线粒体膜ATP敏感钾通道在缺氧性肺动脉平滑肌细胞增殖/凋亡失衡中的作用
摘要
研究背景
迄今为止,肺动脉高压和肺心病的发病率和死亡率仍然居高不下,严重威胁着人类的健康。缺氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)是最常见的类型,其发病机制目前尚未完全阐述清楚,其中缺氧性肺血管收缩(hypoxic pulmonary artery vasoconstriction,HPV)和缺氧性肺血管重建(hypoxic pulmonary vascular remodeling)是被认为是其两大主要的病理生理基础。目前认为,缺氧引起的肺血管收缩并非完全依赖于血管内皮细胞,血管平滑肌细胞也参与了肺血管收缩反应。在影响血管张力的各种因素中,血管平滑肌细胞钾通道和细胞内钙离子发挥着十分重要的作用。研究表明缺氧可抑制肺动脉平滑肌细胞钾通道的活性,引起肺动脉平滑肌细胞(PASMCs)去极化,达到一定阈值后,电压依赖性钙通道开放,引起细胞内钙离子浓度升高,作为重要的第二信使,钙离子促使肺动脉平滑肌细胞收缩、增殖和迁移,也可激活各种蛋白激酶和转录因子,进一步引起缺氧性肺血管收缩和肺血管重建,最终导致肺动脉高压。
有一系列研究表明线粒体膜上ATP敏感的钾通道(MitoK_(ATP))能保护缺血缺氧心肌细胞免受氧化应激的伤害。Takashi等证实线粒体膜上ATP敏感的钾通道的开放能减少心肌细胞缺血/再灌注损伤时的凋亡标记物(细胞色素C的释放,Caspase活性,线粒体膜电位的改变等等)。Akao和他的同事们也研究了mitoK_(ATP)在凋亡中的作用,他们发现线粒体膜上ATP敏感钾通道的开放能抑制H2O2诱导的新生大鼠心肌细胞的凋亡。线粒体mitoK_(ATP)通道的开放为什么能起到保护心肌细胞的机制还不十分清楚,但是目前有三种假设解释mitoK_(ATP)通道与心肌细胞之间的联系:1)线粒体钙离子摄取的下降,Holmuhameclov等证实线粒体mitoK_(ATP)通道的开放能够降低线粒体钙离子摄取的程度。2)线粒体基质肿胀和ATP合成的改变,mitoK_(ATP)的开放能引起线粒体基质肿胀,并激活呼吸链产生更多的ATP,支持心肌细胞的恢复。3)氧自由基(ROS)水平的改变,在心肌细胞的保护中,氧自由基是一把双刃剑。预处理阶段产生的ROS是具有保护作用的,但是在再灌注阶段产生的ROS是有害的。mitoK_(ATP)通道的开放能引起预处理期保护性ROS水平的增加,和再灌注期产生的ROS水平的降低。
尽管这些研究表明线粒体mitoK_(ATP)通道在心血管细胞的增殖凋亡中起很重要的作用,但是线粒体mitoK_(ATP)通道在肺血管中的作用很少有人研究,线粒体mitoK_(ATP)通道是否与缺氧性肺动脉平滑肌细胞增殖/凋亡失衡有关以及其机制目前还没有相关报道。线粒体mitoK_(ATP)通道对动物和人的肺动脉平滑肌细胞增殖/凋亡平衡的调节是否具有一致性也还没有相关报道,但是由于进行人肺血管的体外和体内的实验研究是比较昂贵的和具有局限性的,因此对比动物实验和以人为实验对象的实验是否具有一致性,从而决定是否以动物实验代替以人为实验对象的实验,对以后的实验研究是具有重要意义的。
第一部分线粒体膜ATP敏感钾通道在缺氧性大鼠肺动脉平滑肌细胞增殖/凋亡失衡中的作用
分题一线粒体膜ATP敏感钾通道对缺氧大鼠肺动脉平滑肌细胞细胞内细胞色素C分布的变化及细胞增殖中的作用
目的:研究线粒体膜上ATP敏感的钾通道(mitoK_(ATP))和线粒体膜电位(Δψ_m)在缺氧影响大鼠肺动脉平滑肌细胞内细胞色素C在细胞浆和线粒体内分布的变化及以及其引起细胞增殖变化中的作用,旨在探索缺氧性肺动脉重建和肺动脉高压形成的发生机制,为寻找新的防治方法提供试验依据。
方法:获取大鼠正常肺组织,分离出肺动脉平滑肌细胞(PASMCs)进行常氧或慢性缺氧培养。将标本分为六组:①正常对照组;②MitoK_(ATP)阻断剂5-HD组;③MitoK_(ATP)开放剂Diazoxide组;④慢性缺氧(CH)组;⑤CH+5-HD组;⑥CH+Diazoxide组。利用激光共聚焦显微镜(Leica SP-1 USA)成像检测线粒体膜电位,线粒体/胞浆成分分离试剂盒(BioVision)分离线粒体和胞浆成分后,Western blot法检测两者细胞色素C,流式细胞仪检测细胞周期和MTT法检测细胞增殖情况。
结果:①Diazoxide作用24h后,与正常对照组比较,R-123荧光强度明显增强,线粒体膜电位去极化,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,细胞呈增殖明显增多、凋亡明显减少,与正常对照组相比较均P<0.05;而5-HD作用24h与正常对照组比较,上述指标无明显变化, P>0.05;;②慢性缺氧24h,结果与Diazoxide组相似,与正常对照组比较,R-123荧光强度明显增强,线粒体膜电位去极化,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,细胞呈增殖明显增多、凋亡明显减少,与正常对照组相比较均P<0.05;;CH+Diazoxide组,与缺氧组比较, R-123荧光强度明显增强,线粒体膜电位去极化,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,细胞呈增殖明显增多、凋亡明显减少,与正常对照组相比较均P<0.05; CH+5-HD组,与缺氧组比较,R-123荧光强度明显减弱,线粒体膜电位部分消失,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显升高,细胞增殖明显减少、凋亡明显增多,与正常对照组相比较均P<0.05。
结论:本实验结果显示,缺氧可以引起MitoK_(ATP)的开放以及m的去极化,并进而抑制了细胞色素C从细胞线粒体释放到细胞浆,抑制线粒体凋亡途径,从而参与并影响了肺动脉高压的发生发展。
分题二线粒体膜ATP敏感钾通道对缺氧大鼠肺动脉平滑肌细胞氧自由基的变化及细胞增殖中的作用
目的:研究线粒体ATP敏感性钾通道和线粒体膜电位(Δψ_m)在缺氧影响大鼠肺动脉平滑肌细胞细胞内氧自由基的变化及细胞增殖中的作用,旨在探索缺氧性肺动脉重建和肺动脉高压形成的发生机制,为寻找新的防治方法的提供试验依据。
方法:获取大鼠正常肺组织,分离出肺动脉平滑肌细胞(PASMCs)进行常氧或慢性缺氧培养。将标本分为六组:①正常对照组;②MitoK_(ATP)阻断剂5-HD组;③MitoK_(ATP)开放剂Diazoxide组;④慢性缺氧(CH)组;⑤CH+5-HD组;⑥CH+Diazoxide组。利用激光共焦显微镜(Leica SP-1 USA)成像检测线粒体膜电位,荧光染色检测细胞内氧自由基含量,流式细胞仪检测细胞周期和MTT法检测细胞增殖情况。
结果:①Diazoxide作用24h后,与正常对照组比较,R-123荧光强度明显增强,线粒体膜电位去极化,细胞内氧自由基含量明显增加,细胞呈增殖明显增多、凋亡明显减少,P<0.05;而5-HD作用24h后,上述指标与正常对照组相比较,均无显著性差异, P>0.05;②慢性缺氧24h,结果与Diazoxide组相似,与正常对照组比较,R-123荧光强度明显增强,线粒体膜电位去极化,细胞内氧自由基含量明显增加,细胞呈增殖明显增多、凋亡明显减少,P<0.05;CH+Diazoxide组,与缺氧组比较, R-123荧光强度明显增强,线粒体膜电位去极化,细胞内氧自由基含量明显增加,细胞呈增殖明显增多、凋亡明显减少,P<0.05;CH+5-HD组,与缺氧组比较,R-123荧光强度明显减弱,线粒体膜电位部分消失,细胞内氧自由基含量明显下降,细胞呈增殖明显减少、凋亡明显增多,P<0.05。
