European Journal of Pharmacology 737 (2014) 177–184
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Quaternary ammonium salt of U50488H, a new κ-opioid receptor agonist, protects rat heart against ischemia/reperfusion injury Qianmei Wang a,b,1, Yang Sun c,1, Juan Li b,1, Wenjuan Xing b, Shumiao Zhang b, Xiaoming Gu b, Na Feng b, Lei Zhao b, Rong Fan b, Yuemin Wang b, Wen Yin a,n, Jianming Pei b,n a
Department of Emergency, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, PR China Department of Physiology, National Key Discipline of Cell Biology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, PR China c Department of Geriatrics, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 19 October 2013 Received in revised form 4 May 2014 Accepted 7 May 2014 Available online 20 May 2014
To keep U50488H from going through the blood–brain barrier, U50488H, a selective κ-opioid receptor agonist, was structurally transformed into its quaternary ammonium salt (Q-U50488H). The effect of Q-U50488H on ischemic/reperfused myocardium and its underlying mechanisms were also investigated. U50488H was transformed into Q-U50488H, which was identified with mass spectrometry. The existences of U50488H and Q-U50488H in serum and brain tissue fluid were determined by high performance liquid chromatography (HPLC). SD rats’ hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion in vivo. After reperfusion, myocardial enzymes and free radicals in serum, area of myocardial infarction, cardiomyocyte apoptosis and the expression of Kir6.2 in rats’ myocardium were determined. Molecular weight and solubility of Q-U50488H were higher than those of U50488H. Result of HPLC showed that Q-U50488H existed in serum but not in brain tissue after Q-U50488H intravenous injection. However, U50488H was detected in both serum and brain tissue. Compared with the I/R group, treatment with Q-U50488H significantly attenuated the activity of LDH, CK, AST/GOT and content of MDA in serum, upregulated the activity of SOD, and increased the expression of myocardial Kir6.2. It also reduced myocardial infarct size and cardiomyocyte apoptosis induced by I/R. Moreover, pretreatment with Nor-BNI (a selective κ-opioid receptor antagonist), 5-HD and glibenclamide (KATP antagonists) abolished the effects of Q-U50488H. It is suggested Q-U50488H, a new compound of κ-opioid receptor agonist, which is not able to pass the blood–brain barrier, elicits a protective effect against myocardial ischemia/reperfusion injury. The cardioprotective effect of Q-U50488H is associated with the opening of KATP. & 2014 Elsevier B.V. All rights reserved.
Keywords: Quaternary ammonium of U50488H (Q-U50488H) κ-opioid receptor KATP Ischemia/reperfusion (I/R) injury Myocardial infarction Apoptosis Chemical compounds studied in this article: Quaternary ammonium of U50488H (PubChem CID: Q-U50488H)
1. Introduction Ischemic heart disease is a tremendous threaten to people's health. With the widespread application of internal treatment and surgical interventions such as intra-coronary artery thrombolysis and coronary artery bypass graft surgery, the following emergence of reperfusion injury of cardiomyocytes has aroused wide concern from cardiologists increasingly. It is reported that opioid receptors widely exist in human tissues and organs such as heart, brain, etc. In terms of heart, κ-opioid
n
Corresponding authors. E-mail addresses: [email protected] (J. Pei), [email protected] (W. Yin). 1 These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.ejphar.2014.05.006 0014-2999/& 2014 Elsevier B.V. All rights reserved.
receptor, a predominant isoform of opioid receptor, is of great importance in the regulation of myocardial ischemia/reperfusion (I/R) (Kim et al., 2011; Schultz and Gross 2001; ValtchanovaMatchouganska and Ojewole, 2003). Previous studies have suggested that U50488H, an exogenous selective κ-opioid receptor agonist (Peart et al., 2004; ValtchanovaMatchouganska and Ojewole, 2002), significantly reduces infarct size, cardiomyocyte apoptosis and the incidence of arrhythmia (Chun et al., 2010; Rong et al., 2009; Tong et al., 2011). U50488H can effectively protect the heart against I/R injury; however, taking into account the relatively low molecular weight and poor solubility, U50488H may go through the blood–brain barrier, and its probable addiction imposes restrictions on its clinical application. To keep it from permeating through the blood–brain barrier, U50488H was transformed into its quaternary ammonium salt
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(Q-U50488H). Is Q-U50488H still a kind of κ-opioid receptor agonist? Can it still protect rat heart from I/R injury? These questions were addressed in the present study.
2. Materials and methods 2.1. Reagents U50488H (trans-3,4-dichloro-N-methyl- [2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide), nor-BNI (nor-Binaltorphimine) were purchased from Tocris Bioscience (Ellisville, MO). 5-HD, Glibenclamide, Evan's blue and TTC (2,3,5-triphenyl-tetrazolium chloride) were purchased from Sigma Chemical (St. Louis, MO). HPLC grade methanol was purchased from Concord Technology (Merck, Germany). Creatine kinase (CK), lactate dehydrogenase (LDH), glutamic–oxaloacetic transaminase (AST/GOT), malondialdehyde (MDA) and Superoxide Dismutase (SOD) assay kits were purchased from Jiancheng Reagent Company (Nanjing, China). Terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) using an in situ cell death detection kit was purchased from Roche Molecular Biochemicals (Mannheim, Germany). 2.2. Animals Adult male Sprague-Dawley (SD) rats (250730 g) used for the present study were obtained from the laboratory animal center of the Fourth Military Medical University. All procedures were approved by the Fourth Military Medical University Animal Ethics Committee. 2.3. Structural transform and detection of U50488H Under the condition of polar solvent and catalyst, U50488H, a selective κ-opioid receptor agonist, reacted with CH3I to form Q-U50488H (Fig. 1). Firstly, U50488H was transformed to U50488 by a method of acid-base neutralization reaction.
Secondly, in the condition with N2, U50488 reacted with CH3I to form Q-U50488H, a new compound which was identified to be a quaternary ammonium salt of U50488H. A Quattro Premier XE triple quadruple mass spectrometer (Waters) and Mass Lynx 4.1 SCN 714 (Waters) software were used to perform the detection of Q-U50488H.
Fig. 1. Chemical structure of U50488H (A), U50488 (B) and Q-U50488H (C).
