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Am J Physiol Heart Circ Physiol 285: H1032-H1039, 2003. First published May 1, 2003; doi:10.1152/ajpheart.01004.2002
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Activation of {delta}- and {kappa}-opioid receptors by opioid peptides protects cardiomyocytes via KATP channels

Zhiping Cao,1 Lijuan Liu,1 and Donna M. Van Winkle1,2

1Research and Anesthesiology Services, Veterans Affairs Medical Center; and 2Department of Anesthesiology, Oregon Health Sciences University, Portland, Oregon 97201

Submitted 21 November 2002 ; accepted in final form 29 April 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
To examine the receptor specificity and the mechanism of opioid peptide-induced protection, we examined freshly isolated adult rabbit cardiomyocytes subjected to simulated ischemia. Cell death as a function of time was assessed by trypan blue permeability. Dynorphin B (DynB) and Met5-enkephalin (ME) limitation of cell death (expressed as area under the curve) was sensitive to blockade by naltrindole (NTI, a {delta}-selective antagonist) and 5'-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5{alpha}-epoxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan (GNTI dihydrochloride, a {kappa}-selective antagonist): 85.7 ± 2.7 and 142.9 ± 2.7 with DynB and DynB + NTI, respectively (P < 0.001), 94.1 ± 4.2 and 164.5 ± 7.3 with DynB and DynB + GNTI, respectively (P < 0.001), 111.9 ± 7.0 and 192.1 ± 6.4 with ME and ME + NTI, respectively (P < 0.001), and 120.2 ± 4.3 and 170.0 ± 3.3 with ME and ME + GNTI, respectively (P < 0.001). Blockade of ATP-sensitive K+ channels eliminated DynB- and ME-induced protection: 189.6 ± 5.4 and 139.0 ± 5.4 for control and ME, respectively (P < 0.001), and 210 ± 5.9 and 195 ± 6.1 for 5-HD and ME + 5-HD, respectively (P < 0.001); 136.0 ± 5.7 and 63.4 ± 5.4 for control and ME, respectively (P < 0.001), and 144.6 ± 4.5 and 114.6 ± 7.7 for HMR-1098 and ME + HMR-1098, respectively (P < 0.01); 189.6 ± 5.4 and 139.0 ± 5.4 for control and ME, respectively (P < 0.001), and 210 ± 5.9 and 195 ± 6.1 for 5-HD and ME + 5-HD, respectively (P < 0.001); and 136.0 ± 5.7 and 63.4 ± 5.4 for control and ME, respectively (P < 0.001), and 144.6 ± 4.5 and 114.6 ± 7.7 for HMR-1098 and ME + HMR-1098, respectively (P < 0.01). We conclude that opioid peptide-induced cardioprotection is mediated by {delta}- and {kappa}-receptors and involves sarcolemmal and mitochondrial ATP-sensitive K+ channels.

heart; ischemic preconditioning; hypoxia

THE ENDOGENOUS OPIOID SYSTEM (opioid peptides and opioid receptors) is known to exert important cardiovascular effects. Three precursors encoding three families of endogenous opioid peptides (proopiomelanocortin, proenkephalin, and prodynorphin) have been cloned (6, 19, 32). Likewise, three major opioid receptor types ({delta}, {kappa}, and µ) have been identified (10, 14, 15, 30, 34). Generally, µ- and {delta}-receptors are considered to bind enkephalins and endorphins, and {kappa}-receptors are considered to bind dynorphins, but, on the basis of gene expression studies, promiscuity may exist between the three families of endogenous ligands and the three cloned receptors (1).

The cellular machinery necessary for the local production of endogenous opioid peptides (EOPs) is present within the heart, inasmuch as all three types of opioid peptide precursors are present in mammalian ventricular tissue and cultured cardiomyocytes (13, 52). However, only {delta}- and {kappa}-receptors are present in adult rat hearts. Although binding studies show {kappa}-receptors as the predominant opioid receptor in adult hearts (58), by RT-PCR the {delta}-opioid receptor transcript is the predominant form detected in the adult rat heart (56).

