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Met5-enkephalin protects isolated adult rabbit cardiomyocytes via ␦-opioid receptors YASUSHI TAKASAKI,1 ROGER A. WOLFF,2 GRACE L. CHIEN,1,2 AND DONNA M. VAN WINKLE,1,2 1Department of Anesthesiology, Oregon Health Sciences University, Portland 97201; and 2Research and Anesthesiology Services, Veterans Affairs Medical Center, Portland, Oregon 97201 heart; opioid peptides; hypoxia; cell viability ENDOGENOUS OPIOID PEPTIDES and their receptors are widely distributed throughout the central and peripheral nervous system and are thought to play a neuromodulatory role in many processes, including cardiovascular regulation (14). Endogenous opioid peptides may also affect cardiovascular function through paracrine/ autocrine signaling. The three families of endogenous opioid peptides (enkephalins, endorphins, and dynorphins) are derived from three distinct prohormones (proenkephalin, proopiomelanocortin, and prodynorphin, respectively), which are the result of translation of mRNA from three separate genes (1). The preproenkephalin gene encodes four Met5-enkephalin sequences, one Leu5-enkephalin sequence, and two Met-enkephalin extended peptide sequences; the preproopiomelanocortin gene encodes a single long -endorphin sequence (-endorphin 1–31), which can be posttranslationally processed into the smaller, less active -endorphin 1–27 (Fig. 1) (12). At least three major classes of opioid receptors have been sequenced: µ, ␦, and (27, 31). Generally, µ- and 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. H2442 ␦-receptors bind enkephalins and endorphins and -receptors bind dynorphins (1). The cellular machinery necessary for the local production of endogenous opioid peptides is present within the heart, because all three types of opioid peptide precursors are present in mammalian ventricular tissue and cultured cardiomyocytes (15, 44). Similarly, local transduction of opioidergic signals can occur within the heart, because there is evidence from both binding (, ␦) and functional (, ␦, µ) studies in rats that opioid receptors exist in myocardial tissue (18, 22, 48). Much of the research on opioids in the cardiovascular system has been directed at examining their hemodynamic and contractile effects (17, 30). However, recently it was reported that naloxone blocks the infarctlimiting effect of the protective phenomenon known as ischemic preconditioning (transient sublethal ischemia) (11, 38). Additionally, preischemic administration of morphine to anesthetized open-chest rats and isolated rabbit hearts has been reported to mimic the infarct-limiting effect of ischemic preconditioning (24, 38). Together, these studies suggest that endogenous opioid peptides participate in the cardioprotective phenomenon of ischemic preconditioning. Subsequent studies showed that the ␦-agonist TAN-67 can mimic, and the ␦1-antagonist 7-benzylidenenaltrexone (BNTX) can attenuate, the infarct-limiting effect of ischemic preconditioning (35, 36). This suggests that preconditioning is mediated via ␦1-opioid receptor activation, perhaps by either endorphins or enkephalins. However, there are no published investigations demonstrating cardioprotection after in vivo preischemic administration of any naturally occurring opioid peptide, probably because of the lability of these compounds secondary to their rapid degradation by carboxy- and aminopeptidases (7, 20, 39). To further characterize the role of opioids in preconditioning, we studied isolated adult rabbit cardiac myocytes subjected to simulated ischemia. This model allowed us to apply opioid agonists and antagonists directly to a cardiomyocyte cell suspension. MATERIALS AND METHODS Animals used in these studies were allowed access to food and water ad libitum until anesthesia was induced. With local Institutional Animal Care and Use Committee approval, all animals received humane treatment in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health [DHHS Publication No. (NIH) 85-23, Revised 1985]. Cell isolation. Isolated, calcium-tolerant, adult rabbit cardiomyocytes were isolated by collagenase digestion as previously reported by Downey and colleagues (46). Male New Zealand White rabbits (2.4–3.1 kg) were anesthetized with 30 http://www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on May 7, 2017 Takasaki, Yasushi, Roger A. Wolff, Grace L. Chien, and Donna M. Van Winkle. Met5-enkephalin protects isolated adult rabbit cardiomyocytes via ␦-opioid receptors. