Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jul 18.
Published in final edited form as: Pharmacology. 2012 Jul 18;90(1-2):110–116. doi: 10.1159/000340058

Acute and Chronic Cardioprotection by the Enkephalin Analogue, Eribis Peptide 94, is Mediated via Activation of Nitric Oxide Synthase and Adenosine Triphosphate-Regulated Potassium Channels

Garrett J Gross 1, Anna Hsu 1, Kasem Nithipatikom 1, Adam W Pfeiffer 1, Irina Bobrova 2, Erik Bissessar 2
PMCID: PMC3482344  NIHMSID: NIHMS408625  PMID: 22814415

Abstract

Background/Aims

Eribis peptide 94 (EP 94) is a new enkephalin derivative which potently binds to the μ- and δ-opioid receptor. In this study, we determined the effects of EP 94 and potential mechanism(s) involved in cardioprotection of the rat heart.

Methods and Results

An acute (5 and 10 min into ischemia) and a chronic (24 h prior to ischemia) EP 94 administration produced a similar 30–40 % reduction in IS/AAR and the effects were blocked by the KATP channel antagonists, HMR 1098 and 5-HD. The cardioprotective effects were blocked by a nonselective nitric oxide synthase (NOS) inhibitor (L-NAME) following acute administration and by a selective iNOS inhibitor (1400W) following chronic administration.

Conclusion

These results suggest that EP 94 may have potential for the treatment of ischemic heart disease via a nitric oxide (NO)-KATP mediated mechanism.

Keywords: Opioid peptides, myocardial infarction, nitric oxide, KATP channels

INTRODUCTION

The first evidence that opioids produced potent and efficacious cardioprotective effects via activation of opioid receptors was published by Schultz et al. in 1995 [1]. These experiments were performed in intact anesthetized rats in which the left coronary artery was occluded for 30 min of index ischemia followed by 2 h of reperfusion. Ischemic preconditioning (IPC) was used as a basis of comparison and was produced by either 1 or 3 five-min periods of left coronary occlusion interspersed with 5 min of reperfusion prior to the 30-min index ischemic period. IPC produced an approximately 70 % reduction in infarct size. Similarly, three 5-min infusions of the opioid agonist, morphine, at 100 μg/kg, reduced infarct size approximately 65 %, similar to that of IPC. Interestingly, the effects of IPC and morphine were blocked by the ATP-sensitive potassium (KATP) channel antagonist glibenclamide [2]. These data and several subsequent publications led to a surge of interest in studying the cardioprotective effects of opioids, primarily as they relate to their role in IPC and more recently postconditioning (POC) [3], the receptors involved and their distal signaling pathways.

Eribis peptide 94 (EP 94) is a newly synthesized opioid peptide similar in structure to the naturally occurring enkephalins such as met- and leu-enkephalin [4]. This compound has recently been shown to reduce myocardial infarct size in rat heart and several pig models of ischemia/reperfusion injury by increasing the phosphorylation of eNOS (ser 1177), suggesting an increase of eNOS activity and subsequent release of nitric oxide (NO) that is at least partially responsible for the acute cardioprotective effect of EP 94 in pigs. This is the first paper to demonstrate that opioids produce an increase of eNOS activity and NO release. Based on these intriguing results [5], and evidence that several NOS isoforms mediate cardioprotection [6–8], we extended these studies to determine the NOS isoforms that mediate the acute and chronic effects of EP 94 in a rat model of infarction. Our second objective was to determine the role of the sarc- and mito- KATP channel in mediating the acute and chronic effect of EP 94 to reduce infarct size in the rat heart. This objective was based on our earlier studies where we demonstrated a role for both KATP channels in the cardioprotective effect of the synthetic opioid agonist, morphine in isolated and intact rat hearts and isolated chick myocytes [2,9,10]. The results obtained suggest that NO and both KATP channels are very important in mediating cardioprotection following EP 94 administration in rat hearts subjected to ischemia/reperfusion.