In intact rat and rabbit hearts, the nonselective opioid receptor antagonist naloxone and the selective {delta}-opioid receptor antagonist 7-benzylidenenaltrexone have been shown to inhibit the cardioprotective effect of ischemic preconditioning and morphine-induced cardioprotection (42). Activation of opioid receptors by exogenous enkephalins also has been shown to exert a protective effect in isolated rabbit cardiomyocytes, and the enkephalin-induced cardioprotection was blocked by the {delta}-selective antagonist naltrindole (NTI) (47). Selective activation of {kappa}-receptors with U-50488H improves postischemic recovery of contractile function compared with untreated controls (36), but whether opioid peptide-induced cell salvage can also be elicited by activation of {kappa}-opioid receptors is not known.

Preconditioning due to activation of opioid receptors has been reported to be mediated by a kinase cascade involving protein kinase C (9, 29) and via opening of the ATP-sensitive K+ (KATP) channel (8, 43). However, the signal pathway involved in opioid peptide-induced cardioprotection has not been described. We hypothesized that 1) {delta}- and {kappa}-opioid receptors can elicit ischemic tolerance and 2) opioid peptide-induced cardioprotection is mediated via activation of KATP channels. To test these hypotheses, we studied isolated adult rabbit cardiomyocytes subjected to simulated ischemia. The results indicate that activation of {delta}- and {kappa}-opioid receptors by the opioid peptides Met5-enkephalin (ME) and dynorphin B (DynB) triggers cardioprotection that is mediated by sarcolemmal and mitochondrial KATP channels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals used in these studies 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 Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Research, National Research Council, National Academy Press, 1996).

Cell isolation. Calcium-tolerant, adult rabbit cardiomyocytes were isolated by collagenase digestion as previously reported by Weinbrenner and colleagues (55). Male New Zealand White rabbits (2.4–3.1 kg) were anesthetized with pentobarbital sodium (30 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 left thoracotomy, the heart was rapidly excised and mounted on a nonrecirculating Langendorff apparatus. The heart was perfused for 5 min 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. Hearts were 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 contraction ceased. 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, i.e., for ~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% basic minimum essential amino acids, 1% MEM nonessential amino acids, and 1% basic minimum essential 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 beginning of the experimental protocol, 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. A separate isolate was used for each experiment, and each experimental series consisted of four to 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 37°C for 180 min.

Drugs. DynB was obtained from American Peptide (Sunnyvale, CA), ME from Calbiochem (San Diego, CA), NTI and 5-HD from Sigma (St. Louis, MO), and 5'-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5{alpha}-epoxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan (GNTI dihydrochloride) from Tocris (Ellisville, MO). HMR-1098 was a kind gift from Aventis Pharma. All drugs were dissolved in distilled water, aliquoted, and frozen until use. On the day of the experiment, stock solutions were diluted directly into cell suspensions. Agonists were administered to the cell suspension 15 min before the 180-min pelleting; antagonists were administered to the cell suspension 5 min before agonist treatment.

Determination of cell viability. Cell viability was determined before any experimental maneuvers (baseline), immediately before the 180 min of 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 amobarbital (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 permeability (blue vs. not blue), and the results were averaged for each group (2). 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.

Experimental protocols. We previously demonstrated that exogenous administration of the endogenously produced opioid peptide ME protects isolated cardiomyocytes subjected to simulated ischemia via activation of {delta}-opioid receptors (47). To determine whether dynorphins are similarly protective, we examined a DynB dose response. The groups were control, 1 µM DynB, 10 µM DynB, and 100 µM DynB. To further characterize the receptor specificity of opioid peptide-induced protection, we studied DynB- and ME-induced protection in the presence and absence of selective opioid-receptor antagonists. Two sets of groups per opioid peptide agonist were studied: 1) control, 100 µM DynB or ME, 1 nM GNTI hydrochloride (GNTI; a {kappa}-antagonist), and 100 µM DynB or ME + 1 nM GNTI hydrochloride and 2) control, 100 µM DynB or ME, 10 nM NTI (a {delta}-antagonist), and 100 µM DynB or ME + 10 nM NTI. The expected actions of the drugs are shown in Table 1.


