INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

OPIOID RECEPTORS HAVE BEEN IMPLICATED in the protection against ischemia or hypoxia in several organs, including the heart. In the rat heart, opioid receptor activation appears to be the primary trigger of ischemic preconditioning (PC) (14). In the rabbit, activation of the opioid receptor can also contribute to the initiation of PC (2), and recent results of Takasaki et al. (17) suggest that several proenkephalin products interact with ₁-opioid receptors endogenously to produce opioid-mediated cardioprotection. A recent study by Liang and Gross (8) showed that functional opioid receptors are present on the chick cardiac ventricular myocyte and that activation of these receptors by the nonselective opioid receptor agonist morphine can cause a PC-like effect. That study provided the first demonstration that morphine, likely via activation of an opioid receptor, mediates a cardioprotective effect in isolated cardiac myocytes. The protective effect of morphine in the myocyte was mediated via activation of the ATP-sensitive K⁺ (K_ATP) channel, most likely of mitochondrial origin. However, the identity of the specific subtype of opioid receptor involved and the signaling pathway from the receptor to the mitochondrial K_ATP channel in mediating the cardioprotective effect remain unknown.

Thus the objective of the present study was to determine the subtype of opioid receptor that mediates the direct cardioprotective effect in chick cardiac ventricular myocytes. The main advantage of using the current model of ischemic PC is that the exact concentrations of receptor agonist and antagonist can be determined. This will allow delineation of the subtype of opioid receptor involved. Because these myocytes are a relatively homogenous population of cells, there are unlikely to be any potential confounding effects arising from activation of other opioid receptors in cells other than myocytes. Similarly, the exact concentrations of enzyme inhibitors for protein kinase C (PKC) or the K_ATP channel blocker to which the myocytes are exposed can also be determined and controlled. This study provides direct evidence that the ₁-opioid receptor is present on the cardiac myocyte and mediates a cardioprotective effect via activation of PKC and the mitochondrial K_ATP channel.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Preparation of cultured ventricular myocytes. Cardiac ventricular myocytes were cultured from chick embryos 14 days in ovo according to a previously described procedure (5, 6). Myocytes were cultivated in a humidified 5% CO₂-95% air mixture at 37°C for 3 days, at which time cells grew to confluence and exhibited rhythmic spontaneous contractions. All experiments were performed on day 3 in culture. For PC studies, the medium was changed to a HEPES-buffered medium containing (in mM) 139 NaCl, 4.7 KCl, 0.5 MgCl₂, 0.9 CaCl₂, and 5 HEPES and 2% fetal bovine serum, pH 7.4, at 37°C before exposing cells to various conditions at 37°C. Control cells were maintained in the HEPES-buffered media under room air. Ischemia was simulated by placing the cells in a hypoxic incubator (NuAire) for 90 min, where O₂ was replaced by N₂ and was <1%. The prolonged period of ischemia was then followed by 60 min of reexposure to room air at 37°C. PC was induced by exposing the cells to 5 min of simulated ischemia, termed PC ischemia, before a second 90-min period of ischemia. In studying the ability of the opioid receptor agonist to mimic the protective effect of ischemic PC, cells were exposed to different concentrations of the agonist ()-TAN-67 with or without antagonist for 5 min and incubated in fresh drug-free media for 10 min before being exposed to 90 min of simulated ischemia. Cells not subjected to PC or drug were exposed to 90 min of ischemia only (nonpreconditioned cells). All experiments were performed on myocytes attached to the culture plates as previously described (5-8). Determination of cell injury was made at the end of the 90-min ischemia/60-min reoxygenation period. The extent of the basal level of cell injury was quantitated after parallel incubation of the control cells under normal percentage of O₂.