METHODS

Studies followed the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publications No. 85-23, revised 1996). The protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

Materials

Eribis peptide 94 (EP 94) was obtained from Eribis Pharmaceuticals AB, Uppsala, Sweden. N5-[Imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME) was obtained from Sigma-Aldrich Chemical (St. Louis, MO). (4S)-N-(4-Amino-5[aminoethyl]aminopentyl)-N′-nitroguanidine (nNOS I) was obtained from EMD Millipore (Billerica, MA). N-[[3-(Aminomethyl)phenyl]methyl]-ethanimidamide, dihydrochloride (1400W) and 5-Hydroxydecanoate (5-HD) were obtained from Tocris Bioscience (Ellisville, MO). 1-[[5-[2-(5-Chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea, sodium salt (HMR1098) was obtained from Aventis (Frankfort, Germany). Rabbit polyclonal antibody against p-eNOS (Ser1177) and eNOS, ECL Western blotting detection kit, and BCA protein assay kit were from Peirce Biotechnology (Rockford, IL). Goat anti-rabbit IgG-HRP secondary antibody was obtained from Invitrogen (Camarillo, CA). Mini-Protean gels were obtained from Bio-Rad Laboratories (Hercules, CA).

In vivo surgical preparation

Male Sprague-Dawley rats (250–300 g) were anesthetized with 100 mg/kg of Inactin, placed on a heating pad and a tracheotomy performed and the rat ventilated with room air supplemented with 100 % oxygen (the pO2 was adjusted to approximately 150–200 mmHg). Catheters were placed in the carotid artery and jugular vein for the measurement of systemic blood pressure, heart rate, blood gases and for administration of drugs or vehicle. The heart was exposed by a left thoracotomy and the left coronary artery isolated and a suture placed around it to produce an occlusion followed by reperfusion. The heart was subjected to 30 min of total coronary artery occlusion followed by 2 h of reperfusion at which time the artery was reoccluded and Evans Blue Dye injected into the jugular vein to stain the nonischemic area of the heart blue. Subsequently, the heart was arrested with a bolus of 10 % KCl and removed for the measurement of infarct size. After heart removal, it was washed in buffer and cut in a bread loaf fashion into 5–6 pieces and the blue stained normal region and the ischemic region separated and placed in separate vials containing triphenyltetrazolium chloride staining solution and incubated at 37°C in phosphate buffer at pH 7.4 for approximately 15 min. Subsequently, the pieces of tissue were placed into vials containing formaldehyde and stored overnight at room temperature and infarct size determined by planimetry the following day. Infarct size (IS) was expressed as a percent (%) of the area at risk (IS/AAR, %) as determined by our dual staining technique. In the AAR, the dark red color represents noninfarcted tissue, whereas, the infarcted tissue remains a pale yellow color.

Experimental protocols

The various drugs and protocols are shown in fig. 1. Nineteen groups with 9 animals per group (total 171 rats) were subjected to the 30-min ischemia/2-h reperfusion protocol. In all acute experiments, EP 94 was administered at 5 and 10 min into the ischemic period and the antagonists of the various isoforms of nitric oxide synthase (NOS) (L-NAME and nNOS I) and KATP channels (5-HD or HMR 1098) were administered at 10 min prior to ischemia. In the chronic studies, EP 94 was administered 24 h prior to ischemia by intraperitoneal injection and the selective iNOS inhibitor, 1400W, or 5-HD or HMR 1098 were administered 10 min prior to ischemia. All drugs were dissolved in water in the original stock solutions, except 1400W was dissolved in 95% ethanol and EP 94 was dissolved in saline. Then, they were serially diluted in water to desired concentrations and administered to animals in water as a vehicle.

Figure 1.

Figure 1

Open in a new tab

Schematic diagram of the various experimental protocols used in the present study. (A) Acute experimental protocol and (B) Chronic experimental protocol. Abbreviations: A = Acute treatment; C = Chronic treatment; L-NAME = L-NG-Nitroarginine methyl ester ; 1400W = N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide; nNOS I = (4S)-N-(4-Amino-5[aminoethyl]aminopentyl-N′-nitroguanidine; HMR = HMR 1098; 5-HD = 5-hydroxydecanoic acid.