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H2442–H2450, 1999.—In rats and rabbits, endogenous opioid peptides participate in ischemic preconditioning. However, it is not known which endogenous opioid(s) can trigger cardioprotection. We examined preconditioning-induced and opioid-induced limitation of cell death in isolated, calcium-tolerant, adult rabbit cardiomyocytes. Cells were subjected to simulated ischemia by pelleting and normothermic hypoxic incubation. Preconditioning was elicited with 15 min of simulated ischemia followed by 15 min of resuspension and reoxygenation. All cells underwent 180 min of simulated ischemia. Cell death was assessed by trypan blue permeability. Morphine protected cells, as did preconditioning; naloxone blocked the preconditioning-induced protection. Exogenous Met5-enkephalin (ME) induced protection, but exogenous -endorphin did not. ME-induced protection was blocked by the ␦-selective antagonist naltrindole. Additionally, two other proenkephalin products, Leu5-enkephalin and Met5-enkephalin-Arg-Phe, provided protection equipotent to ME. These data suggest that one or more proenkephalin products interact with ␦-opioid receptors to endogenously trigger opioid-mediated protection. MET5-ENKEPHALIN INDUCES PRECONDITIONING VIA ␦-RECEPTORS H2443 mg/kg pentobarbital sodium via a marginal ear vein. A tracheostomy was performed, and positive pressure ventilation with 100% oxygen was established at a rate of 35 breaths/min. After myocardium was exposed via left thoracotomy, the heart was rapidly excised and mounted on a nonrecirculating Langendorff apparatus. The heart was perfused at 38°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 (⬃3–4 min). The heart was then perfused with calcium-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 the heart ceased to contract. On cessation of contractile activity the heart was 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 heart became dilated and started to soften, ⬃20 min. The heart was then removed from the perfusion apparatus, trimmed of the 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 [in mM: 118.5 NaCl, 24.8 NaHCO3, 10.0 glucose, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 30.0 HEPES, 60.0 taurine, 20.0 creatine, and 0.68 glutamine, plus 1% basal medium Eagle (BME) amino acids, 1% MEM nonessential amino acids, and 1% BME vitamin solution; pH 7.4]. After a 30-min equilibration period, calcium was gradually reintroduced to a final concentration of 1.25 mM. Before the experimental protocol was begun, cells were washed twice (centrifuged at ⬃5 g for 90 s), resuspended in fresh incubation buffer, and gently pipetted in 1-ml aliquots into 1.8-ml microcentrifuge tubes. Isolate yield was sufficient for four experimental groups plus an oxygenated time control. Isolates containing ⬍60% rod-shaped cells were not used. A separate isolate was used for each experiment, and each experimental series consisted of approximately five experiments. 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 38°C for 180 min. For preconditioning, cells were pelleted, layered with mineral oil, and incubated without agitation at 38°C for 15 min. Nonpreconditioned groups were also pelleted and then resuspended in fresh oxygenated incubation buffer and incubated without agitation at 38°C for 15 min. At the end of the 15-min incubation, preconditioned cells were carefully pipetted from beneath the oil layer and resuspended in fresh oxygenated incubation buffer. Nonpreconditioned groups were also resus- Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on May 7, 2017 Fig. 1. Schematic representation of proenkephalin and proopiomelanocortin genes. Arrows indicate cleavage sites for posttranslationally