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  		Table 1. Expected drug effects

 

To determine whether opioid peptide-induced cardioprotection is dependent on activation of KATP channels, we examined two sets of groups per opioid peptide agonist: 1) control, 100 µM DynB or ME, 100 µM 5-HD (a mitochondrial KATP inhibitor), and 100 µM DynB or ME + 100 µM 5-HD and 2) control, 100 µM DynB or ME, 30 µM HMR-1098 (a sarcolemmal KATP inhibitor), and 100 µM DynB or ME + 30 µM HMR-1098. All series were accompanied by an untreated oxygenated time-control group.

Data analysis. Data analysis was performed with a personal computer-based statistical software package (Prism 3.0, GraphPad Software, San Diego, 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 vs. duration of pelleted incubation. The area under these injury curves (AUC) was calculated for each individual experiment. Differences between groups were assessed by one-way ANOVA 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
 DISCLOSURES
 REFERENCES
 
Data were collected from 40 experiments. As shown in Fig. 1, DynB provides dose-dependent protection of isolated cardiomyocytes. All three treatments with different doses of DynB resulted in a decrease in the percentage of dead cells compared with control [AUC data: 191 ± 8.1 for control vs. 124.3 ± 7.6 with 100 µM DynB (P < 0.001), vs. 153.6 ± 3.6 with 10 µM DynB (P < 0.01), and vs. 175.2 ± 6.1 with 1 µM DynB (P > 0.05, n = 5)]. In our previous study, we found that ME-induced protection is sensitive to blockade of {delta}-receptors (47). In the present study, we found that ME-induced protection is abolished by selective {delta}-opioid receptor blockade with a very low dose (10 nM) of NTI [AUC data: 111.9 ± 5.3 with ME vs. 197.0 ± 7.0 for control (P < 0.001), vs. 192.1 ± 6.4 with ME + NTI (P < 0.001), and vs. 192.0 ± 7.1 with NTI (P < 0.001, n = 4; Fig. 2, left)] and also that DynB-induced protection is sensitive to selective {delta}-receptor blockade [AUC data: 142.9 ± 7.1 for control vs. 84.7 ± 2.7 with DynB (P < 0.001), 84.7 ± 2.7 with DynB vs. 142.9 ± 2.7 with DynB + NTI (P < 0.001), and 140.9 ± 5.9 with NTI (P < 0.001, n = 4; Fig. 2, right)]. When we tested whether EOP-induced protection of isolated cardiomyocytes involves activation of {kappa}-opioid receptors, we found that low-dose GNTI dihydrochloride blocks the protective effects of ME and DynB [AUC data: 120.2 ± 4.3 with ME vs. 172.6 ± 10.3 for control (P < 0.001) and vs. 179.0 ± 3.3 with GNTI + ME (P < 0.001); 171.8 ± 4.7 with GNTI vs. GNTI + ME (not significant, n = 4; Fig. 3, left); 94.1 ± 4.2 with DynB vs. 167.9 ± 8.3 for control (P < 0.001) and vs. 164.5 ± 7.3 with GNTI + DynB (P < 0.001); and 164.5 ± 7.3 with GNTI + DynB vs. 168.8 ± 12.0 with GNTI (not significant, n = 4; Fig. 3, right)].



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  		Fig. 1. Dynorphin B (DynB)-induced dose-dependent protection of isolated cardiomyocytes. Top: percentage of cell death at 0, 30, 60, 90, 120, 150, and 180 min of simulated ischemia. Linear regression equations for the 4 experimental groups are as follows: y = 0.22 * x + 12.11, R2 = 0.93 (control), y = 0.18 * x + 13.76, R2 = 0.94 (1 µM DynB), y = 0.14 * x + 13.96, R2 = 0.95 (10 µM DynB), and y = 0.11 * x + 12.97, R2 = 0.83 (100 µM DynB). Differences between slopes were highly significant (P < 0.0001). Bottom: area under the curve (AUC) data. Values are means ± SE (n = 5).