Western blot analysis of p-eNOS (Ser 1177) in rat heart tissues

In this experiment, three rats were used for each group of treatment. Rat heart tissues (without staining) in the control rats (without ischemia/reperfusion), non-ischemic and ischemic areas of rat hearts subjected to 30-min occlusion and 5-min reperfusion were cut into pieces and put in sample tubes containing protease and phosphatase inhibitor cocktail, snapped frozen in liquid nitrogen and stored at −80 °C until analysis. Frozen tissues were homogenized in the additional protease and phosphatase inhibitor cocktail, centrifuged at 10,000 rpm at 4°C for 10 min. The supernatant was determined for protein content using BCA protein assay kit and analyzed for expression of p-eNOS (Ser 1177) and total eNOS (t-eNOS) by Western blot analysis. Proteins were separated on SDS-PAGE (4–15 %) BioRad Mini Protean Gels and transferred to a nitrocellulose membrane. Blots were incubated with p-eNOS (Ser 1177) primary antibody (1:1,000) or t-eNOS primary antibody (1:1,000) or β-actin primary antibody (1,000) at 4 °C overnight, followed by goat anti-rabbit IgG-HRP (1:50,000) at room temperature for 1 h. Protein loading, t-eNOS and β-actin were used as loading controls. Detection was made by using ECL Western Blotting Substrate and captured by Fuji film X-ray (Tokyo, Japan). Band densities were analyzed using Image J software from the NIH.

Statistics

Data were expressed as mean ± SEM. All data were analyzed using GraphPad Prism v.4.0. Comparison between groups in hemodynamics and blood gases was performed using a two-way ANOVA with post-hoc analysis by Bonferroni’s test. A one way ANOVA was used to compare infarct sizes between groups followed by Tukeys post hoc test. A probability value of p < 0.05 was considered statistically significant.

RESULTS

Exclusions

A total of 178 rats were entered into the protocols shown in fig. 1. Seven rats were excluded due to intractable ventricular fibrillation (3 rats); abnormal blood gases (2 rats) and severe hypotension (mean arterial pressure less than 40 mm Hg, 2 rats). Therefore, 171 rats were included in the data analysis (19 groups of 9 rats).

Area at risk to left ventricular weight (AAR/LV, %), hemodynamics and blood gases

The percent of area at risk expressed as a percent of left ventricular weight (AAR/LV, %) was summarized in Table 1. There were no significant differences in AAR/LV (%) as compared with the control group. There were no significant differences in the baseline values for heart rate and mean arterial pressure between all groups and at 30 min of ischemia and 2 h of reperfusion (Data not shown). Similarly, there were no differences in blood gases or body temperature between groups throughout the experiment (Data not shown).

Dose response studies

In the initial series of experiments we performed a dose-response study using doses of EP 94 between 0.1 and 10 μg/kg, iv. EP 94 significantly reduced IS/AAR at 0.5, 1.0 and 2.5 μg/kg as compared to the control group, with the maximal effect occurring at 0.5 and 1.0 μg/kg (fig. 2). Based on these data we chose the 1.0 μg/kg dose of EP 94 in all future experiments examining the role of KATP channels and nitric oxide (NO) in EP 94-induced cardioprotection.

Figure 2.

Figure 2

Open in a new tab

Dose response relationship and the reduction in IS/AAR (%) produced by various doses of EP 94. (n = 9 rats/group). *P < 0.01 vs. Control; **P < 0.001 vs. Control.

Role of KATP channels in EP 94-induced cardioprotection

To investigate a possible downstream pathway which may be mediating the beneficial effect of acute EP 94 (A), the sarcolemmal KATP channel antagonist, HMR 1098, or the mitochondrial KATP channel antagonist, 5-HD, were administered 10 prior to ischemia. Both antagonists completely abolished the cardioprotective effect of EP 94 (fig. 3) which suggests an important role for the KATP channels as a key mediator in the pathway by which EP 94 reduces infarct size similar to that seen with a number of other cardioprotective agents as well as ischemic preconditioning and postconditioning. In addition, both 5-HD and HMR 1098 blocked the cardioprotective effects of EP 94 following chronic EP 94 administration (fig. 4). These results were identical to those obtained in the acute EP 94 studies.

Figure 3.

Figure 3

Open in a new tab

Effects of the selective sarcolemmal (HMR 1098, 6 mg/kg iv) and the mitochondrial selective (5-HD, 10 mg/kg, iv) KATP channel antagonists on the infarct size reducing effect of EP 94 (1.0 μg/kg, iv) in in vivo rat hearts (n = 9/group). **P < 0.001 vs. Control.

Figure 4.