shortened endorphin peptides: -endorphin 1–16 (␣-endorphin), -endorphin 1–17 (␥-endorphin), and -endorphin 1–27. ACTH, adrenocorticotropic hormone; -E, -endorphin; LE, Leu5-enkephalin; LPH, lipotropic hormone; ME, Met5-enkephalin; MEAGL, Met5-enkephalin-Arg6-Gly7-Leu8; MEAP, Met5enkephalin-Arg6-Phe7; MSH, melanocyte-stimulating hormone. H2444 MET5-ENKEPHALIN INDUCES PRECONDITIONING VIA ␦-RECEPTORS against simulated ischemia. Groups were control, ME, Leu5enkephalin (LE), and Met5-enkephalin-Arg6-Phe7 (MEAP). In series 5, we tested whether enkephalin-induced protection could be blocked by a ␦-selective opioid receptor antagonist. Groups studied were control, naltrindole, ME, and naltrindole plus Met5-enkephalin (natrindole ⫹ ME). Last, in series 6, we examined a Met5-enkephalin dose-response. Groups studied were control, 100 µM Met5-enkephalin (ME-100), 10 µM Met5-enkephalin (ME-10), and 1 µM Met5-enkephalin (ME-1). Data analysis. Data analysis was performed with a personal computer-based statistical software package (Crunch 4, Crunch Software, Oakland, CA). The primary measured end point for all series was cell death, defined as uptake of trypan blue. For each group, the percentage of dead cells was plotted versus the duration of pelleted incubation. The area underneath these injury curves (AUC) was calculated for each individual experiment. 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 values ⱕ 0.05. Results are expressed as means ⫾ SE. RESULTS Of 38 experiments attempted, 30 contributed to the final data set. Reasons for exclusion of experiments included technical errors during cell isolation in five experiments and ⬍60% rod-shaped cells at baseline in three experiments. Therefore; n ⫽ 5 experiments for all experimental series. The baseline morphology of isolated cells was 68.3 ⫾ 2.7% rod-shaped cells. In series 1, we assessed whether preconditioning, the adenosine A1-receptor agonist R-PIA, and the nonselective opiate alkaloid agonist morphine protected cells from simulated ischemia. As shown in Fig. 3, all three experimental conditions resulted in a decrease in the percentage of dead cells compared with the control (AUC data: control 125.3 ⫾ 3.6 vs. PC 97.8 ⫾ 3.2, P ⬍ 0.01; control vs. R-PIA 106.2 ⫾ 10.8 and MS 109.3 ⫾ 3.1, both P ⬍ 0.05). R-PIA and morphine provided protection nearly equivalent to that of preconditioning [AUC data: PC vs. R-PIA and MS, P ⫽ nonsignificant (NS)]. When we tested the sensitivity of preconditioning to opioid receptor blockade in series 2, we found that naloxone completely abolished the protective effect of preconditioning (AUC data: control 123.1 ⫾ 7.6 vs. PC 95.2 ⫾ 7.6, P ⬍ 0.001; naloxone 117.7 ⫾ 6.7 vs. naloxone ⫹ PC 122.4 ⫾ 6.6, P ⫽ NS; Fig. 4), although naloxone by itself had little influence on cell death. We next addressed the question in series 3 of which class of endogenous opioid peptides (endorphin vs. enkephalin) is involved in the protection against simulated ischemia. Dynorphins were not addressed in this study because of whole animal experiments that suggest that the relevant opioid receptor is the ␦-receptor (36), which has greater affinity for endorphins and enkephalins compared with dynorphins (9). As shown in Fig. 5, Met5-enkephalin produced significant protection of the isolated myocytes, but -endorphin did not (AUC data: control 122.5 ⫾ 5.1, ME 103.5 ⫾ 7.7, -E 124.7 ⫾ 5.3; P ⬍ 0.01 for ME vs. control and -E). Because -endorphin 1–27 is 10 times less potent than the -endorphin 1–31 used in the experiments, Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on May 7, 2017 pended in fresh oxygenated incubation buffer. All groups were incubated in oxygenated buffer for 15 min before being subjected to the final 180-min pelleting described above. Drugs. The agents used in this study were -endorphin 1–31, Leu5-enkephalin, Met5-enkephalin, Met5-enkephalinArg6-Phe7, morphine sulfate (morphine), naloxone, naltrindole, and (⫺)-N6-(2-phenylisopropyl)adenosine (R-PIA). Opioid peptides were obtained from Peninsula Laboratories (Belmont, CA). All other drugs were obtained from RBI (Natick, MA). Morphine, R-PIA, and