25 1C and delivered at a flow rate of 1.0 ml/min for chromatographic separation. The detection wavelength was 290 nm (Singh et al., 2011). 12 SD rats were anesthetized with pentobarbital sodium (45 mg/ kg, i.p.). Supplemental doses of pentobarbital sodium were given when needed in order to maintain uniform anesthesia. An intratracheal cannula was inserted and the animals were placed in intermittent positive pressure ventilation with room air. The left jugular vein and the right jugular artery were cannulated to administer drugs and take blood samples respectively. After the preparation, 6 rats were administrated with U50488H (2 mg/kg), and the other 6 ones with Q-U50488H (10 mg/kg). It should be mentioned that in order to investigate whether U50488H and Q-U50488H were able to go through the blood–brain barrier or not, both amounts of administration were larger than those used in the subsequent animal experiment. And then, 5 min after the administration, blood and brains were removed. In order to know that status during the entire 150 min period, we have performed an additional experiment. Brains were also harvested following 30 min of ischemia and 120 min of reperfusion. Brain tissue fluid was prepared by adding purified water to brain tissue at ratio of 2:1 (ml:g), and then homogenized and centrifuged at 12,000 rpm for 120 min. Serum was harvested by centrifuging the blood at 3000 rpm for 20 min. Blank, drug-free serum and brain tissue fluid samples were collected from adult male cleaning SD rats as described above as well. Blank serum and brain tissue fluid samples, levels of U50488H and Q-U50488H in serum and brain tissue fluid samples were measured by HPLC (Singh et al., 2011; Fang et al., 2013). 2.4. I/R experimental protocols
All standards and samples were analyzed by a Shimadzu HPLC system (Kyoto, Japan) equipped with a LC-20 AT VP pump, SIL-20 AC injector and a SPD-20A UV-VTS detector. Chromatographic separation was performed on Inertsil ODS-3 column (4.6 250 mm, 5.0 μm). The system was run on isocratic mode with mobile phase consisting of methanol (Merck, Germany) and purified water in ratio of 30:70, v/v. Mobile phase was degassed ultrasonically for 15 min at
SD rats were anesthetized with pentobarbital sodium (45 mg/kg, i.p.). An intratracheal cannula was inserted and the animals were placed in intermittent positive pressure ventilation with room air. The left jugular vein was cannulated to administer drugs. The chest was opened through a left intercostals thoracotomy (between the fourth and fifth costa), and the heart was exposed by removing pericardium. Myocardial ischemia was produced by exteriorizing the
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heart with a left thoracic incision followed by a slipknot (5–0 silk) around the left anterior descending coronary artery (LAD). Significant ECG changes including widening of the QRS complex and elevation of ST segment, and color changes of the area at risk were considered as successful coronary occlusion. After 30 min of ischemia, the slipknot was released and the animal received 120 min of reperfusion. After the experiment, blood and hearts were harvested. SD rats were randomly assigned to one of the following five experimental groups. There were 12 rats in each group: (1) Sham group: silk was drilled underneath the LAD but the LAD was not ligated; (2) I/R group: LAD was ligated for 30 min and then allowed 120 min of reperfusion with receiving vehicle (0.9% NaCl, i.v.); (3) U50488HþI/R group: U50488H (1.5 mg/kg, i.v.), a selective κ-opioid receptor agonist, was administered just 10 min before ischemia (Zhang et al., 2010); (4) Q-U50488HþI/R group: Q-U50488H (5 mg/kg, i.v.) was administered just 10 min before ischemia; (5) Nor-BNIþ Q-U50488Hþ I/R group: nor-BNI (2 mg/kg, i.v.), a selective κ-opioid receptor antagonist, was administered 10 min before Q-U50488H. Other procedures were the same as the Q-U50488HþI/R group. All the following determinations were performed in a double-blind manner. 2.5. Detection of serum CK, LDH, AST/GOT activity, MDA content and SOD activity At the end of reperfusion, blood samples were collected from the heart chambers to measure the myocardial enzyme leakage including CK, LDH, AST/GOT and free radical such as MDA. Blood samples were centrifuged and then obtained serums were analyzed by using the CK, LDH, AST/GOT and MDA assay kits (Nanjing Jiancheng Bioengineering Institute) according to the procedures. After reperfusion, blood samples were taken from the carotid artery and the serum was collected by centrifugation. SOD assay kit was used (Nanjing Jiancheng Bioengineering Institute) to detect serum SOD activity according to the procedures. 2.6. Determination of myocardial infarct size The area of the infarct size was measured using Evan's Blue-TTC (2,3,5-triphenyltetrazolium chloride, TTC) double-staining technique. In brief, LAD was reoccluded (except the Sham group) and 1 ml of a 20 g/L Evan's blue solution was administered into the heart via a catheter at the end of 120 min reperfusion. The heart was then removed and immediately frozen at 20 1C. The heart was cut into five transverse slices from the apex with a thickness of about 2 mm. Heart slices were incubated with 10 g/L solution of TTC in phosphate buffer (pH 7.4) at 37 1C for 15 min and then fixed overnight in 4% paraformaldehyde solution. The images of heart slices were captured by a high-resolution CCD camera and the area at risk (AAR) and infarct size (IS) were digitally measured using OPTIMAS software. The heart consisted of three parts as follows. Non-I/R myocardium which is stained blue by Evan's blue, AAR which is not stained by Evan's blue, but by TTC; and infarct myocardium which is stained neither by Evan's blue nor by TTC. Myocardial infarct sizes (IS/AAR %) were then calculated. 2.7. Evaluation of myocardial apoptosis At the end of reperfusion, frozen slices were obtained instantly. Then myocardial apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) using an in situ cell death detection kit (Roche). A double-staining technique was used, in brief, TUNEL staining for apoptotic cell nuclei and 40 ,6diamino-2-phenylindole (DAPI) staining for all myocardial cell nuclei. The index of apoptosis was expressed by the number of positively
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stained apoptotic cardiomyocytes/the total number of cardiomyocytes counted 100%. 2.8. Determination of myocardial Kir6.2 expression At the end of reperfusion, rats’ pericardial tissues were harvested and washed with ice-cold normal saline and homogenized on ice in phosphate buffered saline. After centrifuged at 3000 rpm for 5 min, the supernatant was discarded. The homogenate was incubated in lysis buffer including protease inhibitor, PMSF, etc. for 20 min with vortex-mixing at 4 1C, filtered and centrifuged at 12,000g for 20 min. The resulting supernatants were the proteins, of which the concentrations were determined by Bradford's method, with bovine serum albumin serving as a calibrator. Lysate proteins were mixed with loading buffer with the ratio of 1:4 and denatured at 100 1C for 9 min. Then 30 to 40 μg proteins from denatured total extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with a molecular weight marker, and transferred to a NC membrane. After washing in Tris-buffered saline containing 0.01% Tween-20 (TBST) for 5 min, saturating with 30 g/L bovine serum albumin (BSA) in TBST for 1 h at room temperature, the membranes were incubated with the following primary antibodies overnight at 4 1C: polyclonal rabbit GAPDH (1:10,000, Sigma-Aldrich, St. Louis, MO, USA) or polyclonal rabbit anti-Kir6.2 (1:1000, Santa Cruz Biotechnology, St. Cruz, CA, USA) diluted in TBST. The membranes were then washed six times in TBST for 5 min and incubated with the following horseradish peroxidase HRP-labeled secondary antibodies, goat anti-mouse (1:4000, Santa Cruz Biotechnology, St. Cruz, CA, USA), for 1 h at room temperature. After washing six times in TBST for 5 min and incubating with ECL-Plus, the pixel intensities of the immunoreactive bands were quantified using the percentage adjusted volume feature of Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Data are expressed as the ratio of the band intensity of the protein of interest to the loading control protein band (GAPDH). 2.9. Statistical analysis Data were presented as means 7S.E.M. The significance of differences among groups were evaluated by Dunnett's t-test for multiple comparisons preceded by one-way analysis of variance (ANOVA). A value of P o0.05 was considered as statistically significant.
3. Results 3.1. Chromatography of U50488H and Q-U50488H in serum and brain tissue The quaternary ammonium salt of U50488H, Q-U50488H, was a kind of faint yellow powder. Q-U50488H was proved to be C20H29Cl2IN2O by mass spectrometry (Fig. 2). Q-U50488H, with the molecular weight of 511.2695, had a solubility of no less than 5 mg/ml; however, U50488H, with the molecular weight of 405.7895, had a solubility of about 2 mg/ml. This fact indicated that Q-U50488H was a new compound with increased molecular weight and solubility than U50488H. Typical chromatograms of physiological saline, standards of U50488H and Q-U50488H (dissolved in physiological saline) are shown in Fig. 3A. The peak between 6 min and 8 min indicated U50488H and Q-U50488H respectively. Chromatograms of serum samples are shown in Fig. 3B. Compared to the blank serum, U50488H and Q-U50488H could be detected in serum after intravenous injection.