Figure 4

Open in a new tab

Effect of EP 94 when administered 24 h prior to index ischemia and the subsequent effect of the selective iNOS inhibitor, 1400W (0.1 mg/kg, iv) or the sarc KATP or mito KATP channel antagonists on the infarct size reducing effect of EP 94 (1.0 μg/kg, iv) when given 10 min prior to index ischemia in rats previously treated with EP 94 for 24 h (n = 9/group). **P < 0.001 vs. Control.

Role of NO in acute (A) and chronic (C) EP 94-induced cardioprotection

Finally, we investigated the roles of nitric oxide (NO) and eNOS in mediating the acute cardioprotection produced by EP 94 and inducible NO synthase (iNOS) in mediating the chronic cardioprotection produced by EP 94. The chronic administration of EP 94 produced a reduction in IS/AAR equal to that observed following its acute administration and that this effect was blocked by 1400W, a selective iNOS inhibitor, administered 24 h after EP 94 administration (fig. 4). Interestingly, 1400W did not block the acute effect of EP 94 which is not surprising since eNOS is a more likely target for the early phase of protection. Our experiments with the nonselective NOS inhibitor, L-NAME and the selective nNOS inhibitor, nNOS I (fig. 5) and the selective inhibitor, 1400W, support a major role for eNOS in the acute cardioprotection produced by EP 94 since both the selective nNOS inhibitor and selective iNOS inhibitor did not block the protection produced by EP 94 acutely, whereas, L-NAME, the nonselective NOS inhibitor, blocked the acute beneficial effect of EP 94 (figs. 4 and 5).

Figure 5.

Figure 5

Open in a new tab

The effect of two NOS inhibitors, L-NAME (nonselective NOS inhibitor) and nNOS I (selective neuronal NOS inhibitor) on the acute (A) cardioprotective effect of EP 94 (1.0 μg/kg, iv) in in vivo rat hearts (n = 9/group). **P < 0.001 vs. Control.

Effect of ischemia/reperfusion and EP 94 on the phosphorylated-eNOS (Ser 1177)

Western blot analysis of rat heart tissues obtained from the control rats, non-ischemic areas and ischemic areas of rat hearts subjected to 30-min occlusion and 5-min reperfusion with or without acute administration of EP 94 (1.0 μg/kg) showed an increase of the ratio of p-eNOS (Ser 1177) to t-eNOS. A three-fold increase and a two-fold increase of the ratio of p-eNOS (Ser 1177) to t-eNOS were observed in the ischemic areas and non-ischemic areas, respectively (fig. 6A). Interestingly, EP 94 produced a 12-fold increase of the ratio of p-eNOS (Ser 1177) to t-eNOS in the ischemic areas of rat heart tissues and only about a two-fold increase in the non-ischemic areas as compared with the heart tissues of the control rats (fig. 6B). The intensity of the immunoreactive bands of t-eNOS of the ischemic area tissues appeared to be higher than the control and non-ischemic area tissues due to the protein loading as indicated by the band intensities of β-actin in these samples (figs. 6A&B).

Figure 6.

Figure 6

Open in a new tab

Western blot analysis of rate heart tissues obtained from nonischemic control rats, non-ischemic areas and ischemic areas of rats subjected to 30- min occlusion and 5-min reperfusion. (A) Examples of immunoreactive bands of p-eNOS (Ser 1177) and t-eNOS of the control, non-ischemic areas and ischemic areas of rat heart tissues (left panel). Also shown are the immunoreactive bands of β-actin from the same membranes for t-eNOS. The average of the ratio of p-eNOS to t-eNOS as normalized to the control rat tissues (right panel). (n = 3 rats); *, significantly different from control, P < 0.05. (B) Examples of immunoreactive bands of p-eNOS (Ser 1177) and t-eNOS of the control (no treatment and occlusion and reperfusion), ischemic areas and non-ischemic areas of rats administered EP 94 (left panel). Also shown are the immunoreactive bands of β-actin from the same membranes for t-eNOS. The average of the ratio of p-eNOS to t-eNOS as normalized to the control rat tissues (right panel). (n = 3 rats); *, significantly different from control, P < 0.05.