Quantitative determination of extent of myocyte injury. The extent of hypoxia-induced injury to the ventricular cell was quantitatively determined by the percentage of cells killed and the amount of creatine kinase (CK) released into the media according to previously described methods (5). To quantitate the percentage of cells killed, cells were detached after exposure to a trypsin-EDTA-Hanks' balanced salt solution for 10 min for detachment. Viable cells were sedimented by centrifugation (300 g for 10 min) and resuspended in culture media for counting in a hemocytometer. Only live cells sedimented, and the cells that were counted represented those that survived. None of the sedimented cells subsequently counted included trypan blue. Control experiments carried out in prior studies indicated that trypsin treatment, reexposure to Ca²⁺-containing media, or 300-g sedimentation did not cause any significant damage to the control, normoxia-exposed cells (5, 6). The cell viability assay clearly separated the control healthy cells from the hypoxia-exposed damaged cells. In support of the notion that 90-min hypoxia caused significant cell injury and loss of membrane integrity, there was also marked release of lactate dehydrogenase from cells incubated under prolonged hypoxia (5, 6). Parallel changes in the amount of CK released into the media and in the percentage of cells killed under every experimental condition studied further validated the cell viability assay. The amount of CK was measured as enzyme activity (in U/mg), and increases in CK activity above the control level were determined. The percentage of cells killed was calculated as the number of cells obtained from the control group (representing cells not subjected to any hypoxia or drug treatment) minus the number of cells from the treatment group divided by the number of cells in the control group multiplied by 100%.

Materials. The ₁-opioid receptor agonist ()-TAN-67 and the ₁-opioid receptor antagonist 7-benzylidenenaltrexone (BNTX) were the generous gifts of Dr. Hiroshi Nagase of Toray Industries, Kanagawa, Japan. The PKC inhibitor chelerythrine chloride, the ₂-opioid receptor antagonist naltriben methanesulfonate (NTB), the -opioid receptor antagonist nor-binaltorphimine dihydrochloride (nor-BNI), and the K_ATP channel inhibitors glibenclamide and 5-hydroxydecanoic acid (5-HD) were purchased from Research Biochemicals International (Natick, MA). Embryonic chick eggs were purchased from Spafas (Storrs, CT).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

₁-opioid receptor agonist ()-TAN-67 mimics cardioprotective effect of ischemic PC. A 5-min exposure to ()-TAN-67, the ₁-opioid receptor agonist, followed by a 10-min drug-free period protected cardiac ventricular myocytes against injury induced by the subsequent prolonged ischemia (Fig. 1A). The brief exposure to ()-TAN-67 caused a significant decrease in the number of cells killed or CK released in a concentration-dependent manner, reaching a maximum effect at 1 µM. To further confirm that the ₁-opioid receptor is the opioid receptor subtype that mediates the cardioprotective effect, the ₁-opioid receptor-selective antagonist BNTX was used to inhibit the cardioprotective effect mediated by ()-TAN-67. When BNTX was present during preexposure to ()-TAN-67, it abolished the protective effect of the ₁-opioid receptor agonist (Fig. 1B). These data further support the notion that the ₁-opioid receptor can mediate a cardioprotective effect in isolated cardiac myocytes. Additional data using the ₂-opioid receptor-selective antagonist NBT (Fig. 2, A and B) and the -opioid receptor antagonist nor-BNI (Fig. 2C) showed that neither antagonist was able to block the ()-TAN-67-induced cardioprotective effect. However, at the highest concentration of NTB (10 µM), there was a slight inhibition of ()-TAN-67-mediated cardioprotection (Fig. 2, A and B).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   A: cardioprotective effect of ()-TAN-67 (TAN) in cardiac myocytes. Cardiac ventricular myocytes were preconditioned by exposure to 5-min simulated ischemia or by treatment with 0.1-10 µM ()-TAN-67. Myocytes not subjected to either stimulus were exposed to 90 min of ischemia only (nonpreconditioned cells). The extent of myocyte injury was quantitated as percentage of cells killed and as the amount of creatine kinase (CK) released. Data are the mean and SE of 5 experiments. *Significantly different from cells preconditioned by 5-min ischemia or ()-TAN-67 (1-way ANOVA followed by Student-Newman-Keul's comparison test, P < 0.01). B: 1-opioid receptor antagonist 7-benzylidenenaltrexone (BNTX) reversed the cardioprotective effect of ()-TAN-67. Cardiac ventricular myocytes were treated with the indicated concentrations of BNTX in the presence of 1 µM ()-TAN-67. Data are the mean and SE of 5 experiments. *Significantly different from cells that were preexposed to ()-TAN-67 in the absence of BNTX (1-way ANOVA followed by Student-Newman-Keuls comparison test, P < 0.01).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   A: 2-opioid receptor antagonist naltriben methanesulfonate (NBT) only partially reversed the preconditioning (PC)-like effect of ()-TAN-67 on cell death at 10 µM. Data are the mean and SE of 5 experiments. *Significantly different from cells that were preexposed to ()-TAN-67 in the absence of NBT (1-way ANOVA followed by Student-Newman-Keuls comparison test, P < 0.05). B: 2-opioid receptor antagonist NBT only partially reversed the cardioprotective effect of ()-TAN-67 to reduce CK release at 10 µM. Data are the mean and SE of 5 experiments. Pluses, presence of drugs; minuses, absence of drugs. *Significantly different from cells that were preexposed to ()-TAN-67 in the absence of naltriben (1-way ANOVA followed by Student-Newman-Keuls comparison test, P < 0.05). C: effect of -opioid receptor antagonist nor-binaltorphimine on the cardioprotective response to ()-TAN-67. The -opioid receptor antagonist had no effect on the cardioprotective effect of ()-TAN-67 at concentrations up to 10 µM. Data are the mean and SE of 5 experiments.