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Fig. 2. Mass spectrometry of Q-U50488H. Species in the new compound was determined by mass spectrometry which was identified to be Q-U50488H, and the peak fit well with m/z of Q-U50488H.
Chromatograms of brain tissue fluid samples are shown in Fig. 3C. Compared to the blank brain tissue fluid, U50488H could be detected in brain tissue fluid. However, Q-U50488H was not detected. In addition, as shown in Fig. 3D, chromatograms of brain samples were harvested at 0 min, 5 min and 150 min after the administration of Q-U50488H. It was shown that Q-U50488H could be detected neither at 5 min nor at 150 min in the brain tissue after intravenous injection of Q-U50488H. This result further strengthened that Q-U50488H was not able to go through the blood–brain barrier. Taken together, U50488H was detected in both serum and brain tissue fluid of the rats after intravenous injection by the HPLC method. However, Q-U50488H was detected only in the serum and not in the brain tissue. 3.2. Q-U50488H lowered the activity of serum CK, LDH, AST/GOT and the level of MDA in rats subjected to I/R As shown in Fig. 4, activity of intracellular myocardial enzymes such as CK, LDH, AST/GOT and level of MDA in serum increased significantly following I/R. Administration of Q-U50488H prior to I/R significantly reduced levels of the myocardial enzymes and MDA. No difference was found in these parameters when compared with the U50488HþI/R group. The effects of Q-U50488H mentioned above were blocked by Nor-BNI, a selective κ-opioid receptor antagonist. 3.3. Q-U50488H increased the activity of serum SOD in rats subjected to I/R
(Po0.01), and no difference was found when compared with rats in group administered with U50488H. The effect of Q-U50488H was abolished by Nor-BNI. In order to investigate whether KATP plays a role in Q-U50488Hinduced myocardial protection, KATP blockers such as 5-HD (10 mg/ kg, i.v.) and glibenclamide (1 mg/kg, i.p.) were administrated 10 min before ischemia (Abdallah et al., 2011; Kai et al., 2013; Taliyan et al., 2010). As shown in Fig. 6, the effect of Q-U50488H was abolished by 5-HD and glibenclamide. 3.5. Q-U50488H reduced myocardial apoptosis in rats subjected to I/R A very low level of TUNEL-positive staining was detected in the cardiomyocytes of the Sham group. A great number of TUNELpositive cells were observed in myocardial tissue from rats subjected to I/R. And the cardiomyocyte apoptosis was dramatically alleviated by the administration of Q-U50488H prior to I/R (Po0.01). No significantly statistical difference was found when compared with the U50488Hþ I/R group. The effect of Q-U5088H was blocked by Nor-BNI (Fig. 7). In order to investigate whether KATP plays a role in Q-U50488Hinduced myocardial protection, KATP blockers such as 5-HD and glibenclamide were administrated 10 min before ischemia. As shown in Fig. 7, the effect of Q-U50488H was abolished by 5-HD and glibenclamide. 3.6. Q-U50488H increased the expression of Kir6.2 in the ischemia area of the heart subjected to I/R
I/R induced a low activity of SOD in serum, whereas Q-U50488H increased the activity of SOD in serum significantly when administrated prior to I/R. No difference was found when compared with the U50488Hþ I/R group (Fig. 5). The effect of Q-U50488H mentioned above was blocked by Nor-BNI.
As shown in Fig. 8, the expression of Kir6.2 was increased by the administration Q-U50488H prior to I/R (P o0.01), and no difference was found when compared with the U50488Hþ I/R group. The effect of Q-U50488H was blocked by Nor-BNI.
3.4. Q-U50488H reduced myocardial infarction area in rats subjected to I/R
4. Discussion
The I/R injury of cardiomyocytes caused a significant myocardial infarction (Fig. 6). Compared with the I/R group, the Q-U50488Hþ I/R group showed significantly reduced myocardial infarct size
In the present study, U50488H, a selective κ-opioid receptor agonist, for the first time, was transformed into Q-U50488H by chemical methods. Q-U50488H was a faint yellow powder with
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Fig. 3. Comparison of chromatography of U50488H and Q-U50488H in rat serum and brain tissue. A: Chromatography of physiological saline (I), standards of U50488H (II) and Q-U50488H (III) (dissolved in physiological saline). B: Chromatography of blank serum (I), U50488H (II) and Q-U50488H (III) in serum. C: Chromatography of blank brain tissue fluid (I), U50488H (II) and Q-U50488H (III) in brain tissue fluid samples. D: Chromatography of blank brain tissue fluid (I), Q-U50488H 5 min (II) and 150 min after administration (III) in brain tissue fluid samples.
remarkably increased solubility and molecular weight than U50488H. Results of HPLC indicated that Q-U50488H was incapable of going through the blood–brain barrier. U50488H's ability to go through the blood–brain barrier attributes to its poor solubility and low molecular weight, and may lead to probable addiction, which imposes restrictions on its clinical application. To keep U50488H from penetrating into brain tissue, it is necessary to improve its solubility in physiological saline and molecular weight. In the preliminary experiment, we tried to make polyethyleneglycol (PEG)-modification of U50488H. However, U50488H would separate from the compound with PEG soon after intravenous injection. In our present study, we obtained Q-U50488H by structurally transforming U50488H into its quaternary ammonium salt. The latter was stable and not prone to degrade relatively. In addition, Q-U50488H detected by HPLC was not able to go through blood–brain barrier, whereas U50488H was able to penetrate into brain tissue.
The present study also provided the evidence that like U50488H, Q-U50488H could significantly decrease the level of myocardial enzymes such as CK, LDH, AST/GOT, and free radical MDA generation, and enhance SOD activity as well. And these effects of Q-U50488H were blocked by a selective κ-opioid receptor antagonist, Nor-BNI, indicating that Q-U50488H, a new κ-opioid receptor agonist, elicited protective effect on ischemic/ reperfused myocardium. In order to further investigate underlying mechanisms, KATP blockers such as 5-HD and glibenclamide were administered just 10 min before Q-U50488H. We observed that Q-U50488H could significantly decrease of the infarct size and cardiomyocyte apoptosis. Moreover, these cardioprotective effects of Q-U50488H were abolished by KATP blockers. There were a lot of studies that suggest the κ-opioid receptor reduced infarct size and apoptosis through the opening of KATP channels (Chen et al., 2003; Lishmanov et al., 2007; Wang et al., 2001). Our study showed that
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Fig. 4. Comparison of the activity of serum CK, LDH, AST/GOT and the level of MDA in rat subjected to I/R. A: Comparison of the activity of CK in serum. B: Comparison of the activity of LDH in serum. C: Comparison of the activity of AST/GOT in serum. D: Comparison of the level of MDA in serum. n¼ 6 in each group. Nor-BNI: nor-Binaltorphimine. All values are presented as means 7S.E.M. nnPo 0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488H þI/R group.