 


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  		Fig. 2. Naltrindole (NTI) blocks Met5-enkephalin (ME)- and DynB-induced protection. Top: percentage of cell death at 0, 30, 60, 90, 120, 150, and 180 min of simulated ischemia for ME (left) and DynB (right). Linear regression equations are as follows: y = 0.23 * x + 10.80, R2 = 0.96 (control), y = 0.09 * x + 11.01, R2 = 0.90 (ME), y = 0.20 * x + 12.43, R2 = 0.93 (10 nM NTI), and y = 0.20 * x + 12.99, R2 = 0.91 (100 µM ME + 10 nM NTI) and y = 0.20 * x + 3.99, R2 = 0.96 (control), y = 0.09 * x + 5.63, R2 = 0.92 (DynB), y = 0.19 * x + 4.60, R2 = 0.96 (10 nM NTI), and y = 0.18 * x + 5.57, R2 = 0.97 (100 µM DynB + 10 nM NTI). Differences between slopes were highly significant for control or NTI + ME vs. ME (P < 0.0001) and for control or NTI + DynB vs. DynB (P < 0.0001). NTI and NTI + endogenous opioid peptide (EOP; ME or DynB) slopes were not significantly different from each other. Bottom: AUC data. Values are means ± SE (n = 4). NS, not significant.

 


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  		Fig. 3. 5'-Guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5{alpha}-epoxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan (GNTI) hydrochloride (GNTI) blocks ME- and DynB-induced protection. Top: percentage of cell death at 0, 30, 60, 90, 120, 150, and 180 min of simulated ischemia for ME (left) and DynB (right). Linear regression equations are as follows: y = 0.21 * x + 8.55, R2 = 0.92 (control), y = 0.10 * x + 11.02, R2 = 0.92 (100 µM ME), y = 0.20 * x + 9.53, R2 = 0.96 (1 nM GNTI), and y = 0.19 * x + 10.96, R2 = 0.96 (100 µM ME + 1 nM GNTI) and y = 0.22 * x + 6.53, R2 = 0.95 (control), y = 0.08 * x + 8.46, R2 = 0.93 (100 µM DynB), y = 0.20 * x + 8.03, R2 = 0.93 (1 nM GNTI), and y = 0.20 * x + 7.44, R2 = 0.96 (100 µM DynB + 1 nM GNTI). Differences between slopes were highly significant for control or GNTI + ME vs. ME (P < 0.0001) and control or GNTI + DynB vs. DynB (P < 0.0001). GNTI and GNTI + EOP (ME or DynB) slopes were not significantly different from each other. Bottom: AUC data. Values are means ± SE (n = 4).

 

The opening of mitochondrial KATP channels is a key contributor to cardioprotection conferred by a variety of pharmacological agents, as well as ischemic preconditioning. However, it is becoming increasingly appreciated that, under some conditions, sarcolemmal KATP channels also play a role. In our studies, we found that ME- and DynB-induced protection is completely blocked by 5-HD [AUC data: 139.0 ± 5.4 with ME vs. 189.6 ± 5.4 for control (P < 0.001) vs. 210.0 ± 5.9 with 5-HD (P < 0.001) and vs. 195.0 ± 6.1 with 5-HD + ME (P < 0.001); 5-HD + ME vs. 5-HD (P > 0.05, n = 5; Fig. 4, left); and 98.8 ± 4.1 with DynB vs. 187.2 ± 6.3 with control (P < 0.001) vs. 186.6 ± 6.3 with 5-HD (P < 0.001) and vs. 189.9 ± 9.1 with 5-HD + DynB (P < 0.001); 5-HD + ME vs. 5-HD (P > 0.05, n = 4; Fig. 4, right)]. Similarly, inhibition of sarcolemmal KATP channels with HMR-1098 attenuated EOP-induced protection [AUC data: 112.5 ± 4.9 with ME vs. 186.1 ± 3.6 for control (P < 0.001) vs. 194.3 ± 3.2 with HMR-1098 (P < 0.001) and vs. 168.9 ± 7.7 with HMR-1098 + ME (P < 0.001); HMR-1098 + ME vs. HMR-1098 (P < 0.01, n = 6; Fig. 5, left); and 101.2 ± 3.8 with DynB vs. 178.0 ± 5.7 for control (P < 0.001) vs. 183.5 ± 3.8 with HMR-1098 (P < 0.001) and vs. 176.6 ± 6.2 with HMR-1098 + DynB (P < 0.001); HMR-1098 + DynB vs. HMR-1098 (P > 0.05, n = 4; Fig. 5, right)].