Role of PKC in mediating cardioprotective effect of ()-TAN-67. PKC has been shown to be a mediator of the cardioprotective effect of PC, acting downstream of a number of receptors (5-7), including the opioid receptor (10). It is unknown whether PKC also acts downstream of the ₁-opioid receptor on the cardiac myocyte. That a 5-min exposure to ()-TAN-67 was able to induce a cardioprotective effect suggests that activation of the ₁-opioid receptor can trigger the process of PC. When the PKC inhibitor chelerythrine was present during the brief exposure to ()-TAN-67, it abolished the ₁-opioid receptor agonist-induced cardioprotective response (Fig. 3). The dose of chelerythrine used was previously shown to completely block phorbol ester-stimulated PKC activation, by using phosphorylation myristolated alanine-rich C kinase substrate as an index of intact cell PKC activity (6). These data suggest that PKC acts downstream of the ₁-opioid receptor to trigger its cardioprotective response.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   The effect of the protein kinase C inhibitor chelerythrine on the cardioprotective response to ()-TAN-67. Chelerythrine produced a concentration-dependent (0.01-1 µM) inhibition of the cardioprotective effect of ()-TAN-67 to reduce cell death and CK release. Data are the mean and SE of 5 experiments. Pluses, presence of ()-TAN-67; minuses, absence of chelerythrine. *Significantly different from cells that were preexposed to ()-TAN-67 in the absence of chelerythrine (1-way ANOVA followed by Student-Newman-Keuls comparison test, P < 0.01).