Fig. 5. Comparison of the activity of SOD in serum. n¼6 in each group. Nor-BNI: nor-Binaltorphimine. All values are presented as means 7 S.E.M. nnP o0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488H þI/R group.
the cardioprotective effect of Q-U50488H mentioned above was abolished by 5-HD and glibenclamide. This fact holds potential promises that the effect of Q-U50488H against myocardial I/R injury may be associated with the opening of KATP. ATP-sensitive potassium channel (KATP), a specific type of potassium channel, exists in heart and vascular smooth muscle.
Fig. 6. Comparison of myocardial infarct sizes (IS/AAR %). n¼ 6 in each group. NorBNI: nor-Binaltorphimine; 5-HD: 5-hydroxydecanoate; Gli: glibenclamide. All values are presented as means 7 S.E.M. nnP o0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488H þ I/R group.
KATP is named for the reason that it is inhibited by intracellular ATP and adenine nucleotides. The opening of KATP dilates smooth muscle effectively, lowers blood pressure and produces specific
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Fig. 8. Comparison of the expression of Kir6.2 in precordial region. A: Representative photographs of Kir6.2 expression were determined by Western blots. B: Expressions of Kir6.2 were determined by Quantity One software. n ¼6 in each group. Nor-BNI: nor-Binaltorphimine. All values are presented as means 7 S.E.M. nn Po 0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488H þ I/R group.
Fig. 7. Comparison of myocardial apoptosis in rat heart subjected to I/R. A: Myocardial apoptosis was determined by TUNEL staining. TUNEL staining (green) indicates apoptotic nuclei; DAPI counterstaining (blue) indicates total nuclei. B: TUNEL-positive nuclei were expressed as a percentage of the total number of nuclei automatically counted and calculated. n¼ 6 in each group. NorBNI: nor-Binaltorphimine; 5-HD: 5-hydroxydecanoate; Gli: glibenclamide. All values are presented as means 7 S.E.M. nnPo 0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488Hþ I/R group.
cardiovascular protection; it has become the typical target of drugs applied in the research of protecting ischemic heart. U50488H, a selective κ-opioid receptor agonist, activates KATP and plays its protective role in the ischemia preconditioning (IPC) of heart (Eleftherianos et al., 2011; Wu et al., 2011). According to Schultz et al. (1998) and Schultz and Gross (2001), κ-opioid-induced cardioprotection and IPC seem to share a common pathway, KATP channel, and possibly the mitochondrial KATP channel appears to be involved in the beneficial effects observed. Activation of KATP channels during ischemia is an important adaptive mechanism for protecting the myocardium when blood flow to the tissue is compromised (Cole et al., 1991; Lee et al., 2008).In the present study, it was found that 5-HD and glibenclamide, both KATP channel blockers, were able to abolish the beneficial effects of Q-U50488H. However, the intracellular signaling pathways related to the mechanisms of opioid-receptor stimulation and the possible end-effector, perhaps the mitochondrial KATP channel, are less well understood. Several mechanisms have been proposed to explain the process of involvement of the KATP up-regulation or KATP opening in its cardioprotective effect. The major one involved is that activation of KATP channels can prevent calcium entry via L-type voltage-gated Ca2 þ channels and alleviate calcium overload induced by I/R (Chun et al., 2010; Shi et al., 2013). However, it is still unclear how this observation relates to κ-opioid receptors and cardioprotection
mediated by κ-opioid receptors. Further study is needed to investigate the underlying mechanism. Q-U50488H, a new κ-opioid receptor agonist, is incapable of going through the blood–brain barrier. It protects the heart from I/R injury, which can be used in scientific research. In addition, it is a potential drug, which may be employed in the prevention and treatment of ischemic heart disease clinically. Q-U50488H, therefore, is of great scientific research value and clinical application prospect.
5. Conclusions We have demonstrated that Q-U50488H is a new κ-opioid receptor agonist, which is not able to pass the blood–brain barrier. Importantly, Q-U50488H has a protective effect against myocardial I/R injury. The cardioprotective effect of Q-U50488H is associated with the opening of KATP.
Acknowledgments This work was supported by grants from National New Drug Research Major Project (2009ZX09301–009-BD11), and grants from the National Natural Science Foundation of China (Nos. 81270402, 31200875, 30971060, 30900535, and 81070063) and grants from Shaanxi Province of China (Nos. 2013K132-02-06, 2011JM4001).
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Chun, K.J., Jang, Y.H., Kim, J.H., Kim, J., Park, Y.H., 2010. Cardioprotection by kappaopioid receptor agonist U50488H is mediated by opioidergic regulation but not by calcium current modulation. Korean J. Anesthesiol. 58, 162–168. Cole, W.C., McPherson, C.D., Sontag, D., 1991. ATP-regulated K þ channels protect the myocardium against ischemia/reperfusion damage. Circ. Res. 69, 571–581. Eleftherianos, I., Won, S., Chtarbanova, S., Squiban, B., Ocorr, K., Bodmer, R., Beutler, B., Hoffmann, J.A., Imler, J.L., 2011. ATP-sensitive potassium channel (K(ATP))dependent regulation of cardiotropic viral infections. Proc. Natl. Acad. Sci. USA 108, 12024–12029. Fang, W., Lv, P., Geng, X., Shang, E., Yang, Q., Sha, L., Li, Y., 2013. Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation. Neurochem. Int. 62, 23–30. Kai, Z., Yongbo, W., Lin, Z., Jie, G., Daoqun, J., Zhiqiang, C., 2013. Exendin-4 attenuates ischemia-induced ventricular arrhythmias in rats. Cardiol. J. 20, 29–33. Kim, J.H., Jang, Y.H., Chun, K.J., Kim, J., Park, Y.H., Kim, J.S., Kim, J.M., Lee, M.Y., 2011. Kappa-opioid receptor activation during reperfusion limits myocardial infarction via ERK1/2 activation in isolated rat hearts. Korean J. Anesthesiol. 60, 351–356. Lee, T.M., Lin, M.S., Chang, N.C., 2008. Effect of ATP-sensitive potassium channel agonists on ventricular remodeling in healed rat infarcts. J. Am. Coll. Cardiol. 51, 1309–1318. Lishmanov, A.Y., Maslov, L.N., Lasukova, T.V., Crawford, D., Wong, T.M., 2007. Activation of kappa-opioid receptor as a method for prevention of ischemic and reperfusion arrhythmias: role of protein kinase C and K(ATP) channels. Bull. Exp. Biol. Med. 143, 187–190. Peart, J.N., Gross, E.R., Gross, G.J., 2004. Effect of exogenous kappa-opioid receptor activation in rat model of myocardial infarction. J. Cardiovasc. Pharmacol. 43, 410–415. Rong, F., Peng, Z., Ye, M.X., Zhang, Q.Y., Zhao, Y., Zhang, S.M., Guo, H.T., Hui, B., Wang, Y.M., Liang, C., Gu, C.H., Tao, C., Cui, Q., Yu, S.Q., Yi, D.H., Pei, J.M., 2009. Myocardial apoptosis and infarction after ischemia/reperfusion are attenuated by kappa-opioid receptor agonist. Arch. Med. Res. 40, 227–234. Schultz, J.E., Gross, G.J., 2001. Opioids and cardioprotection. Pharmacol. Ther. 89, 123–137.
