Download AHEART December 46/6 - AJP

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. Effects of 2,3-butanedione
monoxime (BDM) on contracture and injury of isolated rat
myocytes following metabolic inhibition and ischemia. J. Mol.
Cell. Cardiol. 23: 1001–1014, 1991.
5. Barron, B. A., H. Gu, J. F. Gaugl, and J. L. Caffrey. Screening
for opioids in dog heart. J. Mol. Cell. Cardiol. 24: 67–77, 1992.
6. Barron, B. A., L. X. Oakford, J. F. Gaugl, and J. L. Caffrey.
Methionine-enkephalin-Arg-Phe immunoreactivity in heart tissue. Peptides 16: 1221–1227, 1995.
7. Bausback, H. H., and P. E. Ward. Degradation of low-molecularweight opioid peptides by vascular plasma membrane aminopeptidase M. Biochim. Biophys. Acta 882: 437–444, 1986.
8. Bolling, S. F., T.-P. Su, K. F. Childs, X.-H. Ning, N. Horton, K.
Kilgore, and P. R. Oeltgen. The use of hibernation induction
triggers for cardiac transplant preservation. Transplantation 63:
326–329, 1997.
9. Borsodi, A., and G. Toth. Characterization of opioid receptor
types and subtypes with new ligands. Ann. NY Acad. Sci. 265:
339–352, 1995.
10. Chien, G. L., K. Mohtadi, R. A. Wolff, and D. M. Van Winkle.
Naloxone blockade of myocardial ischemic preconditioning does
not require central nervous system participation. Basic Res.
Cardiol. 94: 136–143, 1999.
11. Chien, G. L., and D. M. Van Winkle. Naloxone blockade of
myocardial ischaemic preconditioning is stereoselective. J. Mol.
Cell. Cardiol. 28: 1895–1900, 1996.
12. Dores, R. M., H. Akil, and S. J. Watson. Strategies for studying
opioid peptide regulation at the gene, message and protein levels.
Peptides 5, Suppl. 1: 9–17, 1984.
13. Forman, L. H., and O. Bagasra. Demonstration by in situ
hybridization of the proopiomelanocortin gene in the rat heart.
Brain Res. Bull. 28: 441–445, 1992.
14. Holaday, J. W. Cardiovascular effects of endogenous opiate
systems. Annu. Rev. Pharmacol. Toxicol. 23: 541–594, 1983.
15. Howells, R. D., D. L. Kilpatrick, L. C. Bailey, M. Noe, and S.
Udenfriend. Proenkephalin mRNA in rat heart. Proc. Natl.
Acad. Sci. USA 83: 1960–1963, 1986.
16. Kevelaitis, E., J. Peynet, C. Mouas, J.-M. Launay, and P.
Menasché. Opening of potassium channels. The common cardioprotective link between preconditioning and natural hibernation? Circulation 99: 3079–3085, 1999.
17. Kindman, L. A., R. E. Kates, and R. Ginsburg. Opioids
potentiate contractile response of rabbit myocardium to the beta
adrenergic agonist isoproterenol. J. Cardiovasc. Pharmacol. 17:
61–67, 1991.
18. Lee, A. Y. S., C. Y. Zhan, and T. M. Wong. Effects of ␤-endorphin on the contraction and electrical activity of the isolated
perfused rat heart. Int. J. Pept. Protein Res. 24: 525–528, 1984.
19. Liang, B. T., and G. J. Gross. Direct preconditioning of cardiac
myocytes via opioid receptors and KATP channels. Circ. Res. 84:
1396–1400, 1999.
20. Llorens-Cortes, C., H. Huang, P. Vicart, J.-M. Gasc, D.
Paulin, and P. Corvol. 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. The signaling pathways used by the enkephalins to elicit this preconditioning-like effect are currently unknown but likely involve protein kinase C and
ATP-sensitive potassium channels, and they deserve
future investigation.
H2449
H2450
21.
22.
23.
24.
25.
27.
28.
29.
30.
31.
32.
33.
34.
neutral endopeptidase in endothelial cells from venous or arterial origins. J. Biol. Chem. 267: 14012–14018, 1992.
Lou, L. G., and G. Pei. Modulation of protein kinase C and
cAMP-dependent protein kinase by delta-opioid. Biochem. Biophys. Res. Commun. 236: 626–629, 1997.
Maeda, S., J. Nakamae, and R. Inoki. Inhibition of cardiac
Na⫹-K⫹-ATPase activity by dynorphin-A and ethylketocyclazocine. Life Sci. 42: 461–468, 1988.
Maslov, L. N., and Y. B. Lishmanov. Change in opioid peptide
level in the heart and blood plasma during acute myocardial
ischaemia complicated by ventricular fibrillation. Clin. Exp.
Pharmacol. Physiol. 22: 812–816, 1995.
Miki, T., M. V. Cohen, and J. M. Downey. Opioid receptor
contributes to ischemic preconditioning through protein kinase C
activation in rabbits. Mol. Cell. Biochem. 186: 3–12, 1998.
Millington, W. R., V. R. Evans, C. N. Battie, O. Bagasra, and
L. H. Forman. Proopiomelanocortin-derived peptides and mRNA
are expressed in rat heart. Ann. NY Acad. Sci. 680: 575–578,
1993.
Millington, W. R., V. R. Evans, L. J. Forman, and C. N.
Battie. Characterization of ␤-endorphin and ␣-MSH related
peptides in rat heart. Peptides 14: 1141–1147, 1993.
Minami, M., and M. Satoh. Molecular biology of the opioid
receptors: structures, functions and distributions. Neurosci. Res.
23: 121–145, 1995.
Mulder, A. H., G. Wardeh, F. Hogenboom, and A. L.
