| Background Administration of morphine or fentanyl, opioid receptor (OR) agonist mimics the cardioprotective effect of ischemic preconditioning (PC) in anesthetized open-chest rats. Protective effects of morphine or fentanyl on postischemic myocardioum injury are via 5 and k- ORs. Remifentanil is a new ultra-short-acting phenylpiperidine opioid analgesic agent, which binds preferentially toμ-opioid receptor as well as □δ- and □κ-ORs. It has an analgesic potency slightly less than fentanyl but are 100 times greater than morphine. When compared with a standard fentanyl-based technique, the use of remifentanil has changed our present anesthetic regimens. However, effect of remifentanil on postischemic myocardium is still unknown. We hypothesize that like morphine and fentanyl, remifentanil confers cardioprotection.Methods The study was performed in vivo, in vitro and myoctyes models respectively. In vivo: Male Sprague-Dawley rats or New Zealand rabbits were anesthetized and the chest opened. All animals were subjected to intermittent occlusion of the left coronary artery (5-min occlusion, 5-min reperfusion x 3) and 2 hours of reperfusion. In vitro: male Sprague-Dawley rats or New Zealand rabbits were killed. The heart was removed immediately and mounted to Langendorff apparatus and perfused retrogradely at 100cmH2O with Krebs-Ringer solution. Isolated myocyte model: Cardiac ventricular myocytes were isolated from the hearts of adult male Spragur-Dawely rats, using a collagenase perfusion method. A spectrofluorometric method with fura-2/AM as the Ca~2+ indicator was used for measurement of [Ca~2+]i. Metaolic inhibition(MIP)and anoxia will achieved with a glucose-free Krebs solution containing2-deoxy-D-glusocse(2-DOG). Study Group and experimental protocols: The study includes 3 series experiments. Firstly, the objective of series experiment 1 is to determine the protective effect of RPC on postischemic myocardium injury and dose-dependent relationship between RPC and the effects. Animals or myoctyes were divided into three groups: control (CON), remifentanil preconditioning group (RPC) and ischemia preconditioning group (IPC), respectively. To study RPC dose-related effect, pre treatment with remifentanil was performed with regime (3 x 5-min infusions) using 0.2, 0.6, 2, 6 or 20(ig.kg"1.min"1 intravenously in vivo; perfused with remifentanil (RPC) using four drug concentrations: 10, 50, lOOand ISOug.L'1 in vitro; and ventricular myocytes were superfused with ICrebs solution containing remifentanil 0.1 -, 0.3 > U3> ^30> 100^300andl000^g.L■1. In the second series of experiments, in which the effects of IPC or RPC It was determined which OR subtypes is mediated the effects of RPC or IPC, the experiments were performed with nor-Binaltorphimine (nor-BNI), Naltrindole (NTD) or CTOP opioid receptor antagonists administered before remifentanil-induced PC or IPC respectively in vivo or in vitro. Finally, the third series of experiment was performed to determine the involvement of PKC, the KAtp channel or MAPK in the cardioprotection of RPC. The experiments were also repeated with Chelerythrine and GF109203X (both PKC inhibitor), glibenclamide, 5-Hydroxydecanoate and HMR-1098(inhibitors of the sarc-KATP channel), PD098059(ERK inhitor) and SB 203580 (p38 MAPK inhibitor) and pre-RPC IPC or post-RPC IPC respectively. Infarct size (IS), as a percentage of the area at risk (AAR), was determined by triphenyltetrazolium staining. Hemodynamic data were recorded in vivo and vitro experiments. The coronary effluent was collected at 5 and 10 min post-reperfusion for determination of myocardial injury via lactate dehydrogenase (LDH) efflux in vitro. To measure the electrically induced [Ca2+]j transient, myoctyes were electrically stimulated at 0.2Hz in myoctyes model. Western blot analyses of p38 MAPK and pJNK were performed in vivo preparation. Data were expressed as mean±SD.Hemodynamics were analyzed using 2-way analysis of variance (ANOVA) with Bonferroni post-hoc test for multiple comparisons if significant F ratios were obtained. IS (expressed as percentage of the area at risk) were analyzed between groups using ANOVA with a Student-Newman-Keuls post-hoc test for multiple comparisons. Statistical differences were considered significant if P<0.05.Results Similar to IPC, there was a dose related reduction in IS/AAR after treatment with remifentanil. The reduction in IS in groups subjected to remifentanil PC in the range of 0.6 - 6 ug-kg^-min"1 were dose related with a peak reduction at 6 |j.g.kg"1.min"1 in vivo. The ED50 was 2.7 ug-kg^-min"1 according to the sigmoid equation Y=15.18 + 17.76/ [1 + 10("257"x)], r = -0.898. In vitro: IS/AAR after RPC was significantly reduced dose-dependently. The EC50 was 16ug.L"' according to the sigmoid equation Y=26.87+18.35/(l+10"1796"x), r = -0.9113. These results are referred to as series 1. In the second series of experiments, the effect of RPC was prevented or significantly attenuated by co administration of a \x, k or 8-opioid antagonist. The infarct sparing effect of ischemic preconditioning was abolished by blockade of 5- or Dk-OR, but not Du-OR in vivo. However, this effect was stopped by pretreatment with NTD and nor-BNI but not CTOP in vitro. RPC also reduced the release of LDH during reperfusion. In the third series of experiments, both studies in vivo and vitro have similar results on PKC and KAtp channel. Chelerythrine and GF109203X, both PKC inhibitors, abolished the effects of RPC or IPC on IS/AAR. Unlike HMR-1098 (a selective inhibitor of the sacrolemmal KAtp channel), 5-Hydroxydecanoate (a selective mito-K.ATP channel blocker) could abolish the cardioprotection of RPC and IPC pre- or post RPC or IPC. PD098059(ERK inhitor) and SB 203580 (p38 MAPK inhibitor) abolished the effects of RPC when used at ischemia 30 min. About study role of PKC and Katp channel in RPC or IPC, results of in vitro were similar to that in vivo. In myoctyes model: remifentanil dose-dependently reduced myoctyes electrically induced [Ca2+]i transient. The EC50 was 0.4ug.L"' according to the sigmoid equation Y=78.49...
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