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  		Fig. 4. 5-HD blocks ME- and DynB-induced protection. Top: percentage of cell death at 0, 30, 60, 90, 120, 150, and 180 min of simulated ischemia. Linear regression equations are as follows: y = 0.23 * x + 9.74, R2 = 0.95 (control), y = 0.13 * x + 11.89, R2 = 0.88 (100 µM ME), y = 0.26 * x + 10.02, R2 = 0.96 (100 µM 5-HD), and y = 0.24 * x + 9.83, R2 = 0.96 (100 µM ME + 100 µM 5-HD) and y = 0.23 * x + 8.60, R2 = 0.96 (control), y = 0.07 * x + 9.98, R2 = 0.90 (100 µM DynB), y = 0.19 * x + 11.98, R2 = 0.92 (100 µM 5-HD), and y = 0.20 * x + 11.75, R2 = 0.93 (100 µM DynB + 100 µM 5-HD). Differences between slopes were highly significant for control or 5-HD + ME vs. ME (P < 0.0001) and control or 5-HD + DynB vs. DynB (P < 0.0001). 5-HD and 5-HD + EOP (ME or DynB) slopes were not significantly different from each other. Bottom: AUC data. Values are means ± SE (n = 4–5).

 


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  		Fig. 5. HMR-1098 attenuates ME- and DynB-induced protection. Top: percentage of cell death at 0, 30, 60, 90, 120, 150, and 180 min of simulated ischemia. Linear regression equations are as follows: y = 0.22 * x + 9.56, R2 = 0.97 (control), y = 0.09 * x + 10.52, R2 = 0.83 (100 µM ME), y = 0.19 * x + 13.28, R2 = 0.93 (30 µM HMR-1098), and y = 0.16 * x + 12.37, R2 = 0.87 (100 µM ME + 30 µM HMR-1098) and y = 0.24 * x + 6.16, R2 = 0.96 (control), y = 0.10 * x + 7.70, R2 = 0.90 (100 µM DynB), y = 0.20 * x + 9.88, R2 = 0.96 (30 µM HMR-1098), and y = 0.20 * x + 9.45, R2 = 0.92 (100 µM DynB + 30 µM HMR-1098). Differences between slopes were highly significant for control or HMR-1098 + ME vs. ME (P < 0.0001) and for control or HMR-1098 + DynB vs. DynB (P < 0.0001). HMR-1098 vs. HMR-1098 + ME was significantly different at P < 0.05. HMR-1098 and HMR-1098 + DynB slopes were not significantly different from each other. Bottom: AUC data. Values are means ± SE (n = 4–6).

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The principal findings of the present study are that, in isolated adult rabbit cardiomyocytes, 1) exogenous DynB induces protection against simulated ischemia, 2) ME- and DynB-induced protection are blocked by {kappa}- and {delta}-receptor blockade, and 3) ME- and DynB-induced protection is sensitive to mitochondrial and sarcolemmal KATP channel blockade. Our findings suggest that opioid peptides trigger protection via {delta}- and {kappa}-opioid receptors and that ME-induced protection utilizes a pathway that involves sarcolemmal and mitochondrial KATP channels.

Opioid peptides are produced as the result of proteolytic cleavage of precursor molecules, which are the products of three separate genes. The mRNA for these precursors is present in heart ventricular tissue and in cultured cardiomyocytes (7, 13, 52), and cardiomyocytes are capable of transcribing and translating opioid mRNAs into peptides (31, 45). Interestingly, the heart contains an exceptionally large amount of mRNA compared with the relatively modest peptide content, and all the preproenkephalin mRNA is contained within the ventricles. 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 (13). As reviewed by Akil et al. (1), the prodynorphin gene encodes three dynorphin peptides, all of which begin with a Leu5-enkephalin sequence. The amino-terminal sequence of all enkephalin and dynorphin opioid peptides contains a common motif, Tyr-Gly-Gly-Phe-[Met/Leu], followed by various carboxy-terminal extensions that result in peptides that vary in length from 5 to 31.