结论:本实验结果显示,Diazoxide能够通过开放线粒体膜上ATP敏感的钾通道,引起Δψ_m去极化,Δψ_m的变化影响细胞内氧自由基的含量,氧自由基作为信号分子影响肺动脉平滑肌细胞的增殖/凋亡的平衡。
第二部分线粒体膜ATP敏感钾通道在缺氧性人肺动脉平滑肌细胞增殖/凋亡平衡中的作用
分题一线粒体膜ATP敏感钾通道对缺氧人肺动脉平滑肌细胞细胞内细胞色素C分布的变化及细胞增殖中的作用
目的:为了探索线粒体ATP敏感钾通道(mitochondrial ATP-sensitive K+ channel, MitoK_(ATP))和线粒体膜电位(mitochondrial membrane potential,Δψm)在细胞缺氧信号传导中的作用以及在缺氧性人肺动脉平滑肌细胞细胞色素C在细胞内的分布及细胞增殖中的影响。
方法:本实验将人动脉平滑肌细胞进行常氧或24小时缺氧培养,并将标本分为六组:①对照组;②MitoK_(ATP)阻断剂5-HD组;③MitoK_(ATP)开放剂Diazoxide组;④24小时缺氧组;⑤24小时缺氧合并5-HD组;⑥24小时缺氧合并Diazoxide组。利用激光共聚焦显微镜(Leica SP-1 USA)成像法检测线粒体膜电位;线粒体/胞浆成分分离试剂盒(BioVision)分离线粒体和胞浆成分后,Western blot法检测两者细胞色素C;Western blot法检测细胞中caspase-9的蛋白表达量;MTT法及PI染色后流式细胞仪检测细胞增殖情况。
结果:①Diazoxide作用24 h后,R123荧光明显增强,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞呈明显增殖增多、凋亡减少,与正常对照组相比较均P<0.05;而5-HD作用24 h与正常对照组比较,上述指标无明显变化, P>0.05。②缺氧24 h后,结果与Diazoxide组相似,R123荧光明显增强,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞呈明显增殖增多、凋亡减少,与正常对照组相比较均P<0.05;24小时缺氧合并Diazoxide组与缺氧组相比较,R123荧光明显增强,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞呈明显增殖增多、凋亡减少,P<0.05;而24小时缺氧合并5-HD组与缺氧组比较,R123荧光明显降低,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显升高,caspase-9的蛋白表达显著增加,细胞呈明显增殖减少、凋亡增多,P<0.05。
结论:缺氧可以引起MitoK_(ATP)的开放以及Δψ_m的去极化,并进而抑制了细胞色素C从细胞线粒体释放到细胞浆,抑制线粒体凋亡途径。
分题二线粒体膜ATP敏感钾通道对缺氧人肺动脉平滑肌细胞氧自由基的变化及细胞增殖中的作用
目的:研究线粒体膜上ATP敏感钾通道(MitoK_(ATP))开放剂Diazoxide和线粒体膜电位(Δψ_m)在缺氧影响人肺动脉平滑肌细胞细胞内氧自由基的变化及细胞增殖中的作用,探索缺氧性肺动脉重建和肺动脉高压形成的发生机制,为探索新的防治方法的提供试验依据。
方法:培养人肺动脉平滑肌细胞(HPASMCs),进行常氧或慢性缺氧培养。将标本分为六组:①对照组;②MitoK_(ATP)阻断剂5-HD组;③MitoK_(ATP)开放剂Diazoxide组;④慢性缺氧组;⑤慢性缺氧合并5-HD组;⑥慢性缺氧合并Diazoxide组。利用激光共焦显微镜(Leica SP-1 USA)成像检测线粒体膜电位,荧光染色检测细胞内氧自由基含量,免疫组化法检测增殖细胞核抗原(PCNA)、C-fos及C-jun的表达和MTT法检测细胞增殖情况。
结果:①Diazoxide作用24h后,与正常对照组(R123荧光强度、细胞内氧自由基含量、PCNA、C-fos、C-jun的蛋白的积光度值及MTT的A值分别为74.67±7.02、2771.69±48.66、42.55±1.97%、68.58±21.54、76.18±12.65及0.2368±0.013)比较,R-123荧光强度(105.45±4.38)明显增强,线粒体膜电位去极化,细胞内氧自由基含量(3044.69±126.34)明显增加,PCNA(53.52±2.28%)、C-fos(164.47±53.47)及C-jun(130.25±10.39 )的蛋白表达明显增加,MTT的A值(0.3048±0.022)也明显增加,P<0.05;而5-HD作用24h后,上述指标与正常对照组相比较,均无显著性差异,P>0.05;②慢性缺氧24h,结果与Diazoxide组相似,与正常对照组比较,R-123荧光强度(95.24±12.92)明显增强,线粒体膜电位去极化,细胞内氧自由基含量(3115.88±34.12)明显增加,PCNA(54.55±2.14%)、C-fos (160.95±47.49)及C-jun(127.87±17.44)的表达明显增加,MTT的A值(0.3282±0.078)也明显增加,P<0.05;CH+Diazoxide组,与缺氧组比较, R-123荧光强度(126.47±7.63)明显增强,线粒体膜电位去极化,细胞内氧自由基含量(3236.01±30.86)明显增加, PCNA (66.13±2.59%)、C-fos ( 370.65±90.85)及C-jun (275.97±49.77)的表达明显增加,MTT的A值(0.4398±0.023)也明显增加,P<0.05;CH+5-HD组,与缺氧组比较,R-123荧光强度(70.69±3.73)明显减弱,线粒体膜电位部分消失,细胞内氧自由基含量(2863.08±132.06)明显减少,PCNA(43.48±1.23%)、C-fos (70.87±21.46)及C-jun (79.87±12.75)的表达明显减少,MTT的A值(0.2637±0.045)也明显减少,P<0.05。
结论:Diazoxide能够通过开放线粒体膜上ATP敏感的钾通道,引起Δψ_m去极化,Δψm的变化影响细胞内氧自由基的含量,氧自由基作为信号分子影响肺动脉平滑肌细胞的增殖/凋亡的平衡,从而参与并影响了肺动脉高压的发生发展。
Backgroud