naltrindole were made fresh each day. Peptides were dissolved, aliquoted, and frozen until use. Met5-enkephalin, Leu5-enkephalin, Met5-enkephalin-Arg6-Phe7, naloxone, and naltrindole were dissolved in distilled water. -Endorphin 1–31 was dissolved in 5% acetic acid and adjusted to pH 7.4 with 4 N NaOH immediately before use. Warmed peptide stock solutions were diluted directly into cell suspensions. R-PIA was dissolved directly into the cell suspension buffer. Unless otherwise stated, all drugs were administered as 100 µM final concentration. This dosage was chosen because Armstrong et al. (3) required 100 µM R-PIA to induce cardioprotection in isolated rat myocytes, and we observed complete blockade of ischemic preconditioning in isolated rabbit hearts with 100 µM naloxone but not with a lower dose (10). Agonists were administered to the cell suspension for 15 min before the 180-min pelleting; antagonists were administered to the cell suspension for 5 min before preconditioning or 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 ⫻100 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 (4). 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 were nonviable. Experimental protocols. The general experimental design is shown in Fig. 2. Six different series of experiments were performed. All series were accompanied by a nontreated oxygenated time control group. Series 1 was designed to determine 1) whether our isolated myocyte model demonstrates protection consistent with preconditioning and with exogenous administration of a known initiator of preconditioning (adenosine receptor agonist) and 2) whether activation of opioid receptors triggers preconditioning in rabbit cardiomyocytes. Groups were control, preconditioning (PC), R-PIA, and morphine sulfate (MS). In series 2, we tested whether preconditioning of isolated cardiomyocytes involves activation of opioid receptors by an endogenous ligand. Groups studied were control, naloxone, preconditioning (PC), and naloxone plus preconditioning (naloxone ⫹ PC). In series 3, we examined whether exogenous administration of agonist peptides that endogenously serve as ligands for the µ- and ␦-opioid receptors confer protection against simulated ischemia. Groups studied were control, -endorphin (-E), and Met5enkephalin (ME). In series 4, we studied whether exogenous administration of other agonist peptides that endogenously serve as ligands for the ␦-opioid receptor confer protection MET5-ENKEPHALIN INDUCES PRECONDITIONING VIA ␦-RECEPTORS H2445 and because other posttranslationally processed endorphins are biologically inactive, we did not study endorphins further but instead examined other enkephalin peptides that endogenously serve as ligands for the ␦-opioid receptor. In series 4, we found that all three enkephalin peptides tested (Met5-enkephalin, Leu5enkephalin, and Met5-enkephalin-Arg6-Phe7 ) confer equivalent protection against simulated ischemia (AUC data: control 136.2 ⫾ 4.0, ME 116.2 ⫾ 8.7, LE 114.9 ⫾ 3.9, MEAP 121.0 ⫾ 3.0; P ⬍ 0.05 for all peptides vs. control). These data are presented in Fig. 6. Although Met5-enkephalin binds with roughly equal affinity to both µ- and ␦-opioid receptors, Leu5-enkephalin and Met5-enkephalin-Arg6-Phe7 display a preference for ␦-opioid receptors. Accordingly, in series 5, we examined whether the selective ␦-opioid receptor blockade would eliminate the protection conferred by enkephalins. Figure 7 shows that the ␦-selective opioid antagonist naltrindole alone did not exhibit a proischemic effect but completely blocked the protection afforded by subsequent administration of Met5-enkephalin (AUC data: control 112.9 ⫾ 2.2 vs. ME 91.4 ⫾ 2.9, P ⬍ 0.001; naltrindole 111.9 ⫾ 4.4 vs. naltrindole ⫹ ME 108.2 ⫾ 5.3, P ⫽ NS). Last, in series 6, we examined whether lower doses of Met5-enkephalin, which are known to activate protein kinase C, a putative postreceptor mediator of ischemic preconditioning, also protected cardiomyocytes against simulated ischemia. Met5-enkephalin provided dosedependent protection of isolated cardiomyocytes, with protection by 1 or 10 µM Met5-enkephalin