Schultz, J.J., Hsu, A.K., Nagase, H., Gross, G.J., 1998. TAN-67, a δ1-opioid receptor agonist, reduced infarct size via activation of Gi/o proteins and KATP channels. Am. J. Physiol. 274, H909–H914. Shi, Q.X., Zhang, L.J., Yao, Y., Zhang, Q.Y., Wang, W., Li, J., Shang, Y.L., Bi, H., Zhang, S. M., Guo, H.T., Wang, Y.M., Yu, S.Q., Yi, D.H., Bueno, F.R., Kaye, A.D., Pei, J.M., 2013. κ-opioid receptor activation prevents against arrhythmias by preserving Cx43 protein via alleviation of intracellular calcium. Am. J. Ther. 20, 493–501. Singh, S.P., Wahajuddin, Tewari, D., Patel, K., Jain, G.K., 2011. Permeability determination and pharmacokinetic study of nobiletin in rat plasma and brain by validated high-performance liquid chromatography method. Fitoterapia 82, 1206–1214. Taliyan, R., Singh, M., Sharma, P.L., Yadav, H.N., Sidhu, K.S., 2010. Possible involvement of α1-adrenergic receptor and K(ATP) channels in cardioprotective effect of remote aortic preconditioning in isolated rat heart. J. Cardiovasc. Dis. Res. 1, 145–151. Tong, G., Sun, Z.C., Wei, X.F., Gu, C., Kaye, A.D., Wang, Y.M., Li, J., Zhang, Q.Y., Guo, H. T., Yu, S.Q., Yi, D.H., Pei, J.M., 2011. U50,488H postconditioning reduces apoptosis after myocardial ischemia and reperfusion. Life Sci. 88, 31–38. Valtchanova-Matchouganska, A., Ojewole, J.A., 2002. Involvement of opioid delta (delta)- and kappa (kappa)-receptors in ischemic preconditioning in a rat model of myocardial infarction. Methods Find. Exp. Clin. Pharmacol. 24, 139–144. Valtchanova-Matchouganska, A., Ojewole, J.A., 2003. Mechanisms of opioid delta (delta) and kappa (kappa) receptors’ cardioprotection in ischaemic preconditioning in a rat model of myocardial infarction. Cardiovasc. J. S. Afr. 14, 73–80. Wang, G.Y., Wu, S., Pei, J.M., Yu, X.C., Wong, T.M., 2001. κ- but not δ-opioid receptors mediate effects of ischemic preconditioning on both infarct and arrhythmia in rats. Am. J. Physiol. Heart Circ. Physiol. 280, H384–H391. Wu, Y.N., Yu, H., Zhu, X.H., Yuan, H.J., Kang, Y., Jiao, J.J., Gao, W.Z., Liu, Y.X., Lou, J.S., 2011. Noninvasive delayed limb ischemic preconditioning attenuates myocardial ischemia-reperfusion injury in rats by a mitochondrial K(ATP) channeldependent mechanism. Physiol. Res. 60, 271–279. Zhang, Q.Y., Wang, W., Shi, Q.X., Li, Y.L., Huang, J.H., Yao, Y., Li, J., Zhang, S.M., Fan, R., Zhou, J.J., Guo, H.T., Wang, Y.M., Yin, W., Pei, J.M., 2010. Antiarrhythmic effect mediated by κ-opioid receptor is associated with Cx43 stabilization. Crit. Care. Med. 38, 2365–2376.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Quaternary ammonium salt of U50488H, a new κ-opioid receptor agonist, protects rat heart against ischemia/reperfusion injury Qianmei Wang a,b,1, Yang Sun c,1, Juan Li b,1, Wenjuan Xing b, Shumiao Zhang b, Xiaoming Gu b, Na Feng b, Lei Zhao b, Rong Fan b, Yuemin Wang b, Wen Yin a,n, Jianming Pei b,n a
Department of Emergency, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, PR China Department of Physiology, National Key Discipline of Cell Biology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, PR China c Department of Geriatrics, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 19 October 2013 Received in revised form 4 May 2014 Accepted 7 May 2014 Available online 20 May 2014
To keep U50488H from going through the blood–brain barrier, U50488H, a selective κ-opioid receptor agonist, was structurally transformed into its quaternary ammonium salt (Q-U50488H). The effect of Q-U50488H on ischemic/reperfused myocardium and its underlying mechanisms were also investigated. U50488H was transformed into Q-U50488H, which was identified with mass spectrometry. The existences of U50488H and Q-U50488H in serum and brain tissue fluid were determined by high performance liquid chromatography (HPLC). SD rats’ hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion in vivo. After reperfusion, myocardial enzymes and free radicals in serum, area of myocardial infarction, cardiomyocyte apoptosis and the expression of Kir6.2 in rats’ myocardium were determined. Molecular weight and solubility of Q-U50488H were higher than those of U50488H. Result of HPLC showed that Q-U50488H existed in serum but not in brain tissue after Q-U50488H intravenous injection. However, U50488H was detected in both serum and brain tissue. Compared with the I/R group, treatment with Q-U50488H significantly attenuated the activity of LDH, CK, AST/GOT and content of MDA in serum, upregulated the activity of SOD, and increased the expression of myocardial Kir6.2. It also reduced myocardial infarct size and cardiomyocyte apoptosis induced by I/R. Moreover, pretreatment with Nor-BNI (a selective κ-opioid receptor antagonist), 5-HD and glibenclamide (KATP antagonists) abolished the effects of Q-U50488H. It is suggested Q-U50488H, a new compound of κ-opioid receptor agonist, which is not able to pass the blood–brain barrier, elicits a protective effect against myocardial ischemia/reperfusion injury. The cardioprotective effect of Q-U50488H is associated with the opening of KATP. & 2014 Elsevier B.V. All rights reserved.
Keywords: Quaternary ammonium of U50488H (Q-U50488H) κ-opioid receptor KATP Ischemia/reperfusion (I/R) injury Myocardial infarction Apoptosis Chemical compounds studied in this article: Quaternary ammonium of U50488H (PubChem CID: Q-U50488H)
1. Introduction Ischemic heart disease is a tremendous threaten to people's health. With the widespread application of internal treatment and surgical interventions such as intra-coronary artery thrombolysis and coronary artery bypass graft surgery, the following emergence of reperfusion injury of cardiomyocytes has aroused wide concern from cardiologists increasingly. It is reported that opioid receptors widely exist in human tissues and organs such as heart, brain, etc. In terms of heart, κ-opioid
n
Corresponding authors. E-mail addresses: [email protected] (J. Pei), [email protected] (W. Yin). 1 These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.ejphar.2014.05.006 0014-2999/& 2014 Elsevier B.V. All rights reserved.
receptor, a predominant isoform of opioid receptor, is of great importance in the regulation of myocardial ischemia/reperfusion (I/R) (Kim et al., 2011; Schultz and Gross 2001; ValtchanovaMatchouganska and Ojewole, 2003). Previous studies have suggested that U50488H, an exogenous selective κ-opioid receptor agonist (Peart et al., 2004; ValtchanovaMatchouganska and Ojewole, 2002), significantly reduces infarct size, cardiomyocyte apoptosis and the incidence of arrhythmia (Chun et al., 2010; Rong et al., 2009; Tong et al., 2011). U50488H can effectively protect the heart against I/R injury; however, taking into account the relatively low molecular weight and poor solubility, U50488H may go through the blood–brain barrier, and its probable addiction imposes restrictions on its clinical application. To keep it from permeating through the blood–brain barrier, U50488H was transformed into its quaternary ammonium salt
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(Q-U50488H). Is Q-U50488H still a kind of κ-opioid receptor agonist? Can it still protect rat heart from I/R injury? These questions were addressed in the present study.