So far,the incidence and mortality of pulmonary hypertension and pulmonary heart disease still remain high. Most of pulmonary hypertension is hypoxic pulmonary hypertension (HPH), and the mechanism of hypoxic pulmonary hypertension has still not been fully elucidated. HPH has two main pathophysiological characters: hypoxic pulmonary artery vasoconstriction (HPV) and hypoxic pulmonary vascular remodeling. HPV has been demonstrated not only in perfused lungs but also in pulmonary arterial rings denuded of endothelium and in single pulmonary arterial smooth muscle cells (PASMCs). Recently, the potassium channel and the cytoplasmic free calcium concentration have been considered to play very important role in the regulation of vascular tone. The activity of voltage-gated potassium channels (Kv) controls the cell membrane potential, which subsequently regulates the cytoplasmic free calcium concentration and the proliferation of PASMCs. Studies have shown that acute or chronic hypoxia can inhibit the function of Kv in PASMCs, inducing membrane depolarization and a rise in intracellular free Ca2+ concentration ([Ca~(2+)]_i), that triggers vasoconstriction and stimulates the proliferation of PASMCs.
The mitochondrial ATP-sensitive K+ channel has been implicated in cellular protection against metabolic stress in a variety of tissues, including liver, gut, brain, and kidney, and has been shown to be an essential component of the mechanism of ischemic preconditioning in the heart. It is unclear how the opening of a K+ channel in the mitochondria would lead to cardioprotection. However three hypotheses have emerged to explain the link between mitoK_(ATP) channel opening and cardioprotection: (1) A decrease in the mitochondrial Ca~(2+) uptake. Holmuhamedov et al showed that diazoxide and pinacidil could reduce the magnitude of mitochondrial Ca2+ uptake, and the effect of both can be reversed by 5-HD. This change in mitochondrial Ca2+ uptake was thought to be mediated by a partial depolarization of mitochondrial membrane potential ( m) in response to mitoK_(ATP) opening. (2) Swelling of the mitochondrial matrix and changes in ATP synthesis. It has been known for some time that the opening of mitoK_(ATP) causes mitochondrial matrix swelling, and that this in turn activates the respiratory chain providing more ATP to support the recovering myocardium. (3) Changes in the levels of reactive oxygen species (ROS). Reactive oxygen species are a double-edge sword when it comes to cardioprotection. The ROS generated during the preconditioning period is thought to be protective. However, the ROS that is produced during reperfusion, is detrimental and causes cell death. It is thought that the opening of mitoK_(ATP) results in an increase in the protective ROS produced during preconditioning phase and a decrease in the levels of ROS generated during reperfusion phase.
A study of our lab suggested that the opening of mitoK_(ATP) followed by a depolarization of m in hypoxia might contribute to the alterations in the expression of cell membrane Kv1.5 mRNA and protein leading to the change in the cell membrane potential of hypoxic HPASMCs. This study continue to investige the detail mechanism about the role of mitochondrial ATP-sensitive K+ channel on the unbalance between proliferation and apoptosis of hypoxic rat and human pulmonary artery smooth muscle cells.