DISCUSSION

The results of the present study suggest that the enkephalin derivative, EP 94, possesses potent cardioprotective effects to reduce infarct size in an in vivo rat model of infarction. The optimal effect was observed at 0.5 μg/kg, iv and its effect decreases with higher doses. The maximal effect was approximately a 30–40 % reduction in infarct size (IS) expressed as a % of the area at risk (AAR). To prolong the plasma concentrations, EP 94 was administered as 2 boluses of 0.5 μg/kg, iv at 5 and 10 min after the onset of the 30 min index ischemia period. It is likely that EP 94 did not get to the heart during the ischemic period and its action probably occurs during the early reperfusion period. It would be of interest to determine if EP 94 is equally effective in rat hearts when administered just prior to reperfusion as suggested by recent data obtained in pig hearts [5], since this is the point where the heart is most susceptible to reperfusion-induced injury.

The magnitude of the effect of EP 94 to reduce IS/AAR (%) was equal to that seen with most opioid agonists studied in this model including morphine [2] and TAN-67 [11], although EP 94 is one of the most potent compounds we have investigated in this model, if not the most potent. There were no noticeable effects of EP 94 on peripheral hemodynamics including heart rate and blood pressure. Similarly, no effects were observed on blood gases or body temperature throughout the experiments.

To investigate a possible downstream pathway which may be mediating the beneficial effect of EP 94 we administered either the sarcolemmal KATP channel antagonist, HMR 1098, or the mitochondrial KATP channel antagonist, 5-HD, prior to EP 94 administration. Both antagonists completely abolished the cardioprotective effect of EP 94, which suggests an important role for the KATP channel as a key mediator in the pathway by which acute EP 94 reduces infarct size similar to that seen with a number of other cardioprotective agents [12] as well as ischemic preconditioning and postconditioning [6–8]. Furthermore, both 5-HD and HMR 1098, blocked the delayed cardioprotective effect of EP 94 when administered 24 h post EP 94 treatment similar to the data obtained in the acute experiments.

Finally, we investigated the ability and mechanism by which EP 94 produces the second window of cardioprotection (SWOP) 24 h after EP 94 administration and the role of nitric oxide (NO) and inducible NO synthase (iNOS). We found that chronic EP 94 administration produced a reduction in IS/AAR equal to that observed following its acute administration and that this effect was blocked by a selective iNOS inhibitor, 1400W, administration 24 h after EP 94 administration. These results suggest that EP 94 probably upregualted iNOS upon chronic exposure to EP 94. Detection of iNOS expression at various times during this chronic treatment protocol may provide direct evidence to this hypothesis. This finding that a drug (EP 94) produces an effect 24 h later similar in magnitude to when the drug was administered acutely is unique. Interestingly, 1400W did not block the acute effect of EP 94 which is not surprising since eNOS is a more likely target for the early phase of protection [5]. Similar results have been previously reported by Fryer et al. [13] and Jiang et al. [14] in rats and mice where these investigators found that the selective δ1-opioid receptor agonist TAN-67 and morphine produced a second window of cardioprotection which was mediated by iNOS upregulation. Moreover, a selective inhibitor of nNOS, nNOS I, did not block the acute cardioprotective effect of EP 94 either but the nonselective NOS inhibitor, L-NAME, blocked the acute protective effect. Taken together, these data with selective and nonselective pharmacological blockers and Western blot analysis of p-eNOS (Ser 1177), clearly suggest that eNOS mediates the acute cardioprotective effect of EP 94 in the ischemic/reperfused rat heart, whereas, iNOS mediates the chronic effect of EP 94 which agrees with previous work with other opioid agonists from our laboratory [15,16] and Bolli’s laboratory [8] in rat and mouse hearts. Present results indicate that the NO release and KATP channel openings are the downstream mediators in the acute cardioprotection produced by EP 94 and the activation of a μ opioid receptor is likely involved [17]. It is of interest to understand the down-stream signaling pathway(s) of NO involved in the cardioprotection by EP 94 administration acutely and chronically. The roles of NO in cardioprotection include the activation of Akt and extracellular receptor kinase (ERK) pathways as well as peroxynitrite-matrix metalloproteinase-2 signaling pathway in preconditioning [18–20].