being transient and protection by 100 µM Met5-enkephalin being Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on May 7, 2017 Fig. 2. Experimental time line. Arrows indicate times at which cardiomyocyte viability was assessed. Shaded bars represent periods of simulated ischemia resulting from pelleting and normothermic hypoxic incubation. MS, morphine sulfate; NAL, naloxone; NTI, natrindole; PC, preconditioning; R-PIA, (⫺)-N6(2-phenylisopropyl)adenosine; RR, reoxygenation and resuspension. H2446 MET5-ENKEPHALIN INDUCES PRECONDITIONING VIA ␦-RECEPTORS Fig. 4. Naloxone sensitivity of preconditioning. Naloxone alone (NAL) has no effect on cell death during simulated ischemia but completely abolishes protection conferred by preconditioning. Data are presented as means ⫾ SE. Table (bottom) indicates statistically significant comparisons; no entry indicates P ⫽ NS. AUC data are presented in text. Fig. 5. Opioid peptide-specific induction of protection. Met5-enkephalin (ME) but not -endorphin (-E) limits cardiomyocyte cell death during simulated ischemia. Data are presented as means ⫾ SE. Table (bottom) indicates statistically significant comparisons; no entry indicates P ⫽ NS. AUC data are presented in text. Fig. 6. Enkephalin-mediated cardiomyocyte protection. All enkephalin products tested conferred protection against simulated ischemia. ME, Met5-enkephalin; LE, Leu5-enkephalin; MEAP, Met-enkephalinArg6-Phe7. Data are presented as means ⫾ SE. Table (bottom) indicates statistically significant comparisons; no entry indicates P ⫽ NS. AUC data are presented in text. Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on May 7, 2017 Fig. 3. Preconditioning in rabbit isolated myocytes. Isolated rabbit cardiomyocytes exhibit less cell death at all time points with preconditioning (PC) or when treated with R-PIA or morphine sulfate (MS). Data are presented as means ⫾ SE. Table (bottom) indicates statistically significant comparisons; no entry indicates P ⫽ nonsignificant (NS). Data calculated from area under injury curves (AUC) are presented in text. Con, control. MET5-ENKEPHALIN INDUCES PRECONDITIONING VIA ␦-RECEPTORS antagonist naloxone blocked ischemic preconditioninginduced infarct limitation and that exogenous preischemic administration of the nonselective opiate agonist morphine limited infarct size after acute coronary occlusion-reperfusion in rats. Subsequently, attenuation of ischemic preconditioning-induced infarct limitation by naloxone was reported for isolated and in situ rabbit hearts (10, 11, 24). Recent evidence from a study (41) examining the effect of naloxone on indexes of ischemia after repeated percutaneous transluminal coronary angioplasty (PTCA) balloon inflations suggests that opioid receptor activation participates in ischemic preconditioning in humans as well. Subsequent studies utilizing selective synthetic opioid receptor agonists and antagonists have pointed to the ␦-opioid receptor as the mediator of the opioidpreconditioning effect (35–37, 42). Because both endogenous endorphins and enkephalins bind to and activate the ␦-opioid receptor with similar affinity, these studies suggested that the endogenous opioid peptide involved in preconditioning is either an endorphin or an enkephalin. However, to our knowledge there have been no previous reports investigating which naturally occurring opioid peptides can induce cardioprotection. Interestingly, Liang and Gross (19) reported that maximal morphine-preconditioning of cultured neonatal chick cardiomyocytes occurred at 1 µM. We did not perform a morphine dose-response test to determine sustained throughout the period of simulated ischemia (AUC data: control 121.7 ⫾ 3.0 vs. ME-100 97.6 ⫾ 2.2, P ⬍ 0.001; control vs. ME-10 111.4 ⫾ 1.4, P ⬍ 0.01; control vs. ME-1 116.1 ⫾ 2.7, P ⬍ 0.05; Fig. 8). DISCUSSION The principal findings of the current study are that in isolated adult rabbit cardiomyocytes 1) the nonselective opioid agonist morphine protects against simulated ischemia, and the nonselective opioid receptor antagonist naloxone blocks preconditioning-induced protection; and 2) naturally