Part 1 The role of mitochondrial ATP-sensitive K+ channel on the balance between proliferation and apoptosis of hypoxic rat pulmonary artery smooth muscle cells
Subject 1 The effect of mitochondrial ATP-sensitive K+ channel on distributing of cytochrome C in rat pulmonary artery smooth muscle cells and on proliferation of hypoxic rat pulmonary artery smooth muscle cells
Objective : The objective of this paper is to investigate the contribution of mitochondrial ATP-sensitive K+ channel (MitoK_(ATP)) and mitochondrial membrane potential ( m) to distributing of cytochrome C in rat pulmonary artery smooth muscle cells and on proliferation of rat pulmonary artery smooth muscle cells induced by hypoxia.
Methods:Fresh normal rat lung tissues were obtained. Then rat PASMCs were isolated and cultured, which were divided into 6 groups, as follow:①control group: cultured under normoxia;②diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP);③5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP);④c hronic hypoxia group: cultured under hypoxia;⑤chronic hypoxia+diazoxide group;⑥chronic hypoxia +5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. Western blot technique was used to trace the expression of cytochrome C protein in cell plasma and mitochondria respectively. The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.
Results:After exposed to diazoxide for 24h, the intensity of R-123 fluorescence in normoxic rat PASMCs were significantly increased as compared with control group (P<0.05), but there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h (P>0.05); chronic hypoxia or chronic hypoxia+diazoxide could markedly increase the intensity of R-123 fluorescence in rat PASMC as compared with control group (P<0.05), the changes were more significant in chronic hypoxia +diazoxide group than those of chronic hypoxia group (P<0.05); 5-HD could partly weaken the effect of hypoxia on the intensity of R-123 fluorescence (P<0.05) too, After exposed to diazoxide for 24h, the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria were significantly decreased as compared with control group (P<0.05), The A value were significantly increased as compared with control group(P<0.05),and the apoptosis of rat PASMCs were significantly decreased as compared with control group(P<0.05) . But there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h; After exposed to hypoxia or hypoxia+diazoxide for 24h, the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria were significantly decreased as compared with control group (P<0.05), the A value were significantly increased as compared with control group (P<0.05), and the apoptosis of rat PASMCs were significantly decreased as compared with control group (P<0.05); The changes were more significant in hypoxia +diazoxide group than those of hypoxia group (P<0.05). 5-HD could partly weaken the effect of hypoxia on the changes of the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria in rat pulmonary artery smooth muscle cells and on the proliferation of rat PASMCs induced by hypoxia (P<0.05).
Conclusion:The results suggested that the opening of MitoK_(ATP) followed by a depolarization of m might contribute to the inhibition of the release of cytochrome C from cell mitochondria to cell plasma in rat PASMCs and proliferation of rat PASMCs induced by hypoxia. This might be a mechanism of the development of hypoxic pulmonary hypertension.
Subject 2 The effect of mitochondrial ATP-sensitive K+ channel on changes of reactive oxygen species in rat pulmonary artery smooth muscle cells and on proliferation of hypoxic rat pulmonary artery smooth muscle cells
Objective : The objective of this paper is to investigate the contribution of mitochondrial ATP-sensitive K+ channel (MitoK_(ATP)) and mitochondrial membrane potential ( m) to changes of H2O2 in rat pulmonary artery smooth muscle cells and on proliferation of hypoxic rat pulmonary artery smooth muscle cells.
Methods:Fresh normal rat lung tissues were obtained. Then rat PASMCs were isolated and cultured, which were divided into 6 groups, as follow:①control group: cultured under normoxia;②diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP);③5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP);④c hronic hypoxia group: cultured under hypoxia;⑤chronic hypoxia+diazoxide group;⑥chronic hypoxia +5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. The level of H2O2 in rat PASMCs were tested with chemiluminescence method. The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.
Results:After exposed to diazoxide for 24h, the intensity of R-123 fluorescence in normoxic rat PASMCs were significantly increased as compared with control group (P<0.05), but there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h (P>0.05); chronic hypoxia or chronic hypoxia+diazoxide could markedly increase the intensity of R-123 fluorescence in rat PASMC as compared with control group (P<0.05), the changes were more significant in chronic hypoxia +diazoxide group than those of chronic hypoxia group (P<0.05); 5-HD could partly weaken the effect of hypoxia on the intensity of R-123 fluorescence (P<0.05) too, After exposed to diazoxide for 24h, the level of H2O2 in rat PASMCs were significantly increased as compared with control group (P<0.05), The A value were significantly increased as compared with control group(P<0.05),and the apoptosis of rat PASMCs were significantly decreased as compared with control group(P<0.05) . But there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h; After exposed to hypoxia or hypoxia+diazoxide for 24h, the level of H2O2 in rat PASMCs were significantly increased as compared with control group (P<0.05), the A value were significantly increased as compared with control group (P<0.05), and the apoptosis of rat PASMCs were significantly decreased as compared with control group (P<0.05); The changes were more significant in hypoxia +diazoxide group than those of hypoxia group (P<0.05). 5-HD could partly weaken the effect of hypoxia on the changes of the level of H2O2 in rat pulmonary artery smooth muscle cells and on the proliferation of rat PASMCs induced by hypoxia (P<0.05).
Conclusion:The results suggested that the opening of MitoK_(ATP) followed by a depolarization of m might contribute to the the increasing of the level of H2O2 in rat PASMCs and proliferation of rat PASMCs induced by hypoxia. This might be a mechanism of the development of hypoxic pulmonary hypertension.
