| Background:Ischemic heart disease is a tremendous threaten to people’s health. With thewidespread application of internal treatment and surgical interventions such asintra-coronary artery thrombolysis and coronary artery bypass graft surgery, thefollowing emergence of reperfusion injury of cardiomyocytes has aroused wide concernfrom cardiologists increasingly.It is reported that opioid receptors widely exist in human tissues and organs such asheart, brain and so on. In terms of the heart, the predominant opioidreceptor——κ-opioid receptor is of great importance in the regulation of myocardialischemia/reperfusion (I/R). A lot of evidence from animal experiments has demonstratedthat U50488H, an exogenous selective κ-opioid receptor agonist, can significantly reduceinfarct size, cardiomyocyte apoptotic death and the incidence of arrhythmia, improvingthe function of heart and coronary microcirculation. Previous studies have also found thatU50488H is able to pass the blood-brain barrier for its poor solubility and smallmolecular weight. Although U50488H can effectively protect the heart against I/R injury, these properties impose restrictions on its clinical application.ATP-sensitive potassium channel (KATP), a specific type of potassium channel, existsin heart and vascular smooth muscle. KATPwas named for the reason that it is inhibitedby intracellular ATP and adenine nucleotides. The opening of KATPdilates smooth muscleeffectively, lowers blood pressure and gets spasmolysis, producing specificcardiocascular protection effects. And it has become the typical target of drugs applied inthe research of protecting ischemic heart. U50488H, a selective κ-opioid receptor agonist,activates KATPand plays its protective role in the ischemia preconditioning (IPC) ofheart.In the present study, we obtained a new compound called quarternary ammonium ofU50488H by changing the structure of U50488H, a selective κ-opioid receptor agonist.Consequently, the new compound was not able to pass the blood brain barrier.Meanwhile, the quarternary ammonium of U50488H continues to protect the ischemicheart in the periphery. The validity that KATPserves as the target of quarternaryammonium of U50488H was also tested. This new opioid which protects the heart can beused in scientific research. In addition, it is a potential drug which may be employed inthe prevention and treatment of ischemic heart disease clinically. The new drug, therefore,is of great scientific research value and clinical application prospect.Aims:The present study was designed to structurally transform U50488H to its quaternaryammonium type to make it incapable of passing the blood-brain barrier. In addition, therole of this new compound in I/R injury of the heart was tested.Methods:(1) Under the condition of polar solvent and catalyst, U50488H, a selective κ-opioidreceptor agonist, reacted with CH3I to form its quaternary ammonium salt.(2) The existence of U50488H and its quaternary ammonium salt in the serum andbrain tissue after intravenous injection were determined by high performance liquidchromatography.(3) The I/R model of rats was established. Male Sprague-Dawley (SD) rats (220~280g) were randomly divided into several groups to form the model of I/R byligating the left anterior descending coronary artery. In the process of the experiment, thechanges of heart rate (HR), arterial blood pressure (ABP)，left ventricular pressure (LVP)and±dp/dtmaxwere observed continuously. After the experiment, blood and hearts wereharvested.(4) To measure the area of myocardial infarction in rats, an Evan’s Blue-TTCdouble-staining technique was used.(5) To determine the expression of Kir6.2in rats’ myocardium, western blotting wasused.(6) To detect the activity of LDH、CK、AST/GOT and SOD in the serum of rats,several kinds of kits were used.(7) To detect the content of MDA in the serum of rats, the method of TBA was used.(8) To evaluate cardiomyocyte apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was used.Results:The structurally transforming U50488H to its quaternary ammonium saltAfter the structural transformation, the quaternary ammonium salt of U50488H wasproved to be C20H29Cl2IN2O by mass spectrometry for its molecular weight of parent ionwas about386, which was keeping in line with the peak value of the chart.Chromatography detection of U50488H and the quaternary ammonium salt ofU50488HU50488H was able to pass the blood-brain barrier, but its structural quaternaryammonium salt was not able to do that.The effect of the quaternary ammonium salt of U50488H on myocardial infarctionThe quaternary ammonium salt of U50488H could significantly decrease of theinfarct size (P<0.01), and no difference was found compared with U50488H. The effectmentioned above was able to be blocked by Nor-BNI, a selective κ-opioid receptorantagonist, and KATPantagonists such as5-HD and glibenclamide.The effect of the quaternary ammonium salt of U50488H on the expression of Kir6.2The expression level of Kir6.2was reversed by the administration of quaternaryammonium salt of U50488H prior to I/R (P<0.01), and no difference was foundcompared with U50488H. The effect mentioned above was able to be blocked byNor-BNI, a selective κ-opioid receptor antagonist, and KATPantagonists such as5-HDand glibenclamide.The effect of the quaternary ammonium salt of U50488H on the myocardialenzymes and free radicals in plasmaThe leakage of intracellular myocardial enzymes such as CK、LDH and AST/GOTwere significantly decreased with the administration of quaternary ammonium salt ofU50488H prior to I/R. The generation of MDA was decreased and the activity of SODwas increased at the same time. No difference was found compared with U50488H. Theeffect mentioned above was able to be blocked by Nor-BNI, a selective κ-opioid receptorantagonist, and KATPantagonists such as5-HD and glibenclamide.The effect of the quaternary ammonium salt of U50488H on the cardiomyocyteapoptosisThe cardiomyocyte apoptosis was dramatically alleviated by the administration ofquarternary ammonium salt of U50488H prior to I/R. No significantly statisticaldifference was found compared with U50488H. The effects mentioned above were ableto be blocked by Nor-BNI, a selective κ-opioid receptor antagonist, and KATPantagonistssuch as5-HD and glibenclamide.Conclusions:(1) U50488H, a selective κ-opioid receptor agonist, for the first time, is transformedto its quaternary ammonium salt by mature methods in chemistry successfully.(2) The quaternary ammonium salt of U50488H is a new κ-opioid receptor agonistwhich has a protective effect on ischemic myocardium. But it was not able to pass theblood-brain barrier.(3) The cardioprotective effect of quaternary ammonium salt of U50488H onischemic myocardium may be associated with the up-regulation or opening of KATP. |
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