occurring enkephalin peptides can induce preconditioning via ␦-opioid receptors. The naturally occurring opioid product of proopiomelanocortin, -endorphin 1–31, was not protective. Of the three enkephalin peptides tested, Met5-enkephalin, Leu5enkephalin, and Met5-enkephalin-Arg6-Phe7, all provided equipotent protection against simulated ischemia. Although Met5-enkephalin binds to and activates both µ- and ␦-opioid receptors, Leu5-enkephalin and Met5-enkephalin-Arg6-Phe7 are predominantly ␦-opioid receptor agonists, suggesting that the protective effect is mediated via the ␦-opioid receptor. This conclusion was supported by the finding that the protective effect of Met5-enkephalin was fully abolished by the ␦-receptorselective antagonist naltrindole. Opioids and ischemic preconditioning. Opioid-induced preconditioning was first reported by Gross and colleagues (38), who found that the nonselective opioid Fig. 8. Met5-enkephalin-induced protection dose response. Met5enkephalin at 1 (ME-1) and 10 µM doses (ME-10) produced a transient cytoprotective effect, whereas Met5-enkephalin at 100 µM dose (ME-100) produced a sustained decrease in cell death. Data are presented as means ⫾ SE. Table (bottom) indicates statistically significant comparisons; no entry indicates P ⫽ NS. AUC data are presented in text. Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on May 7, 2017 Fig. 7. Met5-enkephalin-induced protection mediated by ␦-opioid receptors. Met5-enkephalin-induced protection against simulated ischemia is completely abolished by ␦-selective opioid receptor antagonist naltrindole (NTI). Data are presented as means ⫾ SE. Table (bottom) indicates statistically significant comparisons; no entry indicates P ⫽ NS. AUC data are presented in text. H2447 H2448 MET5-ENKEPHALIN INDUCES PRECONDITIONING VIA ␦-RECEPTORS tent with the relative abundance of these sequences in proenkephalin) (5). Cardiac Met5-enkephalin immunoreactivity has been reported to increase during myocardial ischemia in rats (23). Our results showing that enkephalin peptides confer protection against simulated ischemia in isolated cardiac myocytes are consistent with reports that synthetic enkephalin analogs such as D-Ala2-D-Leu5enkephalin (DADLE) improve cardiac function after prolonged hypothermic ischemic storage of excised rabbit hearts (8). Additionally, our observation that the enkephalin-induced protection is mediated by ␦-opioid receptors is consistent with the data of Schultz et al. (35–37), who reported that ischemic preconditioning in rats is blocked by ␦-opioid-selective antagonists and mimicked by preischemic administration of the ␦-receptor-selective agonist TAN-67. The present data are also consistent with the data of Liang and Gross (19), who recently reported that the ␦1-opioid-selective antagonist BNTX blocks morphine-induced protection in myocytes cultured from chick embryos. The current results suggest that opioid-induced preconditioning is a direct cardiomyocyte effect rather than an indirect effect such as inhibition of neutrophil activation, as proposed by Wang et al. (45). Furthermore, our observation that opioid-induced protection can occur in isolated cardiomyocytes indicates that the effect is not dependent on vascular elements (e.g., due to recruitment of collateral blood flow such as that which might occur in situ). In the current study we did not investigate postreceptor signal transduction mechanisms after administration of endogenous enkephalin peptides. However, preconditioning caused by activation of opioid receptors has been reported to be mediated by a kinase cascade involving protein kinase C (24) and via opening of the ATP-sensitive potassium channel (34, 37). Additionally, micromolar amounts of the enkephalin analog D-Pen2-DPen5-enkephalin have been reported to activate protein kinase C in a pertussis toxin-sensitive dose-dependent manner in NG 108-15 cells (21). It is notable in our study that 1, 10, and 100 µM Met5-enkephalin provided protection against simulated ischemia, although the protection was more robust at the higher dose. Finally, the ␦-receptor-selective enkephalin analog DADLE recently has been shown (16) to preserve postischemic contractile function in isolated rat hearts in a glibenclamide-sensitive manner, indicating participation of ATPsensitive potassium channels in the protection provided by this enkephalin analog. On the basis of the work of Gross and colleagues (35), who demonstrated that ischemic preconditioning is mediated via ␦-opioid receptors, we initially chose to examine -endorphin and Met5-enkephalin for their ability to induced protection in isolated cardiomyocytes. Both are produced in the heart, and both display roughly equal affinity to both µ- and ␦-receptors. However, it is possible that there are other naturally occurring opioid peptides in the heart that may interact with ␦-receptors to induce cardioprotection. Brain dynorphin A-(1–8) is reported to display high affinity for Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on May 7, 2017 the maximal effective dose for morphine preconditioning in isolated adult rabbit cardiomyocytes. However, in our adult rabbit isolated cardiomyocytes, maximal enkephalin preconditioning was achieved at 100 µM. We do not know the reason for the difference in the maximally effective dose of opioid between these two studies. Opioid peptides in the heart. In vivo, the three classes of opioid peptides (endorphins, enkephalins, and dynorphins) are produced as the result of proteolytic cleavage of precursor molecules, which are the products of three separate genes. The endorphins are derived from proopiomelanocortin, and the enkephalins are derived from proenkephalin (refer to Fig. 1). mRNA for these precursors is present in heart ventricular tissue and in cultured cardiac myocytes (13, 15, 44), and cardiomyocytes are capable of transcribing and translating opioid mRNAs into peptides (26, 40). Interestingly, the heart contains an exceptionally large amount of mRNA in comparison to the relatively modest peptide content; this may be explained by the absence of secretory granules in ventricular myocytes so that the pool of mRNA acts as an autocrine production reservoir for the rapidly degraded peptides (15). The proopiomelanocortin gene encodes a single -endorphin 1–31 peptide. However, this peptide may itself undergo posttranslational modifications, including COOH-terminal proteolysis and/or NH2 acetylation. In the heart, -endorphin 1–31 accounts for ⬃16% of -endorphin immunoreactivity, with the predominant peptide product being N-acetyl--endorphin-(1–31) (36%). The remainder of the -endorphin immunoreactivity is associated with ␣-NH2-acetylated and/or COOHterminally shortened -endorphins (25, 26). -Endorphin 1–31 is the most potent of the endorphin products, with the COOH-terminally shortened products being ⬃10-fold less potent and the NH2-acetylated forms inactive at opioid receptors (12). In our study we used -endorphin 1–31; the absence of protection with this peptide, which is the most potent endorphin but which comprises a minor portion of the endorphin peptide pool, suggests that endorphins in vivo are not responsible for mediating preconditioning. This conclusion is supported by recent data obtained from knockout mice deficient in all endorphin products (32) that retain the ability to limit infarct size after ischemic preconditioning (43). The preproenkephalin gene encodes four Met5enkephalin sequences, one Leu5-enkephalin sequence, and two extended enkephalin sequences (Met5-enkephalin-Arg-Phe and Met5-enkephalin-Arg6-Gly7-Leu8 ). Recent immunocytochemistry studies suggest that Met5enkephalin-Arg6-Phe7 is the predominant enkephalin produced locally in heart ventricles, with Met5-enkephalin-Arg6-Phe7 immunoreactivity being ⬃25 times greater than Met5-enkephalin immunoreactivity (6). Met5enkephalin-Arg6-Gly7-Leu8 does not appear to be a major product of proenkephalin in the heart and therefore was not studied in our experiments (ratio of Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity to Met5-enkephalin immunoreactivity is ⬃1:3–4, consis- MET5-ENKEPHALIN INDUCES PRECONDITIONING VIA ␦-RECEPTORS We gratefully thank Drs. James Downey