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).