Role of K_ATP channel in cardioprotective effect mediated by ₁-opioid receptor. In a previous study, we showed that the K_ATP channel, most likely the mitochondrial K_ATP channel, is a downstream effector of morphine in mediating its cardioprotective effect (8). Whether the channel is in fact a downstream effector from the ₁-opioid receptor was examined in the present study. Concomitant presence of either glibenclamide (Fig. 4A) or the mitochondrial selective K_ATP channel antagonist 5-HD (Fig. 4B) during the 5-min exposure to ()-TAN-67 (1 µM) abolished its cardioprotective effect. This was manifested by an increase in the percentage of cells killed (Fig. 4) and the amount CK released (data not shown) in the presence of the K_ATP channel blockers. The antagonistic effect of the K_ATP channel blockers was significant at 1 µM. These data indicate that the mitochondrial K_ATP channel is a very sensitive and important downstream cardioprotective effector of the ₁-opioid receptor.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   A: glibenclamide reversed the PC-like effect of ()-TAN-67. Cardiac ventricular myocytes were treated with the indicated concentrations of glibenclamide in the presence of ()-TAN-67. Data are the mean and SE of 5 experiments. *Significantly different from cells that were preexposed to ()-TAN-67 in the absence of glibenclamide (1-way ANOVA followed by Student-Newman-Keuls comparison test, P < 0.01). B: 5-hydroxydecanoic acid (5-HD) reversed the cardioprotective effect of ()-TAN-67. Cardiac ventricular myocytes were treated with the indicated concentrations of 5-HD in the presence of ()-TAN-67. Data are the mean and SE of 5 experiments. *Significantly different from cells that were preexposed to ()-TAN-67 in the absence of 5-HD (1-way ANOVA followed by Student-Newman-Keuls comparison test, P < 0.01).

Role of 1-opioid receptor in ischemic PC. The present data indicate that the cardioprotective effect of the ₁-opioid receptor signaling pathway exists in the cardiac myocyte. However, blockade of the ₁-opioid receptor only partially attenuated the cardioprotective effect of ischemic PC (Fig. 5). Concomitant presence of the ₁-opioid receptor antagonist BNTX during the 5-min exposure to simulated ischemia had only a small effect on the protection induced by the 5-min PC period. This was manifested by a partial reduction in the percentage of cells killed or the amount of CK release caused by PC.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   7-Benzylidenenaltrexone (BNTX) does not significantly block the cardioprotective effect of ischemic PC. Cardiac ventricular myocytes were preconditioned by exposure to 5-min simulated ischemia in the presence or the absence of BNTX (10 µM) as described in MATERIALS AND METHODS. Myocytes not subjected to ischemic PC were exposed to 90 min of ischemia in the presence or absence of BNTX. Data are the mean and SE of 5 experiments. Percentage of cells killed and the amount of CK released were somewhat higher in the presence of BNTX but not statistically higher. BNTX was present during ischemic PC or during the 90-min simulated ischemia. Pluses, presence of indicated treatment; minuses, absence of indicated treatment. *Significantly different from nonpreconditioned myocytes in the presence or absence of BNTX (1-way ANOVA followed by Student-Newman-Keuls comparison test, P < 0.01).

Signaling function of K_ATP channel and PKC during 90-min ischemic period. The question arises regarding whether K_ATP channel or PKC activation is needed during the 90-min ischemia to exert the actual cardioprotective effect induced by the reexposure to the ₁-opioid receptor agonist. Figure 6 shows that inhibition of the K_ATP channel during the 90-min simulated ischemia was able to block the cardioprotective effect of ()-TAN-67. In this study, a 5-min exposure to 1 µM ()-TAN-67 protected the cardiac myocytes during a subsequent, 90-min period of simulated ischemia. The presence of 5-HD during the 90-min period of ischemia abolished, in a dose-dependent manner, the protective effect of ()-TAN-67. In addition, the presence of a PKC inhibitor (1 µM chelerythrine) during the 90-min ischemia also blocked the protective effect that resulted from a previous 5-min exposure to 1 µM ()-TAN-67. The percentage of cardiac cells killed with chelerythrine was 22.3 ± 1 (n = 4; ±SE) compared with the percentage of cardiac cells without chelerythrine, 11.4 ± 1% (n = 4).