Part 2 The role of mitochondrial ATP-sensitive K+ channel on the balance between proliferation and apoptosis of hypoxic human pulmonary artery smooth muscle cells
Subject 1 The effect of mitochondrial ATP-sensitive K+ channel on distributing of cytochrome C in human pulmonary artery smooth muscle cells and on proliferation of hypoxic human pulmonary artery smooth muscle cells
Objective : The objective of this paper is to investigate the contribution of mitochondrial ATP-sensitive K+ channel (MitoK_(ATP)) and mitochondrial membrane potential (Δψ_m) to distributing of cytochrome C in human pulmonary arterial smooth muscle cells (HPASMCs) and to proliferation of HPASMCs induced by hypoxia.
Methods:HPASMCs were divided into several groups, as following:①control group: cultured under normoxia;②diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP);③5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP);④2 4-hours hypoxia group: cultured under hypoxia for 24 hours;⑤2 4-hours hypoxia+diazoxide group, cultured under hypoxia with diazoxide for 24 hours;⑥2 4-hours hypoxia+5-HD group, cultured under hypoxia with 5-HD for 24 hours. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. Western blot technique was used to trace the expression of cytochrome C protein in cell plasma and mitochondria respectively. The expression of cell caspase-9 protein was determined with western blot technique, too. The proliferation of HPASMCs was examined by cell cycle analysis and MTT colorimetric assay.
Results:After exposed to diazoxide for 24 h, the intensity of R-123 fluorescence in normoxic HPASMCs was significantly increased as compared with control group (P<0.05), but there was no significant change of the intensity of R-123 fluorescence after the HPASMCs had been exposed to 5-HD for 24 h; 24-hours hypoxia or 24-hours hypoxia+diazoxide could markedly increase the intensity of R-123 fluorescence in HPASMC as compared with control group (P<0.05), the change was more significant in 24-hours hypoxia +diazoxide group than that of 24-hours hypoxia group (P<0.05); 5-HD could weaken the effect of 24-hours hypoxia on the intensity of R-123 fluorescence. After exposed to diazoxide for 24 h, the ratio of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein was significantly decreased as compared with control group (P<0.05), the expression of caspase-9 protein was significantly decreased as compared with control group (P<0.05), the percentage of S phase and A value of MTT were significantly increased as compared with control group (P<0.05). But there were no significant changes of these tests after the HPASMCs had been exposed to 5-HD for 24 h (P>0.05). After exposed to hypoxia or hypoxia+diazoxide for 24 h, the rate of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein and the expression of caspase-9 protein were significantly decreased as compared with control group (P<0.05), the percentage of S phase and A value of MTT were significantly increased as compared with control group (P<0.05). These changes were more significant in 24-hours hypoxia +diazoxide group than those of 24-hours hypoxia group (P<0.05). 5-HD could weaken the effect of hypoxia on the changes of distributing of cytochrome C, the expression of caspase-9 in human pulmonary arterial smooth muscle cells and the proliferation of HPASMCs induced by hypoxia (P<0.05).
Conclusion:All these results suggested that the opening of MitoK_(ATP) followed by a depolarization ofΔψ_m induced by hypoxia might contribute to the inhibition of the release of cytochrome C from cell mitochondria to cell plasma in HPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension. The signal transduction pathway of mitochondria might play an important role in the relationship betweenΔψ_m and apoptosis of HPASMCs.
Subject 2 The effect of mitochondrial ATP-sensitive K+ channel on changes of reactive oxygen species in human pulmonary artery smooth muscle cells and on proliferation of hypoxic human pulmonary artery smooth muscle cells
Objective:The objective of this paper is to investigate the contribution of diazoxide, an opener of mitochondrial ATP-sensitive K+ channel (MitoK_(ATP)), and mitochondrial membrane potential(Δψ_m) to changes of reactive oxygen species in human pulmonary arterial smooth muscle cells (HPASMCs) and to proliferation of HPASMCs induced by hypoxia.
Methods:HPASMCs were divided into several groups, as follow:①control group: cultured under normoxia;②diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP);③5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP);④chronic hypoxia group: cultured under hypoxia for 24 hours;⑤chronic hypoxia+diazoxide group;⑥chronic hypoxia+5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. The level of H2O2 in HPASMCs was tested with chemiluminescence method. The proliferation of HPASMCs was examined by examing the expression of PCNA, c-fos and c-jun proteins, and by MTT colorimetric assay.
Results:After exposed to diazoxide for 24h, the intensity of R-123 fluorescence in normoxic HPASMCs(105.45±4.38) was significantly increased as compared with control group (74.67±7.02) (P<0.05), but there was no significant change in this test after the HPASMCs had been exposed to 5-HD for 24h; chronic hypoxia (95.24±12.92)or chronic hypoxia +diazoxide (126.47±7.63)could markedly increase the intensity of R-123 fluorescence in HPASMC as compared with control group (P<0.05), the change was more significant in chronic hypoxia +diazoxide group than that of hypoxia group (P<0.05); CH+5-HD(70.69±3.73) could weaken the effect of hypoxia on the intensity of R-123 fluorescence. After exposed to diazoxide for 24h, the level of H2O2(3044.69±126.34)in HPASMCs was significantly increased as compared with control group (2771.69±48.66) (P<0.05), the expression of PCNA(53.52±2.28%), c-fos(164.47±53.47) and c-jun(130.25±10.39 ) proteins were significantly increased as compared with control group(PCNA 42.55±1.97%,c-fos 68.58±21.54 and c-jun 76.18±12.65 ) (P<0.05) too, The A value ( 0.3048±0.022 ) was significantly increased as compared with control group(0.2368±0.013) (P<0.05). But there were no significant changes in these tests after the HPASMCs had been exposed to 5-HD for 24h; After exposed to chronic hypoxia for 24h, the level of H2O2(3115.88±34.12) in HPASMCs was significantly increased as compared with control group (P<0.05), the expression of PCNA(54.55±2.14%), c-fos (160.95±47.49)and c-jun (127.87±17.44)proteins were significantly increased as compared with control group (P<0.05) too, The A value (0.3282±0.078)was significantly increased as compared with control group(P<0.05). These changes were more significant in chronic hypoxia +diazoxide group( the level of H2O2 3236.01±30.86,PCNA 66.13±2.59%, C-fos 370.65±90.85,C-jun 275.97±49.77 and A value 0.4398±0.023) than those of hypoxia group (P<0.05); 5-HD could weaken the effect of hypoxia on the changes of the level of H2O2 (2863.08±132.06) in human pulmonary arterial smooth muscle cells and on the proliferation of HPASMCs ( PCNA 43.48±1.23%,C-fos 70.87±21.46 , C-jun 79.87±12.75 and A value 0.2637±0.045) induced by hypoxia (P<0.05).