Conclusions

In summary, the results of the present study suggest that the newly synthesized enkephalin derivative, EP 94, has a potent cardioprotective effect to reduce infarct size in a rat model of ischemia reperfusion injury. We also discovered that EP 94 exerts its beneficial effect by opening sarc- and mito-KATP channels in the heart and has an acute effect to enhance NO release most likely via eNOS phosphorylation in the heart. Of great interest is the novel observation that EP 94 exerts a cardioprotective effect 24 h post drug treatment via upregulation of iNOS and enhanced NO release. Based on earlier results published by Frassdorf et al. [9], it is likely that the increase in iNOS activity results from activation of the nuclear transcription factor NF-κB in rats. Future experiments are needed in animal models or humans to test this possibility. Nevertheless, EP 94 may be a novel new treatment for the acute and/or chronic treatment of patients with an acute coronary syndrome, particularly if its side effect profile is less serious as compared to standard treatment with opioids such as morphine.

Acknowledgments

These experiments were supported by funds obtained from Eribis Pharmaceuticals AB and NIH Grants HL074314 and HL111392 (GJG).

Nonstandard Abbreviations

1400W

N-[[3-(Aminomethyl) methyl)phenyl]methyl]-ethanimdamide dihydrochloride

AAR

area at risk

EP 94

Eribis peptide 94

5-HD

5-hydroxydecanoic acid

HMR 1098

(1-[[5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-methylthiourea), Na+ salt