View larger version (10K): [in this window] [in a new window]   Fig. 6.   The presence of 5-HD during the 90-min ischemia blocked the cardioprotective effect of ()-TAN-67. Cardiac ventricular myocytes were pharmacologically preconditioned by treatment with 1 µM ()-TAN-67. For some of the ()-TAN 67-treated myocytes, 5-HD (at the indicated concentrations) was present during the 90-min prolonged ischemia. The extent of myocyte injury was quantitated as percentage of cells killed and amount of CK released. Data were the mean and SE of 4 experiments. *Significantly different from cells not exposed to 5-HD during the 90-min ischemia (1-way ANOVA followed by Student-Newman-Keuls comparison test, P  0.01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A brief episode of ischemia before a second sustained period of ischemia can protect the myocardium against infarction (1). This clinically observed phenomenon is called ischemic PC. Previous studies (4-8) have demonstrated that adenosine receptor activation on cardiac myocytes can elicit this cardioprotective phenomenon. Opioid receptors are another class of G protein-coupled receptors that have demonstrated the ability to elicit this response (8, 12, 13). Morphine hydrochloride, a nonselective opioid receptor agonist, has been shown to induce a cardioprotective effect in previous studies from our laboratory (8, 12, 13). The purpose of the present study was to determine the identity of the opioid receptor/receptors involved in opioid receptor-mediated cardioprotection and the signaling pathways involved. ()-TAN-67, a highly selective ₁-opioid receptor agonist, was used to induce a cardioprotective effect at the level of the cardiac myocytes. The observed protective effect of ()-TAN-67 was reversed by the highly selective ₁-opioid receptor antagonist BNTX. The ability of BNTX to attenuate the effect of ()-TAN-67 in a concentration-dependent manner demonstrates that the ₁-opioid receptor subtype is the major opioid receptor involved in opioid receptor-mediated cardioprotection. To further characterize receptor identity, cells treated with ()-TAN-67 were concomitantly exposed to different concentrations of a ₂-opioid receptor antagonist, NTB. At 1 µM, NTB had no effect on the ()-TAN-67-induced decrease in the number of viable cells after prolonged ischemia. At the 10 µM concentration, there was a small increase in the percentage of cells killed. However, the observed increase did not approach total abolition of the cardioprotective effect, as was the case with BNTX. This demonstrates that BNTX is a much more potent blocker of this cardioprotective pathway compared with the ₂-opioid receptor antagonist. Moreover, at the 10 µM concentration, it is possible that there may be some crossover inhibition of the ₁-opioid receptor by NBT.

Another class of opioid receptors present on myocytes are -opioid receptors, and activation of these receptors by a selective -opioid receptor agonist, U50,488H, has been shown by Wu et al. (21) to protect isolated rat ventricular myocytes against metabolic inhibition-induced damage. The compound nor-BNI is a -opioid receptor-selective antagonist, being 168-fold and 153-fold selective versus the µ- and -opioid receptors, respectively (11,18). The addition of this compound did not attentuate the cardioprotective effects of ()-TAN-67 at any concentration, which strongly suggests that -opioid receptors are not involved in this pathway. The data gathered throughout these experiments demonstrate that the ₁-opioid receptor subtype is primarily involved in opioid-mediated myocyte protection.

Interestingly, BNTX only marginally blocked the cardioprotective effect of simulated ischemic PC in this model. This is in contrast to intact rat and rabbit hearts, where the opioid receptor antagonist naloxone completely blocked the cardioprotective effect of ischemic PC. There may be several reasons why BNTX did not completely block the cardioprotective effects of simulated ischemia in this model. It may be that not enough endogenous opioids are released during the hypoxic period to precondition the myocytes. Alternatively, perhaps more than one opioid receptor may be involved in PC by hypoxia. In this regard, Wu et al. (21) presented data from the rat myocyte that suggested that the -opioid receptor was responsible for the delayed cardioprotective effect observed after metabolic inhibition. Thus, even though the ₁-opioid receptor appears to be primarily involved in acute cardioprotection in the chick myocyte after treatment with selective opioid agonists and antagonists, we cannot rule out the possibility that other opioid receptors or other G protein-coupled receptors, such as adenosine, might also contribute to the cardioprotective effect of simulated ischemic PC (4, 16).