2. Materials and methods 2.1. Reagents U50488H (trans-3,4-dichloro-N-methyl- [2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide), nor-BNI (nor-Binaltorphimine) were purchased from Tocris Bioscience (Ellisville, MO). 5-HD, Glibenclamide, Evan's blue and TTC (2,3,5-triphenyl-tetrazolium chloride) were purchased from Sigma Chemical (St. Louis, MO). HPLC grade methanol was purchased from Concord Technology (Merck, Germany). Creatine kinase (CK), lactate dehydrogenase (LDH), glutamic–oxaloacetic transaminase (AST/GOT), malondialdehyde (MDA) and Superoxide Dismutase (SOD) assay kits were purchased from Jiancheng Reagent Company (Nanjing, China). Terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) using an in situ cell death detection kit was purchased from Roche Molecular Biochemicals (Mannheim, Germany). 2.2. Animals Adult male Sprague-Dawley (SD) rats (250730 g) used for the present study were obtained from the laboratory animal center of the Fourth Military Medical University. All procedures were approved by the Fourth Military Medical University Animal Ethics Committee. 2.3. Structural transform and detection of U50488H Under the condition of polar solvent and catalyst, U50488H, a selective κ-opioid receptor agonist, reacted with CH3I to form Q-U50488H (Fig. 1). Firstly, U50488H was transformed to U50488 by a method of acid-base neutralization reaction.
Secondly, in the condition with N2, U50488 reacted with CH3I to form Q-U50488H, a new compound which was identified to be a quaternary ammonium salt of U50488H. A Quattro Premier XE triple quadruple mass spectrometer (Waters) and Mass Lynx 4.1 SCN 714 (Waters) software were used to perform the detection of Q-U50488H.
Fig. 1. Chemical structure of U50488H (A), U50488 (B) and Q-U50488H (C).
25 1C and delivered at a flow rate of 1.0 ml/min for chromatographic separation. The detection wavelength was 290 nm (Singh et al., 2011). 12 SD rats were anesthetized with pentobarbital sodium (45 mg/ kg, i.p.). Supplemental doses of pentobarbital sodium were given when needed in order to maintain uniform anesthesia. An intratracheal cannula was inserted and the animals were placed in intermittent positive pressure ventilation with room air. The left jugular vein and the right jugular artery were cannulated to administer drugs and take blood samples respectively. After the preparation, 6 rats were administrated with U50488H (2 mg/kg), and the other 6 ones with Q-U50488H (10 mg/kg). It should be mentioned that in order to investigate whether U50488H and Q-U50488H were able to go through the blood–brain barrier or not, both amounts of administration were larger than those used in the subsequent animal experiment. And then, 5 min after the administration, blood and brains were removed. In order to know that status during the entire 150 min period, we have performed an additional experiment. Brains were also harvested following 30 min of ischemia and 120 min of reperfusion. Brain tissue fluid was prepared by adding purified water to brain tissue at ratio of 2:1 (ml:g), and then homogenized and centrifuged at 12,000 rpm for 120 min. Serum was harvested by centrifuging the blood at 3000 rpm for 20 min. Blank, drug-free serum and brain tissue fluid samples were collected from adult male cleaning SD rats as described above as well. Blank serum and brain tissue fluid samples, levels of U50488H and Q-U50488H in serum and brain tissue fluid samples were measured by HPLC (Singh et al., 2011; Fang et al., 2013). 2.4. I/R experimental protocols
All standards and samples were analyzed by a Shimadzu HPLC system (Kyoto, Japan) equipped with a LC-20 AT VP pump, SIL-20 AC injector and a SPD-20A UV-VTS detector. Chromatographic separation was performed on Inertsil ODS-3 column (4.6 250 mm, 5.0 μm). The system was run on isocratic mode with mobile phase consisting of methanol (Merck, Germany) and purified water in ratio of 30:70, v/v. Mobile phase was degassed ultrasonically for 15 min at
SD rats were anesthetized with pentobarbital sodium (45 mg/kg, i.p.). An intratracheal cannula was inserted and the animals were placed in intermittent positive pressure ventilation with room air. The left jugular vein was cannulated to administer drugs. The chest was opened through a left intercostals thoracotomy (between the fourth and fifth costa), and the heart was exposed by removing pericardium. Myocardial ischemia was produced by exteriorizing the
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heart with a left thoracic incision followed by a slipknot (5–0 silk) around the left anterior descending coronary artery (LAD). Significant ECG changes including widening of the QRS complex and elevation of ST segment, and color changes of the area at risk were considered as successful coronary occlusion. After 30 min of ischemia, the slipknot was released and the animal received 120 min of reperfusion. After the experiment, blood and hearts were harvested. SD rats were randomly assigned to one of the following five experimental groups. There were 12 rats in each group: (1) Sham group: silk was drilled underneath the LAD but the LAD was not ligated; (2) I/R group: LAD was ligated for 30 min and then allowed 120 min of reperfusion with receiving vehicle (0.9% NaCl, i.v.); (3) U50488HþI/R group: U50488H (1.5 mg/kg, i.v.), a selective κ-opioid receptor agonist, was administered just 10 min before ischemia (Zhang et al., 2010); (4) Q-U50488HþI/R group: Q-U50488H (5 mg/kg, i.v.) was administered just 10 min before ischemia; (5) Nor-BNIþ Q-U50488Hþ I/R group: nor-BNI (2 mg/kg, i.v.), a selective κ-opioid receptor antagonist, was administered 10 min before Q-U50488H. Other procedures were the same as the Q-U50488HþI/R group. All the following determinations were performed in a double-blind manner. 2.5. Detection of serum CK, LDH, AST/GOT activity, MDA content and SOD activity At the end of reperfusion, blood samples were collected from the heart chambers to measure the myocardial enzyme leakage including CK, LDH, AST/GOT and free radical such as MDA. Blood samples were centrifuged and then obtained serums were analyzed by using the CK, LDH, AST/GOT and MDA assay kits (Nanjing Jiancheng Bioengineering Institute) according to the procedures. After reperfusion, blood samples were taken from the carotid artery and the serum was collected by centrifugation. SOD assay kit was used (Nanjing Jiancheng Bioengineering Institute) to detect serum SOD activity according to the procedures. 2.6. Determination of myocardial infarct size The area of the infarct size was measured using Evan's Blue-TTC (2,3,5-triphenyltetrazolium chloride, TTC) double-staining technique. In brief, LAD was reoccluded (except the Sham group) and 1 ml of a 20 g/L Evan's blue solution was administered into the heart via a catheter at the end of 120 min reperfusion. The heart was then removed and immediately frozen at 20 1C. The heart was cut into five transverse slices from the apex with a thickness of about 2 mm. Heart slices were incubated with 10 g/L solution of TTC in phosphate buffer (pH 7.4) at 37 1C for 15 min and then fixed overnight in 4% paraformaldehyde solution. The images of heart slices were captured by a high-resolution CCD camera and the area at risk (AAR) and infarct size (IS) were digitally measured using OPTIMAS software. The heart consisted of three parts as follows. Non-I/R myocardium which is stained blue by Evan's blue, AAR which is not stained by Evan's blue, but by TTC; and infarct myocardium which is stained neither by Evan's blue nor by TTC. Myocardial infarct sizes (IS/AAR %) were then calculated. 2.7. Evaluation of myocardial apoptosis At the end of reperfusion, frozen slices were obtained instantly. Then myocardial apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) using an in situ cell death detection kit (Roche). A double-staining technique was used, in brief, TUNEL staining for apoptotic cell nuclei and 40 ,6diamino-2-phenylindole (DAPI) staining for all myocardial cell nuclei. The index of apoptosis was expressed by the number of positively