Frankhuyzen. Selectivity of various opioid peptides toward
delta-, kappa-, and mu-opioid receptors mediating presynaptic
inhibition of transmitter release in the brain. Neuropeptides 14:
99–104, 1989.
Norman, J. A., W. L. Autry, and B. S. Barbaz. Angiotensinconverting enzyme inhibitors potentiate the analgesic activity of
[Met]-enkephalin-Arg6-Phe7 by inhibiting its degradation in
mouse brain. Mol. Pharmacol. 28: 521–526, 1985.
Ogutman, C., I. Kaputlu, and G. Sadan. Cardiovascular
effects of intrathecally injected mu, delta and kappa opioid
receptor agonists in rabbits. J. Auton. Pharmacol. 15: 443–450,
1995.
Reisine, R., and G. I. Bell. Molecular biology of opioid receptors. Trends Neurosci. 16: 506–510, 1993.
Rubinstein, M., J. S. Mogil, M. Japon, E. C. Chan, R. G.
Allen, and M. J. Low. Absence of opioid stress-induced analgesia in mice lacking ␤-endorphin by site-directed mutagenesis.
Proc. Natl. Acad. Sci. USA 93: 3995–4000, 1996.
Sanchez-Blazquez, P., J. K. Chang, J. Garzon, and N. M.
Lee. Dynorphin B-29: chemical synthesis and pharmacological
properties in opioid systems in vitro. Neuropeptides 4: 369–374,
1984.
Schultz, J. E. J., A. K. Hsu, and G. J. Gross. Morphine mimics
the cardioprotective effect of ischemic preconditioning via a
glibenclamide-sensitive mechanism in rat heart. Circ. Res. 78:
1100–1104, 1996.
35. Schultz, J. E. J., A. K. Hsu, and G. J. Gross. Ischemic
preconditioning and morphine-induced cardioprotection involve
the delta (␦)-opioid receptor in the intact rat heart. J. Mol. Cell.
Cardiol. 29: 2187–2195, 1997.
36. Schultz, J. E. J., A. K. Hsu, and G. J. Gross. Ischemic
preconditioning in the intact rat heart is mediated by ␦1- but not
µ- or ␬-opioid receptors. Circulation 97: 1282–1289, 1998.
37. Schultz, J. E. J., A. K. Hsu, H. Nagase, and G. J. Gross.
TAN-67, a ␦1-opioid receptor agonist, reduces infarct size via
activation of Gi/o proteins and KATP channels. Am. J. Physiol. 274
(Heart Circ. Physiol. 43): H909–H914, 1998.
38. Schultz, J. E. J., E. Rose, Z. Yao, and G. J. Gross. Evidence for
involvement of opioid receptors in ischemic preconditioning in
rat hearts. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H2157–
H2161, 1995.
39. Schwartz, J.-C. Biological inactivation of enkephalins and the
role of enkephalin-dipeptidyl-carboxypeptidase (‘‘enkephalinase’’) as neuropeptidase. Life Sci. 29: 1715–1740, 1981.
40. Springhorn, J. P., and W. C. Claycomb. Translation of heart
preproenkephalin mRNA and secretion of enkephalin peptides
from cultured cardiac myocytes. Am. J. Physiol. 263 (Heart Circ.
Physiol. 32): H1560–H1566, 1992.
41. Tomai, F., F. Crea, A. Gaspardone, F. Versaci, A. S. Ghini, C.
Ferri, G. Desideri, L. Chiariello, and P. A. Gioffré. Evidence
of naloxone on myocardial ischemic preconditioning in humans.
J. Am. Coll. Cardiol. 33: 1863–1869, 1999.
42. Tsuchida, A., T. Miura, M. Tanno, Y. Nozawa, H. Kita, and K.
Shimamoto. Time window for the contribution of the ␦-opioid
receptor to cardioprotection by ischemic preconditioning in the
rat heart. Cardiovasc. Drugs Ther. 12: 365–373, 1998.
43. Van Winkle, D. M., D. L. Miller, and M. J. Low. ␤-Endorphin
knock-out mice exhibit myocardial ischemic preconditioning
(Abstract). Circulation 98: I-416, 1998.
44. Ventura, C., C. Guarnieri, I. Vaona, G. Campana, G. Pintus,
and S. Spampinato. Dynorphin gene expression and release in
the myocardial cell. J. Biol. Chem. 269: 5384–5386, 1994.
45. Wang, T.-L., H. Chang, C.-R. Hung, and Y.-Z. Tseng. Morphine preconditioning attenuates neutrophil activation in rat
models of myocardial infarction. Cardiovasc. Res. 40: 557–563,
1998.
46. Weinbrenner, C., C. P. Baines, G. S. Liu, C. E. Ganote, A. H.
Walsh, R. E. Honkanen, M. V. Cohen, and J. M. Downey.
Fostriecin, an inhibitor of protein phosphatase 2A, limits myocardial infarct size even when administered after onset of ischemia.
Circulation 98: 899–905, 1998.
47. Wu, S., H. Y. Li, and T. M. Wong. Cardioprotection of preconditioning by metabolic inhibition in the rat ventricular myocyte.
Involvement of ␬-opioid receptor. Circ. Res. 84: 1388–1395, 1999.
48. Zimlichman, R., D. Gefel, H. Eliahou, Z. Matas, B. Rosen, S.
Gass, C. Ela, Y. Eilam, Z. Vogel, and J. Barg. Expression of
opioid receptors during heart ontogeny in normotensive and
hypertensive rats. Circulation 93: 1020–1025, 1996.
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on May 7, 2017
26.
MET5-ENKEPHALIN INDUCES PRECONDITIONING VIA ␦-RECEPTORS