Having elucidated the identity of the cardioprotective opioid receptor, the goal of the study focused on the signaling pathway involved. The initial hypothesis was that the pathway was probably very similar to the pathway involved in the adenosine receptor-mediated cardioprotective effect observed in previous studies. The involvement of a K_ATP channel in the adenosine receptor-mediated cardioprotective effect has also been well characterized (3, 9, 15). To examine whether the same mechanisms are at work here, two different K_ATP channel blockers were administered individually during treatment with ()-TAN-67. The addition of even 1-10 µM concentrations of 5-HD attenuated the cardioprotective effect of opioid receptor activation by ()-TAN-67. At the 100 µM concentration of 5-HD, there appeared to be a complete abolition of the cardioprotective effect of ()-TAN-67. The addition of glibenclamide, another K_ATP channel antagonist, to cells treated with ()-TAN-67 also exhibited a sharp decrease in myocyte viability after prolonged ischemic conditions. Thus both K_ATP channel blockers effectively inhibited the cardioprotective effect mediated by the ₁-opioid receptor. Although 5-HD has been reported to be less potent than glibenclamide, the present data showed that both inhibitors appear to be similarly potent in blocking the protective effect of ()-TAN-67. The reason for this difference is unclear. Possible explanations include a more limited diffusion of the negatively charged 5-HD in the intact heart or a K_ATP channel in the embryonic cardiac myocyte that is more sensitive to blockade by 5-HD compared with the channel in adult myocytes. These data show that a K_ATP channel, most likely the mitochondrial channel, is a downstream cardioprotective effector of the ₁-opioid receptor.

An additional set of experiments was performed to determine whether PKC was involved in the signal transduction pathway. Previous studies by Miki et al. (10) showed that the cardioporotective effect of morphine in isolated rabbit hearts was antagonized by chelerythrine chloride, and Wang and Ashraf (19) and Wang et al. (20) have shown that the mitochondrial K_ATP channel is dependent on PKC for protection against calcium and ischemic-induced injury. Therefore, we used chelerythrine chloride to determine whether blocking PKC would have any effect upon the observed opioid receptor-mediated cardioprotective effect in our myocytes. The data clearly show a concentration-dependent inhibition of the cardioprotective effect of ()-TAN-67. These data implicate PKC in the pathway for this cardioprotective response.

A final series of experiments were designed to address the question of whether the K_ATP channel or PKC activation is necessary during the 90-min ischemic period to mediate the cardioprotective effect of stimulating the ₁-opioid receptor. The presence of 5-HD during the 90-min period of ischemia abolished, in a concentration-dependent manner, the protective effect of ()-TAN-67. In addition, the presence of the PKC inhibitor chelerythrine (1 µM) during the 90-min ischemic period also blocked the protective effect that resulted from a previous 5-min exposure to 1 µM ()-TAN-67. These data are consistent with the hypothesis that both PKC and the K_ATP channel need to be activated during both the initiation of the cardioprotective effect of ₁-opioid receptor activation as well as during the prolonged 90-min ischemic period to achieve the protection induced by prior ()-TAN-67 exposure. These data are also in agreement with those of Wang and Ashraf (19) and Wang et al. (20), who found similar results in isolated rat hearts.

The data summarized in this discussion show a relationship between ₁-opioid receptor activation and a cardioprotective effect. Although this does not seem to be a physiologically relevant event (brief ischemia before prolonged ischemia did not show appreciable decreases in myocyte viability with the addition of specific and nonspecific opioid receptor antagonists; data not shown), the cardioprotective effect exerted by opioid receptor activation may be a secondary or complimentary system to other signaling pathways responsible for PC of myocytes in the intact heart. Nevertheless, myocyte responses to this class of compounds may prove to be an important area of investigation for novel therapeutic development.