced protection against simulated ischemia and Akt phosphorylation. A: percentage of cell death at 0, 30, 60, 90, 120, 150, and 180 min of simulated ischemia. Differences between slopes of lines were highly significant for control vs. ME (P < 0.001) and ME vs. LY-294002 + ME (P < 0.001); LY-294002 and LY-294002 + ME slopes were not significantly different from each other (P > 0.05). B: AUC data from viability experiments. C: densitometric representation of the Western blot results shown in D. Values are means ± SE.

 
Finally, we addressed interrelations among the ME cardioprotection pathway signaling components. ME-induced Akt phosphorylation at Ser473 and ERK1/2 phosphorylation, as well as ME-induced protection against simulated ischemia, were abolished by treatment with the PKC inhibitor chelerythrine (Fig. 7). We found that ME-induced PI3K and ERK activation is an EGFR-mediated event, because the selective EGFR kinase blocker PD-153035 eliminated ME-induced Akt phosphorylation at Ser473 and ERK1/2 phosphorylation (Fig. 8). Additionally, treatment of cells with LY-294002 totally eliminated ME-induced phosphorylation of ERK1/2, indicating that this is a PI3K-mediated event.



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  		Fig. 7. ME-induced protection against simulated ischemia, as well as ME-induced Akt (Ser473) and ERK1/2 phosphorylation, are abolished by inhibition of protein kinase C (PKC) with chelerythrine. A: percentage of cell death at 0, 30, 60, 90, 120, 150, and 180 min of simulated ischemia. Differences between slopes of lines were highly significant for control vs. ME (P < 0.01) and ME vs. chelerythrine + ME (P < 0.01); chelerythrine and chelerythrine + ME slopes were not significantly different from each other (P > 0.05). B: AUC data from viability experiments. C–D and E–F: immunoblot results for Akt and ERK1/2, respectively. Values are means ± SE.

 


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  		Fig. 8. Interrelations among the ME cardioprotection pathway signaling components. ME-induced PI3K and ERK activation is an EGFR-mediated event. A–D: selective EGFR kinase blocker PD-153035 eliminates ME-induced Akt (Ser473) and ERK1/2 phosphorylation. E and F: blockade of PI3K with LY-294002 eliminates ME-induced phosphorylation of ERK1/2. Values are means ± SE.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The principal findings of the present study in isolated adult rabbit cardiomyocytes are as follows: 1) ME-induced protection against simulated ischemia is mediated via EGFR transactivation; 2) EGFR transactivation is Src dependent; 3) the PI3K/Akt pathway is activated in ME-induced cardioprotection; 4) the MEK/ERK1/2 pathway is also activated in ME-induced protection; and 5) PKC is involved in this process. This demonstrates that the endogenous opioid peptide ME can trigger opioid-induced cardioprotection against simulated ischemia in isolated rabbit cardiomyocytes through transactivation of EGFR with subsequent activation of a complex signaling network.

The nonselective opioid antagonist naloxone blocks ischemic preconditioning (IP)-induced infarct limitation, and exogenous preischemic administration of the nonselective opiate agonist morphine limits infarct size after acute coronary occlusion-reperfusion in rats (44). Attenuation of IP-induced infarct limitation by naloxone is also reported for isolated and in situ rabbit hearts (5, 6, 35). Activation of opioid receptors by exogenous enkephalins exerts a protective effect in isolated rabbit cardiomyocytes subjected to simulated ischemia (pelleting and normothermic hypoxia), and enkephalin-induced cardioprotection is blocked by the {delta}-selective antagonist naltrindole (48). Activation of {kappa}-receptors may also be cardioprotective, because the {kappa}-selective agonist U-50488H has been reported to improve postischemic recovery of contractile function (41), reduce infarct size (50), and decrease cell death during metabolic inhibition of isolated cardiomyocytes (53). Our previous studies indicate that opioid-induced cardioprotection is mediated by the mitochondrial and sarcolemmal KATP channels (4). However, details of the signaling pathway involved in opioid-induced cardioprotection have not been fully characterized.

In the present study, we found that ME-induced protection was sensitive to the EGFR kinase inhibition and that ME phosphorylated the Tyr992 and Tyr1068 sites on EGFR, suggesting that EGFR transactivation participates in ME-induced protection. To determine whether transactivation of EGFR was dependent on Src, we used the selective Src inhibitor herbimycin A and found that herbimycin A completely inhibited the ME-induced protection as well as the phosphorylation of Tyr992 and Tyr1068 on EGFR, indicating that Src is a signaling molecule that links the opioid receptor and EGFR phosphorylation.

Second, we investigated the potential involvement of the PI3K and MEK/ERK1/2 pathways in ME-induced cardioprotection. ME treatment was associated with marked activation of Akt and ERK1/2; ME-induced protection of cardiomyocytes and activation of Akt and ERK1/2 were sensitive to blockade of EGFR, PI3K, and MEK1/2, which demonstrates the existence of the PI3K and MEK/ERK1/2 pathways downstream from EGFR transactivation in cardiomyocytes.