IS

infarct size

IS/AAR

infarct size as a % of the area at risk

L-NAME

nitro-L-arginine methyl ester

nNOS I

(4S)-N-(4-Amino-5[aminoethyl]aminopentyl)-N′-nitroguanidine

NOS

nitric oxide synthase

eNOS

endothelial nitric oxide synthase

nNOS

neuronal nitric oxide synthase

iNOS

inducible nitric oxide synthase

TTZ

triphenyltetrazolium chloride

References

  • 1.Schultz JE, Rose E, Yao Z, Gross GJ. Evidence for involvement of opioid receptors in ischemic preconditioning in rat hearts. Am J Physiol. 1995;268:H2157–2161. doi: 10.1152/ajpheart.1995.268.5.H2157. [DOI] [PubMed] [Google Scholar]
  • 2.Schultz JE, Hsu AK, Gross GJ. Morphine mimics the cardioprotective effect of ischemic preconditioning via a glibenclamide-sensitive mechanism in the rat heart. Circ Res. 1996;78:1100–1104. doi: 10.1161/01.res.78.6.1100. [DOI] [PubMed] [Google Scholar]
  • 3.Zatta AJ, Kin H, Yoshishige D, Jiang R, Wang N, Reeves JG, Mykytenko J, Guyton RA, Zhao ZQ, Caffrey JL, Vinten-Johansen J. Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation. Am J Physiol Heart Circ Physiol. 2008;294:H1444–1451. doi: 10.1152/ajpheart.01279.2006. [DOI] [PubMed] [Google Scholar]
  • 4.Barron BA. Cardiac opioids. Proc Soc Exp Biol Med. 2000;224:1–7. doi: 10.1046/j.1525-1373.2000.22358.x. [DOI] [PubMed] [Google Scholar]
  • 5.Karlsson LO, Grip L, Bissessar E, Bobrova I, Gustafsson T, Kavianipour M, Odenstedt J, Wikstrom G, Gonon AT. Opioid receptor agonist eribis peptide 94 reduces infarct size in different porcine models for myocardial ischaemia and reperfusion. Eur J Pharmacol. 2011;651:146–151. doi: 10.1016/j.ejphar.2010.10.069. [DOI] [PubMed] [Google Scholar]
  • 6.Burkard N, Williams T, Czolbe M, Blomer N, Panther F, Link M, Fraccarollo D, Widder JD, Hu K, Han H, Hofmann U, Frantz S, Nordbeck P, Bulla J, Schuh K, Ritter O. Conditional overexpression of neuronal nitric oxide synthase is cardioprotective in ischemia/reperfusion. Circulation. 2010;122:1588–1603. doi: 10.1161/CIRCULATIONAHA.109.933630. [DOI] [PubMed] [Google Scholar]
  • 7.Jones SP, Greer JJ, Kakkar AK, Ware PD, Turnage RH, Hicks M, van Haperen R, de Crom R, Kawashima S, Yokoyama M, Lefer DJ. Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury. Am J Physiol Heart Circ Physiol. 2004;286:H276–282. doi: 10.1152/ajpheart.00129.2003. [DOI] [PubMed] [Google Scholar]
  • 8.Shinmura K, Xuan YT, Tang XL, Kodani E, Han H, Zhu Y, Bolli R. Inducible nitric oxide synthase modulates cyclooxygenase-2 activity in the heart of conscious rabbits during the late phase of ischemic preconditioning. Circ Res. 2002;90:602–608. doi: 10.1161/01.res.0000012202.52809.40. [DOI] [PubMed] [Google Scholar]
  • 9.Frassdorf J, Weber NC, Obal D, Toma O, Mullenheim J, Kojda G, Preckel B, Schlack W. Morphine induces late cardioprotection in rat hearts in vivo: The involvement of opioid receptors and nuclear transcription factor kappab. Anesth Analg. 2005;101:934–941. doi: 10.1213/01.ane.0000172130.70274.84. table of contents. [DOI] [PubMed] [Google Scholar]
  • 10.Liang BT, Gross GJ. Direct preconditioning of cardiac myocytes via opioid receptors and katp channels. Circ Res. 1999;84:1396–1400. doi: 10.1161/01.res.84.12.1396. [DOI] [PubMed] [Google Scholar]
  • 11.Gross ER, Gross GJ. Ischemic preconditioning and myocardial infarction: An update and perspective. Drug Discov Today Dis Mech. 2007;4:165–174. doi: 10.1016/j.ddmec.2007.10.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Gross ER, Gross GJ. Pharmacologic therapeutics for cardiac reperfusion injury. Expert Opin Emerg Drugs. 2007;12:367–388. doi: 10.1517/14728214.12.3.367. [DOI] [PubMed] [Google Scholar]
  • 13.Fryer RM, Wang Y, Hsu AK, Gross GJ. Essential activation of pkc-delta in opioid-initiated cardioprotection. Am J Physiol Heart Circ Physiol. 2001;280:H1346–1353. doi: 10.1152/ajpheart.2001.280.3.H1346. [DOI] [PubMed] [Google Scholar]
  • 14.Jiang X, Shi E, Nakajima Y, Sato S. Inducible nitric oxide synthase mediates delayed cardioprotection induced by morphine in vivo: Evidence from pharmacologic inhibition and gene-knockout mice. Anesthesiology. 2004;101:82–88. doi: 10.1097/00000542-200407000-00014. [DOI] [PubMed] [Google Scholar]
  • 15.Fryer RM, Hsu AK, Eells JT, Nagase H, Gross GJ. Opioid-induced second window of cardioprotection: Potential role of mitochondrial katp channels. Circ Res. 1999;84:846–851. doi: 10.1161/01.res.84.7.846. [DOI] [PubMed] [Google Scholar]
  • 16.Patel HH, Hsu AK, Gross GJ. Cox-2 and inos in opioid-induced delayed cardioprotection in the intact rat. Life Sci. 2004;75:129–140. doi: 10.1016/j.lfs.2003.10.036. [DOI] [PubMed] [Google Scholar]
  • 17.Gross GJ, Hsu A, Nithipatikom K, Bobrova I, Bissessar E. Eribis peptide 94 reduces infarct size in rat hearts via activation of centrally located mu opioid receptors. J Cardiovasc Pharmacol. 2012;59:194–197. doi: 10.1097/FJC.0b013e318241e8c7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Caimmi PP, Molinari C, Uberti F, Micalizzi E, Valente G, Mary DA, Vacca G, Grossini E. Intracoronary levosimendan prevents myocardial ischemic damages and activates survival signaling through ATP-sensitive potassium channel and nitric oxide. Eur J Cardiothorac Surg. 2011;39:e59–67. doi: 10.1016/j.ejcts.2010.11.044. [DOI] [PubMed] [Google Scholar]
  • 19.Peart JN, Hoe LE, Gross GJ, Headrick JP. Sustained ligand-activated preconditioning via δ-opioid receptors. J Pharmacol Exp Ther. 2011;336:274–81. doi: 10.1124/jpet.110.172593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Bencsik P, Kupai K, Giricz Z, Görbe A, Pipis J, Murlasits Z, Kocsis GF, Varga-Orvos Z, Puskás LG, Csonka C, Csont T, Ferdinandy P. Role of iNOS and peroxynitrite-matrix metalloproteinase-2 signaling in myocardial late preconditioning in rats. Am J Physiol Heart Circ Physiol. 2010;299:H512–8. doi: 10.1152/ajpheart.00052.2010. [DOI] [PubMed] [Google Scholar]

RESOURCES