Conclusion:The results suggested that the opening of MitoK_(ATP) followed by a depolarization ofΔψ_m might contribute to the increasing of the level of ROS in HPASMCs. The ROS in HPASMCs play an important role in the prolifeation of HPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension.
迄今为止,肺动脉高压和肺心病的发病率和死亡率仍然居高不下,严重威胁着人类的健康。缺氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)是最常见的类型,其发病机制目前尚未完全阐述清楚,其中缺氧性肺血管收缩(hypoxic pulmonary artery vasoconstriction,HPV)和缺氧性肺血管重建(hypoxic pulmonary vascular remodeling)是被认为是其两大主要的病理生理基础。目前认为,缺氧引起的肺血管收缩并非完全依赖于血管内皮细胞,血管平滑肌细胞也参与了肺血管收缩反应。在影响血管张力的各种因素中,血管平滑肌细胞钾通道和细胞内钙离子发挥着十分重要的作用。研究表明缺氧可抑制肺动脉平滑肌细胞钾通道的活性,引起肺动脉平滑肌细胞(PASMCs)去极化,达到一定阈值后,电压依赖性钙通道开放,引起细胞内钙离子浓度升高,作为重要的第二信使,钙离子促使肺动脉平滑肌细胞收缩、增殖和迁移,也可激活各种蛋白激酶和转录因子,进一步引起缺氧性肺血管收缩和肺血管重建,最终导致肺动脉高压。
有一系列研究表明线粒体膜上ATP敏感的钾通道(MitoK_(ATP))能保护缺血缺氧心肌细胞免受氧化应激的伤害。Takashi等证实线粒体膜上ATP敏感的钾通道的开放能减少心肌细胞缺血/再灌注损伤时的凋亡标记物(细胞色素C的释放,Caspase活性,线粒体膜电位的改变等等)。Akao和他的同事们也研究了mitoK_(ATP)在凋亡中的作用,他们发现线粒体膜上ATP敏感钾通道的开放能抑制H2O2诱导的新生大鼠心肌细胞的凋亡。线粒体mitoK_(ATP)通道的开放为什么能起到保护心肌细胞的机制还不十分清楚,但是目前有三种假设解释mitoK_(ATP)通道与心肌细胞之间的联系:1)线粒体钙离子摄取的下降,Holmuhameclov等证实线粒体mitoK_(ATP)通道的开放能够降低线粒体钙离子摄取的程度。2)线粒体基质肿胀和ATP合成的改变,mitoK_(ATP)的开放能引起线粒体基质肿胀,并激活呼吸链产生更多的ATP,支持心肌细胞的恢复。3)氧自由基(ROS)水平的改变,在心肌细胞的保护中,氧自由基是一把双刃剑。预处理阶段产生的ROS是具有保护作用的,但是在再灌注阶段产生的ROS是有害的。mitoK_(ATP)通道的开放能引起预处理期保护性ROS水平的增加,和再灌注期产生的ROS水平的降低。
尽管这些研究表明线粒体mitoK_(ATP)通道在心血管细胞的增殖凋亡中起很重要的作用,但是线粒体mitoK_(ATP)通道在肺血管中的作用很少有人研究,线粒体mitoK_(ATP)通道是否与缺氧性肺动脉平滑肌细胞增殖/凋亡失衡有关以及其机制目前还没有相关报道。线粒体mitoK_(ATP)通道对动物和人的肺动脉平滑肌细胞增殖/凋亡平衡的调节是否具有一致性也还没有相关报道,但是由于进行人肺血管的体外和体内的实验研究是比较昂贵的和具有局限性的,因此对比动物实验和以人为实验对象的实验是否具有一致性,从而决定是否以动物实验代替以人为实验对象的实验,对以后的实验研究是具有重要意义的。
第一部分线粒体膜ATP敏感钾通道在缺氧性大鼠肺动脉平滑肌细胞增殖/凋亡失衡中的作用
分题一线粒体膜ATP敏感钾通道对缺氧大鼠肺动脉平滑肌细胞细胞内细胞色素C分布的变化及细胞增殖中的作用
目的:研究线粒体膜上ATP敏感的钾通道(mitoK_(ATP))和线粒体膜电位(Δψ_m)在缺氧影响大鼠肺动脉平滑肌细胞内细胞色素C在细胞浆和线粒体内分布的变化及以及其引起细胞增殖变化中的作用,旨在探索缺氧性肺动脉重建和肺动脉高压形成的发生机制,为寻找新的防治方法提供试验依据。
方法:获取大鼠正常肺组织,分离出肺动脉平滑肌细胞(PASMCs)进行常氧或慢性缺氧培养。将标本分为六组:①正常对照组;②MitoK_(ATP)阻断剂5-HD组;③MitoK_(ATP)开放剂Diazoxide组;④慢性缺氧(CH)组;⑤CH+5-HD组;⑥CH+Diazoxide组。利用激光共聚焦显微镜(Leica SP-1 USA)成像检测线粒体膜电位,线粒体/胞浆成分分离试剂盒(BioVision)分离线粒体和胞浆成分后,Western blot法检测两者细胞色素C,流式细胞仪检测细胞周期和MTT法检测细胞增殖情况。
结果:①Diazoxide作用24h后,与正常对照组比较,R-123荧光强度明显增强,线粒体膜电位去极化,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,细胞呈增殖明显增多、凋亡明显减少,与正常对照组相比较均P<0.05;而5-HD作用24h与正常对照组比较,上述指标无明显变化, P>0.05;;②慢性缺氧24h,结果与Diazoxide组相似,与正常对照组比较,R-123荧光强度明显增强,线粒体膜电位去极化,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,细胞呈增殖明显增多、凋亡明显减少,与正常对照组相比较均P<0.05;;CH+Diazoxide组,与缺氧组比较, R-123荧光强度明显增强,线粒体膜电位去极化,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,细胞呈增殖明显增多、凋亡明显减少,与正常对照组相比较均P<0.05; CH+5-HD组,与缺氧组比较,R-123荧光强度明显减弱,线粒体膜电位部分消失,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显升高,细胞增殖明显减少、凋亡明显增多,与正常对照组相比较均P<0.05。
结论:本实验结果显示,缺氧可以引起MitoK_(ATP)的开放以及m的去极化,并进而抑制了细胞色素C从细胞线粒体释放到细胞浆,抑制线粒体凋亡途径,从而参与并影响了肺动脉高压的发生发展。