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stained apoptotic cardiomyocytes/the total number of cardiomyocytes counted 100%. 2.8. Determination of myocardial Kir6.2 expression At the end of reperfusion, rats’ pericardial tissues were harvested and washed with ice-cold normal saline and homogenized on ice in phosphate buffered saline. After centrifuged at 3000 rpm for 5 min, the supernatant was discarded. The homogenate was incubated in lysis buffer including protease inhibitor, PMSF, etc. for 20 min with vortex-mixing at 4 1C, filtered and centrifuged at 12,000g for 20 min. The resulting supernatants were the proteins, of which the concentrations were determined by Bradford's method, with bovine serum albumin serving as a calibrator. Lysate proteins were mixed with loading buffer with the ratio of 1:4 and denatured at 100 1C for 9 min. Then 30 to 40 μg proteins from denatured total extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with a molecular weight marker, and transferred to a NC membrane. After washing in Tris-buffered saline containing 0.01% Tween-20 (TBST) for 5 min, saturating with 30 g/L bovine serum albumin (BSA) in TBST for 1 h at room temperature, the membranes were incubated with the following primary antibodies overnight at 4 1C: polyclonal rabbit GAPDH (1:10,000, Sigma-Aldrich, St. Louis, MO, USA) or polyclonal rabbit anti-Kir6.2 (1:1000, Santa Cruz Biotechnology, St. Cruz, CA, USA) diluted in TBST. The membranes were then washed six times in TBST for 5 min and incubated with the following horseradish peroxidase HRP-labeled secondary antibodies, goat anti-mouse (1:4000, Santa Cruz Biotechnology, St. Cruz, CA, USA), for 1 h at room temperature. After washing six times in TBST for 5 min and incubating with ECL-Plus, the pixel intensities of the immunoreactive bands were quantified using the percentage adjusted volume feature of Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Data are expressed as the ratio of the band intensity of the protein of interest to the loading control protein band (GAPDH). 2.9. Statistical analysis Data were presented as means 7S.E.M. The significance of differences among groups were evaluated by Dunnett's t-test for multiple comparisons preceded by one-way analysis of variance (ANOVA). A value of P o0.05 was considered as statistically significant.
3. Results 3.1. Chromatography of U50488H and Q-U50488H in serum and brain tissue The quaternary ammonium salt of U50488H, Q-U50488H, was a kind of faint yellow powder. Q-U50488H was proved to be C20H29Cl2IN2O by mass spectrometry (Fig. 2). Q-U50488H, with the molecular weight of 511.2695, had a solubility of no less than 5 mg/ml; however, U50488H, with the molecular weight of 405.7895, had a solubility of about 2 mg/ml. This fact indicated that Q-U50488H was a new compound with increased molecular weight and solubility than U50488H. Typical chromatograms of physiological saline, standards of U50488H and Q-U50488H (dissolved in physiological saline) are shown in Fig. 3A. The peak between 6 min and 8 min indicated U50488H and Q-U50488H respectively. Chromatograms of serum samples are shown in Fig. 3B. Compared to the blank serum, U50488H and Q-U50488H could be detected in serum after intravenous injection.
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Fig. 2. Mass spectrometry of Q-U50488H. Species in the new compound was determined by mass spectrometry which was identified to be Q-U50488H, and the peak fit well with m/z of Q-U50488H.
Chromatograms of brain tissue fluid samples are shown in Fig. 3C. Compared to the blank brain tissue fluid, U50488H could be detected in brain tissue fluid. However, Q-U50488H was not detected. In addition, as shown in Fig. 3D, chromatograms of brain samples were harvested at 0 min, 5 min and 150 min after the administration of Q-U50488H. It was shown that Q-U50488H could be detected neither at 5 min nor at 150 min in the brain tissue after intravenous injection of Q-U50488H. This result further strengthened that Q-U50488H was not able to go through the blood–brain barrier. Taken together, U50488H was detected in both serum and brain tissue fluid of the rats after intravenous injection by the HPLC method. However, Q-U50488H was detected only in the serum and not in the brain tissue. 3.2. Q-U50488H lowered the activity of serum CK, LDH, AST/GOT and the level of MDA in rats subjected to I/R As shown in Fig. 4, activity of intracellular myocardial enzymes such as CK, LDH, AST/GOT and level of MDA in serum increased significantly following I/R. Administration of Q-U50488H prior to I/R significantly reduced levels of the myocardial enzymes and MDA. No difference was found in these parameters when compared with the U50488HþI/R group. The effects of Q-U50488H mentioned above were blocked by Nor-BNI, a selective κ-opioid receptor antagonist. 3.3. Q-U50488H increased the activity of serum SOD in rats subjected to I/R
(Po0.01), and no difference was found when compared with rats in group administered with U50488H. The effect of Q-U50488H was abolished by Nor-BNI. In order to investigate whether KATP plays a role in Q-U50488Hinduced myocardial protection, KATP blockers such as 5-HD (10 mg/ kg, i.v.) and glibenclamide (1 mg/kg, i.p.) were administrated 10 min before ischemia (Abdallah et al., 2011; Kai et al., 2013; Taliyan et al., 2010). As shown in Fig. 6, the effect of Q-U50488H was abolished by 5-HD and glibenclamide. 3.5. Q-U50488H reduced myocardial apoptosis in rats subjected to I/R A very low level of TUNEL-positive staining was detected in the cardiomyocytes of the Sham group. A great number of TUNELpositive cells were observed in myocardial tissue from rats subjected to I/R. And the cardiomyocyte apoptosis was dramatically alleviated by the administration of Q-U50488H prior to I/R (Po0.01). No significantly statistical difference was found when compared with the U50488Hþ I/R group. The effect of Q-U5088H was blocked by Nor-BNI (Fig. 7). In order to investigate whether KATP plays a role in Q-U50488Hinduced myocardial protection, KATP blockers such as 5-HD and glibenclamide were administrated 10 min before ischemia. As shown in Fig. 7, the effect of Q-U50488H was abolished by 5-HD and glibenclamide. 3.6. Q-U50488H increased the expression of Kir6.2 in the ischemia area of the heart subjected to I/R
I/R induced a low activity of SOD in serum, whereas Q-U50488H increased the activity of SOD in serum significantly when administrated prior to I/R. No difference was found when compared with the U50488Hþ I/R group (Fig. 5). The effect of Q-U50488H mentioned above was blocked by Nor-BNI.
As shown in Fig. 8, the expression of Kir6.2 was increased by the administration Q-U50488H prior to I/R (P o0.01), and no difference was found when compared with the U50488Hþ I/R group. The effect of Q-U50488H was blocked by Nor-BNI.