and Guang Liu for helpfulness and expert technical advice in the development of the isolated cardiomyocyte preparation. This study was supported by a Veterans Affairs Merit Review grant (to D. M. Van Winkle). Y. Takasaki is a Visiting Fellow from Ehime University, Ehime, Japan. Address for reprint requests and other correspondence: D. M. Van Winkle, Anesthesiology Service, P8ANES, VA Medical Center, 3710 SW US Veterans Hospital Rd., Portland, Oregon 97201 (E-mail: [email protected]). Received 29 July 1999; accepted in final form 26 August 1999. REFERENCES 1. Akil, H., C. Owens, H. Gutstein, L. Taylor, E. Curran, and S. Watson. Endogenous opioids: overview and current issues. Drug Alcohol Depend. 51: 127–140, 1998. 2. American Heart Association. 1999 Heart and Stroke Statistical Update. [Online] American Heart Association. http://www. amhrt.org/statistics/index.html [July 1999] 3. Armstrong, S., and C. E. Ganote. Adenosine receptor specificity in preconditioning of isolated rabbit cardiomyocytes: evidence of A3 receptor involvement. Cardiovasc. Res. 28: 1049–1056, 1994. 4. Armstrong, S. C., and C. E. Ganote. 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Identification and characterization of Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on May 7, 2017 ␦-receptors as well as -receptors (28), and our preliminary experiments with dynorphin A-(1–8) suggest that it, too, protects isolated cardiomyocytes. Dynorphin B, which in hearts has been reported to be the primary peptide product of the prohormone prodynorphin (44), displays high affinity for the -receptor but also shows significant, albeit lower, affinity for ␦-receptors (33). Additionally, a recent study demonstrated that -receptors are involved in late preconditioning in rat cardiomyocytes (47). Thus it is possible that both ␦- and -receptors participate in early cardioprotection, and the potential role of dynorphins in the acute protection of myocardium deserves future investigation. Clinical relevance. Of Americans living today, ⬃6.2 million have angina and ⬃12 million have a history of myocardial infarction or angina (2). These individuals often undergo invasive diagnostic and/or therapeutic procedures and are at risk for suffering ischemic events perioperatively. Additionally, PTCA and cardiopulmonary bypass result in brief periods of myocardial ischemia as an inescapable consequence of the procedure; ⬃2.75 million of these procedures were performed in 1996 (2). Because it may not be possible to eliminate these episodes of myocardial ischemia, minimizing their severity and/or mitigating their deleterious effects is highly desirable. Knowledge of anti- and proischemic tendencies of specific drugs may allow health care providers to tailor medications to limit ischemic damage. For instance, morphine is used clinically for sedation/analgesia and may also be used as the primary anesthetic in cardiac surgery. Our current isolated myocyte data, as well as previous in situ and isolated heart data from the Gross and Downey laboratories (24, 34), show that morphine can induce a cardioprotective effect. Additionally, endogenous cardioprotective opioids (such as the enkephalins) are modulated by clinically used drugs. Most notably, angiotensinconverting enzyme (ACE) degrades enkephalins, and ACE inhibitors have been shown to potentiate enkephalin-induced analgesia (29). Thus it is possible that treatment with ACE inhibitors may also potentiate enkephalin-induced cardioprotection. The interaction of such clinically used drugs with induced cardioprotection is an important topic and merits further investigation. In conclusion, our results are the first to demonstrate a protective effect of naturally occurring opioid peptides when administered to isolated adult rabbit cardiac myocytes. Because enkephalins but not -endorphin exerted this cardioprotective effect, and because the ␦-selective antagonist naltrindole blocked the cardioprotection conferred by Met5-enkephalin, we have concluded that the protective effect is mediated via ␦-opioid receptors. 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