Finally, we investigated the potential involvement of PKC, because it has been reported to participate in cardioprotection in the rat model (24). The data presented here confirm previous evidence that opioid-induced cardioprotection is PKC dependent, because chelerythrine significantly reduced the ME-induced protection. Inhibition of PKC also eliminated activation of Akt and ERK1/2, suggesting that PKC is proximal to these kinases in the ME-cardioprotection signaling pathway.

Our observation that ME induced transactivation of EGFR and subsequent activation of the MEK/ERK1/2 pathway is supported by previous reports from others (17, 21, 24, 30). In contrast, Kramer et al. (31) reported that opioid-mediated activation of the MAPK cascade does not require transphosphorylation of EGFR. However, in that study, experiments were carried out in HEK-293 (human embryonic kidney) cells and C6 glioma cells stably transfected with {delta}-opioid receptors, not in cardiomyocytes (31). It is possible that opioid-mediated signaling is cell type specific. In addition, our finding that ME induces EGFR transactivation via Src is not consistent with a previous report from Gross’s laboratory (22). The reason for the discrepancy may be the experimental model (in vivo rat vs. rabbit cardiomyocytes), the opioid agonist (synthetic {delta}-selective opioid agonists vs. the endogenous peptide ME), or the tyrosine kinase inhibitor (genistein or PP2 vs. AG-1478 and PD-153035). However, it was recently reported that GPCR-induced protection (using acetylcholine) in isolated rabbit cardiomyocytes is mediated by PI3K- and Src kinase-dependent opening of mitochondrial KATP channels, which results in mitochondrial generation of reactive oxygen species and triggering of the preconditioned state (38). These observations are consistent with our present data and support our finding that activation of opioid receptors results in activation of Src kinase and, consequently, transactivation of EGFR. Of course, all studies that employ pharmacological inhibitors are subject to potential nonspecific effects of the antagonists chosen (9); hence, where possible, we have endeavored to use more than one inhibitor or to also employ an inactive congener. Inhibition of the kinase of interest was confirmed by Western blotting.

The PI3K/Akt signaling pathway plays a critical role in mediating survival signals in a wide range of neuronal cell types. Activation of Akt in different cells leads to cell survival, glucose uptake, and regulation of glycogen metabolism (33). Stimulation of the µ-opioid receptor by specific agonists induces the phosphorylation and activation of Akt at Ser473, which is associated with cell survival and translational control (40). The recent identification of a number of substrates for the serine/threonine kinase Akt suggests that it blocks cell death by directly inhibiting the cytoplasmic cell death machinery and by regulating the expression of genes involved in cell death and survival (2, 7). Our data showing protection that is dependent on PI3K and MEK indicate that intracellular signal transduction in ME-induced cardioprotection is similar to the antiapoptotic signaling of insulin-like growth factor I (34).

As noted above, we previously demonstrated that opioid peptide-induced cardioprotection is mediated via activation of opioid receptors and involves activation of sarcolemmal and mitochondrial KATP channels. In the present study, we did not address the location of KATP channel activation within the opioid peptide signaling network. However, on the basis of our data and data of others (27, 28), we postulate that activation of opioid receptors results in opening of sarcolemmal KATP channels, activation of PKC, and transphosphorylation of EGFR, leading to enhancement of PKC signaling. Activation of PKC results in activation of PI3K and subsequent activation of the target of PI3K, Akt, as well activation of the MEK/ERK signaling cascade. Together, these events are expected to elicit opening of mitochondrial KATP channels and to modulate downstream effectors of cardioprotection, as presented schematically in Fig. 9.



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  		Fig. 9. Schematic of proposed signaling pathways. Activation of opioid receptors with ME results in transactivation of EGFR with resultant activation of PKC, the MAPK pathway, and the PI3K pathway. Previous studies showed that opioid-mediated cardioprotection of isolated myocytes also involves opening of sarcolemmal and mitochondrial ATP-sensitive K+ (KATP) channels (sKATP and mKATP). TK, tyrosine kinase; PI-4,5-P2, phosphatidylinositol 4,5-bisphosphate; PI-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PDK, phosphoinositide-dependent kinase.

 
In conclusion, our results demonstrate that the endogenous opioid peptide ME protects isolated rabbit cardiomyocytes via transactivation of EGFR and subsequent activation of the PI3K and MEK/ERK1/2 pathways. ME-induced EGFR transactivation is c-Src dependent. PKC participates in ME-induced protection, acting upstream of PI3K and MEK/ERK.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by a Department of Veterans Affairs Medical Research Service Merit Review Grant to D. M. Van Winkle.


   ACKNOWLEDGMENTS
 
The authors thank Eric Tonsfeldt for assistance in performing the experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. M. Van Winkle, Anesthesiology Service, P3ANES, DVA Medical Center, 3710 SW US Veterans Hospital Rd., Portland, OR 97239-2999 (E-mail: Donna.Vanwinkle{at}med.va.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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