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外周苯二氮##zhuo受体激动剂Ro5--4864抑制大鼠心肌细胞线粒体通透性转换

李敬远, 王俊科, 曾因明

中国医科大学附属第一医院麻醉科.辽宁,沈阳 110001；江苏省麻醉医学研究所,徐州医学院江苏省麻醉学重点实验室.江苏,徐州 221002

摘要

线粒体通透性转换（mitochondrial permeability transition，MPT）导致线粒体氧化应激性损伤。近年研究认为，位于线粒体外膜的外周苯二氮##zhuo受体（peripheral benzodiazepine receptor，PBR）参与了线粒体的重要生理功能。该研究在心肌细胞线粒体水平探讨激动PBR能否抑制Ca~(2+)诱发的MPT。分离Sprague--Dawley大鼠心肌细胞线粒体，将PBR激动剂Ro5--4864（50、100、200#mu#mol／L）和线粒体孵育，利用150#mu#mol／L Ca~(2+)诱发MPT，部分线粒体在与100#mu#mol／L Ro5--4864孵育前5min加入MPT孔道开放剂苍术苷（atractyloside，ATR）。采用分光光度法观察线粒体膨胀情况：Western blot检测线粒体细胞色素C（cytochrome C，Cyto C）释放；利用荧光探针JC--1在激光共聚集显微镜下观察线粒体膜电位的变化。50、100、200#mu#mol／L Ro5--4864均显著抑制Ca~(2+)诱发的520nm处线粒体吸光度的下降，而且抑制Ca~(2+)引起的线粒体Cyto C释放和线粒体膜电位下降，但ATR可阻断R05--4864的上述作用。结果提示，PBR激动剂可抑制大鼠心肌MPT，保持线粒体Cyto C含量和稳定线粒体膜电位，减轻线粒体损伤。PBR的激活可能成为减轻心肌细胞应激性损伤及心肌保护的新方法。

关键词： 外周苯二氮zhuo受体; Ro5-4864; 线粒体通透性转换

Peripheral benzodiazepine receptor agonist Ro5--4864 inhibits mitochondrial permeability transition in rat heart

Li Jingyuan, Wang Junke, Zeng Yinming

Department of Anesthesiology, the First Affiliated Hospital, China Medical University.Shenyang 110001,Liaoning；China

Abstract

Opening of mitochondrial permeability transition （MPT） pores leads to mitochondrial injury during oxidative stress. The peripheral benzodiazepine receptor （PBR） located at mitochondrial outer-membrane has been shown to be involved in several mitochondrial functions. In the present study, we used Ro5-4864, a PBR agonist, to test if activation of PBR could prevent MPT pore opening during Ca~(2+) overloading. Cardiac mitochondria isolated from Sprague-Dawley rats were treated by 150 #mu#mol/L Ca~(2+) to induce MPT. Ro5-4864 （50, 100 and 200 #mu#mol/L） was added into incubation buffer before adding 150 #mu#mol/L Ca~(2+). In additional group, atractyloside （ATR, 20 #mu#mol/L）, an opener of MPT pores was added 5 min before the addition of 100 #mu#mol/L Ro5-4864. The change of absorbance at 520 nm was monitored with a spectrophotometer at 30℃ for 10 min. Western blot was used to detect cytochrome C loss. The mitochondrial membrane potential was monitored with the fluorescence dye JC-1. Ro5-4864 inhibited the decrease of absorbance at 520 nm compared to that in the untreated Ca~(2+) group （P〈0.01, P〈0.05）. In the presence of ATR, Ro5-4864 was not able to prevent MPT anymore. Opening of MPT pores by Ca~(2+) decreased the content of cytochrome C in mitochondria, but increased cytochrome C content in cytosol. Ro5- 4864 preserved cytochrome C content in mitochondria and led to less cytochrome C release to cytosol. ATR treatment reversed the protective effect of Ro5-4864 on cytochrome C content. Opening of MPT pores led to mitochondrial depolarization, whereas Ro5-4864 treatment maintained mitochondrial membrane potential. Thus, prevention of MPT by activation of PBR during calcium overloading maintains mitochondrial cytochrome C content and membrane potential. Activation of PBR during cardiac ischemia and reperfusion may be an alternative way for cardioprotection.

Key words: peripheral benzodiazepine receptor;Ro5-4864;mitochondrial permeability transition

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引用本文：

李敬远, 王俊科, 曾因明. 外周苯二氮##zhuo受体激动剂Ro5--4864抑制大鼠心肌细胞线粒体通透性转换[J]. 生理学报 2007; 59 (1): 13-18.

Li Jingyuan, Wang Junke, Zeng Yinming. Peripheral benzodiazepine receptor agonist Ro5--4864 inhibits mitochondrial permeability transition in rat heart. Acta Physiol Sin 2007; 59 (1): 13-18 (in Chinese with English abstract).