分题二线粒体膜ATP敏感钾通道对缺氧大鼠肺动脉平滑肌细胞氧自由基的变化及细胞增殖中的作用
目的:研究线粒体ATP敏感性钾通道和线粒体膜电位(Δψ_m)在缺氧影响大鼠肺动脉平滑肌细胞细胞内氧自由基的变化及细胞增殖中的作用,旨在探索缺氧性肺动脉重建和肺动脉高压形成的发生机制,为寻找新的防治方法的提供试验依据。
方法:获取大鼠正常肺组织,分离出肺动脉平滑肌细胞(PASMCs)进行常氧或慢性缺氧培养。将标本分为六组:①正常对照组;②MitoK_(ATP)阻断剂5-HD组;③MitoK_(ATP)开放剂Diazoxide组;④慢性缺氧(CH)组;⑤CH+5-HD组;⑥CH+Diazoxide组。利用激光共焦显微镜(Leica SP-1 USA)成像检测线粒体膜电位,荧光染色检测细胞内氧自由基含量,流式细胞仪检测细胞周期和MTT法检测细胞增殖情况。
结果:①Diazoxide作用24h后,与正常对照组比较,R-123荧光强度明显增强,线粒体膜电位去极化,细胞内氧自由基含量明显增加,细胞呈增殖明显增多、凋亡明显减少,P<0.05;而5-HD作用24h后,上述指标与正常对照组相比较,均无显著性差异, P>0.05;②慢性缺氧24h,结果与Diazoxide组相似,与正常对照组比较,R-123荧光强度明显增强,线粒体膜电位去极化,细胞内氧自由基含量明显增加,细胞呈增殖明显增多、凋亡明显减少,P<0.05;CH+Diazoxide组,与缺氧组比较, R-123荧光强度明显增强,线粒体膜电位去极化,细胞内氧自由基含量明显增加,细胞呈增殖明显增多、凋亡明显减少,P<0.05;CH+5-HD组,与缺氧组比较,R-123荧光强度明显减弱,线粒体膜电位部分消失,细胞内氧自由基含量明显下降,细胞呈增殖明显减少、凋亡明显增多,P<0.05。
结论:本实验结果显示,Diazoxide能够通过开放线粒体膜上ATP敏感的钾通道,引起Δψ_m去极化,Δψ_m的变化影响细胞内氧自由基的含量,氧自由基作为信号分子影响肺动脉平滑肌细胞的增殖/凋亡的平衡。
第二部分线粒体膜ATP敏感钾通道在缺氧性人肺动脉平滑肌细胞增殖/凋亡平衡中的作用
分题一线粒体膜ATP敏感钾通道对缺氧人肺动脉平滑肌细胞细胞内细胞色素C分布的变化及细胞增殖中的作用
目的:为了探索线粒体ATP敏感钾通道(mitochondrial ATP-sensitive K+ channel, MitoK_(ATP))和线粒体膜电位(mitochondrial membrane potential,Δψm)在细胞缺氧信号传导中的作用以及在缺氧性人肺动脉平滑肌细胞细胞色素C在细胞内的分布及细胞增殖中的影响。
方法:本实验将人动脉平滑肌细胞进行常氧或24小时缺氧培养,并将标本分为六组:①对照组;②MitoK_(ATP)阻断剂5-HD组;③MitoK_(ATP)开放剂Diazoxide组;④24小时缺氧组;⑤24小时缺氧合并5-HD组;⑥24小时缺氧合并Diazoxide组。利用激光共聚焦显微镜(Leica SP-1 USA)成像法检测线粒体膜电位;线粒体/胞浆成分分离试剂盒(BioVision)分离线粒体和胞浆成分后,Western blot法检测两者细胞色素C;Western blot法检测细胞中caspase-9的蛋白表达量;MTT法及PI染色后流式细胞仪检测细胞增殖情况。
结果:①Diazoxide作用24 h后,R123荧光明显增强,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞呈明显增殖增多、凋亡减少,与正常对照组相比较均P<0.05;而5-HD作用24 h与正常对照组比较,上述指标无明显变化, P>0.05。②缺氧24 h后,结果与Diazoxide组相似,R123荧光明显增强,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞呈明显增殖增多、凋亡减少,与正常对照组相比较均P<0.05;24小时缺氧合并Diazoxide组与缺氧组相比较,R123荧光明显增强,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞呈明显增殖增多、凋亡减少,P<0.05;而24小时缺氧合并5-HD组与缺氧组比较,R123荧光明显降低,细胞胞浆细胞色素C与线粒体细胞色素C的比值明显升高,caspase-9的蛋白表达显著增加,细胞呈明显增殖减少、凋亡增多,P<0.05。
结论:缺氧可以引起MitoK_(ATP)的开放以及Δψ_m的去极化,并进而抑制了细胞色素C从细胞线粒体释放到细胞浆,抑制线粒体凋亡途径。
分题二线粒体膜ATP敏感钾通道对缺氧人肺动脉平滑肌细胞氧自由基的变化及细胞增殖中的作用
目的:研究线粒体膜上ATP敏感钾通道(MitoK_(ATP))开放剂Diazoxide和线粒体膜电位(Δψ_m)在缺氧影响人肺动脉平滑肌细胞细胞内氧自由基的变化及细胞增殖中的作用,探索缺氧性肺动脉重建和肺动脉高压形成的发生机制,为探索新的防治方法的提供试验依据。
方法:培养人肺动脉平滑肌细胞(HPASMCs),进行常氧或慢性缺氧培养。将标本分为六组:①对照组;②MitoK_(ATP)阻断剂5-HD组;③MitoK_(ATP)开放剂Diazoxide组;④慢性缺氧组;⑤慢性缺氧合并5-HD组;⑥慢性缺氧合并Diazoxide组。利用激光共焦显微镜(Leica SP-1 USA)成像检测线粒体膜电位,荧光染色检测细胞内氧自由基含量,免疫组化法检测增殖细胞核抗原(PCNA)、C-fos及C-jun的表达和MTT法检测细胞增殖情况。