Although the three major opioid receptor types have been studied extensively, there is little agreement regarding the one-to-one correspondence between three families of endogenous opioid peptides and three cloned receptors. All three receptors share extensive sequence homologies. Because of the structural homology of opioid receptors and the common Tyr-Gly-Gly-Phe-[Met/Leu] motif of opioid peptides, promiscuity between opioid peptides and opioid receptors can occur (26). For instance, DynB, which is the primary active peptide product of prodynorphin in the heart (52), displays high affinity for the {kappa}-receptor but also shows significant affinity for {delta}-receptors (39). Furthermore, recent studies demonstrate that opioid receptors exist in oligomeric complexes, including heterooligomers of {delta}-{kappa}- and µ-{delta}-receptor complexes (21). The {delta}-{kappa}-receptor complexes display novel pharmacological properties, including decreased selective agonist affinity, cooperative agonist binding, enhanced signaling, and lack of receptor endocytosis (37). Thus, although binding affinity studies of cloned opioid receptors overexpressed in COS-1 cells show that DynB displays selectivity for {kappa}-receptors over {delta}-receptors [inhibition constant (Ki) = 0.45 ± 0.12 and 3.48 ± 0.70 nM, respectively] and ME displays selectivity for {delta}-receptors over {kappa}-receptors (Ki = 0.45 ± 0.03 and 47.44 ± 11.99 nM, respectively) (26), the binding of EOPs to opioid receptor heteromers in vivo may be significantly different. Interestingly, ligand binding assays in rat cardiac tissue suggest a single ME binding site with a receptor affinity of 6.8 ± 0.3 nM (27), significantly different from that reported for any cloned receptor subtype.

Opioid-induced cardioprotection was first demonstrated by Schultz and colleagues (44), who found that the nonselective opioid antagonist naloxone blocked ischemic preconditioning-induced 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 (3, 4, 29). Recent evidence from a study examining the effect of naloxone on indexes of ischemia after repeated percutaneous transluminal coronary angioplasty balloon inflations suggests that opioid receptor activation participates in ischemic preconditioning in humans as well (50).

Subsequent studies utilizing selective synthetic opioid receptor agonists and antagonists have pointed to the {delta}-opioid receptor as a mediator of opioid-induced cardioprotection (41–43, 51). Because endogenous endorphins and enkephalins bind to and activate the {delta}-opioid receptor with similar affinity, these studies suggested that the endogenous opioid peptide involved in preconditioning is an endorphin or an enkephalin. We previously reported that, in the isolated rabbit cardiomyocyte model, enkephalins induce cardioprotection, which can be blocked by NTI, suggesting that enkephalins and the {delta}-opioid receptor play important roles in cardioprotection (47).

Our present study demonstrates that DynB also elicits protection of isolated rabbit cardiomyocytes and that ME and DynB can trigger the cardioprotective effect via {delta}- and {kappa}-opioid receptors. Our finding that {kappa}-opioid receptors participate in cardioprotection agrees with reports previously published by other laboratories (53, 57). The finding that {delta}-receptor blockade abrogates EOP-induced cardioprotection and that {kappa}-receptor blockade also eliminates EOP-induced cardioprotection suggests that {kappa}- and {delta}-opioid receptors work together to produce acute cardioprotection. This conclusion is supported by reports indicating that the early phase of cardioprotection can be induced by the {delta}1-opioid agonist TAN-67 and blocked by the {kappa}-opioid receptor antagonist norbinaltorphine (43, 57).