3.4. Q-U50488H reduced myocardial infarction area in rats subjected to I/R
4. Discussion
The I/R injury of cardiomyocytes caused a significant myocardial infarction (Fig. 6). Compared with the I/R group, the Q-U50488Hþ I/R group showed significantly reduced myocardial infarct size
In the present study, U50488H, a selective κ-opioid receptor agonist, for the first time, was transformed into Q-U50488H by chemical methods. Q-U50488H was a faint yellow powder with
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Fig. 3. Comparison of chromatography of U50488H and Q-U50488H in rat serum and brain tissue. A: Chromatography of physiological saline (I), standards of U50488H (II) and Q-U50488H (III) (dissolved in physiological saline). B: Chromatography of blank serum (I), U50488H (II) and Q-U50488H (III) in serum. C: Chromatography of blank brain tissue fluid (I), U50488H (II) and Q-U50488H (III) in brain tissue fluid samples. D: Chromatography of blank brain tissue fluid (I), Q-U50488H 5 min (II) and 150 min after administration (III) in brain tissue fluid samples.
remarkably increased solubility and molecular weight than U50488H. Results of HPLC indicated that Q-U50488H was incapable of going through the blood–brain barrier. U50488H's ability to go through the blood–brain barrier attributes to its poor solubility and low molecular weight, and may lead to probable addiction, which imposes restrictions on its clinical application. To keep U50488H from penetrating into brain tissue, it is necessary to improve its solubility in physiological saline and molecular weight. In the preliminary experiment, we tried to make polyethyleneglycol (PEG)-modification of U50488H. However, U50488H would separate from the compound with PEG soon after intravenous injection. In our present study, we obtained Q-U50488H by structurally transforming U50488H into its quaternary ammonium salt. The latter was stable and not prone to degrade relatively. In addition, Q-U50488H detected by HPLC was not able to go through blood–brain barrier, whereas U50488H was able to penetrate into brain tissue.
The present study also provided the evidence that like U50488H, Q-U50488H could significantly decrease the level of myocardial enzymes such as CK, LDH, AST/GOT, and free radical MDA generation, and enhance SOD activity as well. And these effects of Q-U50488H were blocked by a selective κ-opioid receptor antagonist, Nor-BNI, indicating that Q-U50488H, a new κ-opioid receptor agonist, elicited protective effect on ischemic/ reperfused myocardium. In order to further investigate underlying mechanisms, KATP blockers such as 5-HD and glibenclamide were administered just 10 min before Q-U50488H. We observed that Q-U50488H could significantly decrease of the infarct size and cardiomyocyte apoptosis. Moreover, these cardioprotective effects of Q-U50488H were abolished by KATP blockers. There were a lot of studies that suggest the κ-opioid receptor reduced infarct size and apoptosis through the opening of KATP channels (Chen et al., 2003; Lishmanov et al., 2007; Wang et al., 2001). Our study showed that
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Fig. 4. Comparison of the activity of serum CK, LDH, AST/GOT and the level of MDA in rat subjected to I/R. A: Comparison of the activity of CK in serum. B: Comparison of the activity of LDH in serum. C: Comparison of the activity of AST/GOT in serum. D: Comparison of the level of MDA in serum. n¼ 6 in each group. Nor-BNI: nor-Binaltorphimine. All values are presented as means 7S.E.M. nnPo 0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488H þI/R group.
Fig. 5. Comparison of the activity of SOD in serum. n¼6 in each group. Nor-BNI: nor-Binaltorphimine. All values are presented as means 7 S.E.M. nnP o0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488H þI/R group.
the cardioprotective effect of Q-U50488H mentioned above was abolished by 5-HD and glibenclamide. This fact holds potential promises that the effect of Q-U50488H against myocardial I/R injury may be associated with the opening of KATP. ATP-sensitive potassium channel (KATP), a specific type of potassium channel, exists in heart and vascular smooth muscle.
Fig. 6. Comparison of myocardial infarct sizes (IS/AAR %). n¼ 6 in each group. NorBNI: nor-Binaltorphimine; 5-HD: 5-hydroxydecanoate; Gli: glibenclamide. All values are presented as means 7 S.E.M. nnP o0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488H þ I/R group.
KATP is named for the reason that it is inhibited by intracellular ATP and adenine nucleotides. The opening of KATP dilates smooth muscle effectively, lowers blood pressure and produces specific
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Fig. 8. Comparison of the expression of Kir6.2 in precordial region. A: Representative photographs of Kir6.2 expression were determined by Western blots. B: Expressions of Kir6.2 were determined by Quantity One software. n ¼6 in each group. Nor-BNI: nor-Binaltorphimine. All values are presented as means 7 S.E.M. nn Po 0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488H þ I/R group.
Fig. 7. Comparison of myocardial apoptosis in rat heart subjected to I/R. A: Myocardial apoptosis was determined by TUNEL staining. TUNEL staining (green) indicates apoptotic nuclei; DAPI counterstaining (blue) indicates total nuclei. B: TUNEL-positive nuclei were expressed as a percentage of the total number of nuclei automatically counted and calculated. n¼ 6 in each group. NorBNI: nor-Binaltorphimine; 5-HD: 5-hydroxydecanoate; Gli: glibenclamide. All values are presented as means 7 S.E.M. nnPo 0.01 vs. the Sham group; ♯♯Po 0.01 vs. the I/R group; $$Po 0.01 vs. the Q-U50488Hþ I/R group.
cardiovascular protection; it has become the typical target of drugs applied in the research of protecting ischemic heart. U50488H, a selective κ-opioid receptor agonist, activates KATP and plays its protective role in the ischemia preconditioning (IPC) of heart (Eleftherianos et al., 2011; Wu et al., 2011). According to Schultz et al. (1998) and Schultz and Gross (2001), κ-opioid-induced cardioprotection and IPC seem to share a common pathway, KATP channel, and possibly the mitochondrial KATP channel appears to be involved in the beneficial effects observed. Activation of KATP channels during ischemia is an important adaptive mechanism for protecting the myocardium when blood flow to the tissue is compromised (Cole et al., 1991; Lee et al., 2008).In the present study, it was found that 5-HD and glibenclamide, both KATP channel blockers, were able to abolish the beneficial effects of Q-U50488H. However, the intracellular signaling pathways related to the mechanisms of opioid-receptor stimulation and the possible end-effector, perhaps the mitochondrial KATP channel, are less well understood. Several mechanisms have been proposed to explain the process of involvement of the KATP up-regulation or KATP opening in its cardioprotective effect. The major one involved is that activation of KATP channels can prevent calcium entry via L-type voltage-gated Ca2 þ channels and alleviate calcium overload induced by I/R (Chun et al., 2010; Shi et al., 2013). However, it is still unclear how this observation relates to κ-opioid receptors and cardioprotection
mediated by κ-opioid receptors. Further study is needed to investigate the underlying mechanism. Q-U50488H, a new κ-opioid receptor agonist, is incapable of going through the blood–brain barrier. It protects the heart from I/R injury, which can be used in scientific research. In addition, it is a potential drug, which may be employed in the prevention and treatment of ischemic heart disease clinically. Q-U50488H, therefore, is of great scientific research value and clinical application prospect.
5. Conclusions We have demonstrated that Q-U50488H is a new κ-opioid receptor agonist, which is not able to pass the blood–brain barrier. Importantly, Q-U50488H has a protective effect against myocardial I/R injury. The cardioprotective effect of Q-U50488H is associated with the opening of KATP.
Acknowledgments This work was supported by grants from National New Drug Research Major Project (2009ZX09301–009-BD11), and grants from the National Natural Science Foundation of China (Nos. 81270402, 31200875, 30971060, 30900535, and 81070063) and grants from Shaanxi Province of China (Nos. 2013K132-02-06, 2011JM4001).
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