The complete blockade of ME- and DynB-induced protection by {delta}- or {kappa}-receptor antagonists could theoretically occur if cardiac opioid receptors were exclusively {delta}/{kappa} heteromers; however, {delta}- and {kappa}-opioid receptors are known to form homo- and heterooligomers (17, 21). Alternatively, the antagonists employed may not have been completely selective for their desired target (NTI for {delta}-receptors and GNTI for {kappa}-receptors). However, these antagonists are reported to be highly selective (NTI ~200-fold more selective for {delta}- than for {kappa}-receptors and GNTI ~800-fold more selective for {kappa}- than for {delta}-receptors), and the doses used in the present study were selected to be slightly above the published Ki for the receptor to be blocked and well below the Ki for other opioid receptors (16, 28). Finally, it is also possible that, in rabbit cardiomyocytes, activation of both opioid receptor subtypes is necessary to trigger cardioprotection. Cohen and colleagues (5) suggested that protection conferred by ischemic preconditioning occurs in a similar fashion, whereby multiple triggers elaborated by preconditioning ischemia result in suprathreshold activation of a critical signaling step, and blockade of any one trigger may reduce the summed signaling step activation level to below threshold levels and, thereby, completely block protection.

There is a wealth of data suggesting that the salutary effects of ischemic preconditioning and many cardioprotective drugs are mediated via KATP channels; yet whether protection is conferred solely through the actions of mitochondrial KATP channels or through sarcolemmal and mitochondrial KATP channels is still controversial (11). However, recent experiments show that sarcolemmal and mitochondrial KATP channels contribute to ischemic preconditioning in dogs and mice (38, 46) and volatile anesthetic-induced cardioprotection (desflurane) (49). Our experiments also demonstrate that opening of mitochondrial and sarcolemmal KATP channels mediates ME- and DynB-induced cardioprotection. In contrast, Fryer and colleagues (8) reported that opioid-induced infarct limitation in rats is mediated solely via mitochondrial KATP channels. The reason for the different results between our study and that of Fryer et al. (9) is not known but may be model related [rats vs. rabbits, in situ vs. isolated cardiomyocytes, or synthetic (TAN-67) vs. naturally occurring (ME or DynB) opioid].

The protection conferred by sarcolemmal KATP channel opening was initially ascribed to shortening of action potential duration and, thus, a metabolic sparing effect; more recently, it has been hypothesized that sarcolemmal KATP channel opening hastens cessation of contractile activity during ischemia and, thus, may prevent cytosolic calcium overload (46). However, in the present study, the isolated cardiomyocytes were quiescent, and thus neither of these mechanisms would be called into play. Nonetheless, hyperpolarization induced by sarcolemmal KATP channel opening may facilitate mitochondrial KATP channel opening via a phospholipase D -> diacylglycerol -> protein kinase C pathway (23, 40, 54). Alternatively, it has been suggested that opening of sarcolemmal KATP channels may reduce cytosolic levels of long-chain acyl-CoA, an endogenous inhibitor of the mitochondrial KATP channel (35, 48). Regardless of the mechanism, cytoprotection by sarcolemmal KATP channel opening has been reported in quiescent or unstimulated cells (18, 22, 24).

Opening of mitochondrial KATP channels is thought to be protective by preservation of mitochondrial integrity, by dissipation of mitochondrial membrane potential and consequent reduction of calcium overload and apoptotic cell death (12, 25) via mitochondrial volume homeostasis and preservation of the architecture of the intermembrane space (20), or by generation of reactive oxygen species, which then activate specific elements in protective signaling pathways (33).

In conclusion, our results are the first to demonstrate a protective effect of the naturally occurring opioid peptide DynB when administered to isolated adult rabbit cardiomyocytes. Additionally, the results obtained from our studies indicate that naturally occurring opioid peptides (ME and DynB) in the heart may interact with {delta}- and {kappa}-opioid receptors to confer cardioprotection. Finally, opioid peptide-induced protection of cardiomyocytes involves opening of mitochondrial and sarcolemmal KATP channels.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This study was supported by a Department of Veterans Affairs Merit Review Grant (D. M. Van Winkle).


   ACKNOWLEDGMENTS
 
The authors thank Matthew Quinn and Jensen Huffman for assistance in performing the experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. M. Van Winkle, Anesthesiology Service, P3ANES, VA Medical Center, 3710 SW US Veterans Hospital Rd., Portland, OR 97201 (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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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