结果:①Diazoxide作用24h后,与正常对照组(R123荧光强度、细胞内氧自由基含量、PCNA、C-fos、C-jun的蛋白的积光度值及MTT的A值分别为74.67±7.02、2771.69±48.66、42.55±1.97%、68.58±21.54、76.18±12.65及0.2368±0.013)比较,R-123荧光强度(105.45±4.38)明显增强,线粒体膜电位去极化,细胞内氧自由基含量(3044.69±126.34)明显增加,PCNA(53.52±2.28%)、C-fos(164.47±53.47)及C-jun(130.25±10.39 )的蛋白表达明显增加,MTT的A值(0.3048±0.022)也明显增加,P<0.05;而5-HD作用24h后,上述指标与正常对照组相比较,均无显著性差异,P>0.05;②慢性缺氧24h,结果与Diazoxide组相似,与正常对照组比较,R-123荧光强度(95.24±12.92)明显增强,线粒体膜电位去极化,细胞内氧自由基含量(3115.88±34.12)明显增加,PCNA(54.55±2.14%)、C-fos (160.95±47.49)及C-jun(127.87±17.44)的表达明显增加,MTT的A值(0.3282±0.078)也明显增加,P<0.05;CH+Diazoxide组,与缺氧组比较, R-123荧光强度(126.47±7.63)明显增强,线粒体膜电位去极化,细胞内氧自由基含量(3236.01±30.86)明显增加, PCNA (66.13±2.59%)、C-fos ( 370.65±90.85)及C-jun (275.97±49.77)的表达明显增加,MTT的A值(0.4398±0.023)也明显增加,P<0.05;CH+5-HD组,与缺氧组比较,R-123荧光强度(70.69±3.73)明显减弱,线粒体膜电位部分消失,细胞内氧自由基含量(2863.08±132.06)明显减少,PCNA(43.48±1.23%)、C-fos (70.87±21.46)及C-jun (79.87±12.75)的表达明显减少,MTT的A值(0.2637±0.045)也明显减少,P<0.05。
结论:Diazoxide能够通过开放线粒体膜上ATP敏感的钾通道,引起Δψ_m去极化,Δψm的变化影响细胞内氧自由基的含量,氧自由基作为信号分子影响肺动脉平滑肌细胞的增殖/凋亡的平衡,从而参与并影响了肺动脉高压的发生发展。
Backgroud
So far,the incidence and mortality of pulmonary hypertension and pulmonary heart disease still remain high. Most of pulmonary hypertension is hypoxic pulmonary hypertension (HPH), and the mechanism of hypoxic pulmonary hypertension has still not been fully elucidated. HPH has two main pathophysiological characters: hypoxic pulmonary artery vasoconstriction (HPV) and hypoxic pulmonary vascular remodeling. HPV has been demonstrated not only in perfused lungs but also in pulmonary arterial rings denuded of endothelium and in single pulmonary arterial smooth muscle cells (PASMCs). Recently, the potassium channel and the cytoplasmic free calcium concentration have been considered to play very important role in the regulation of vascular tone. The activity of voltage-gated potassium channels (Kv) controls the cell membrane potential, which subsequently regulates the cytoplasmic free calcium concentration and the proliferation of PASMCs. Studies have shown that acute or chronic hypoxia can inhibit the function of Kv in PASMCs, inducing membrane depolarization and a rise in intracellular free Ca2+ concentration ([Ca~(2+)]_i), that triggers vasoconstriction and stimulates the proliferation of PASMCs.
The mitochondrial ATP-sensitive K+ channel has been implicated in cellular protection against metabolic stress in a variety of tissues, including liver, gut, brain, and kidney, and has been shown to be an essential component of the mechanism of ischemic preconditioning in the heart. It is unclear how the opening of a K+ channel in the mitochondria would lead to cardioprotection. However three hypotheses have emerged to explain the link between mitoK_(ATP) channel opening and cardioprotection: (1) A decrease in the mitochondrial Ca~(2+) uptake. Holmuhamedov et al showed that diazoxide and pinacidil could reduce the magnitude of mitochondrial Ca2+ uptake, and the effect of both can be reversed by 5-HD. This change in mitochondrial Ca2+ uptake was thought to be mediated by a partial depolarization of mitochondrial membrane potential ( m) in response to mitoK_(ATP) opening. (2) Swelling of the mitochondrial matrix and changes in ATP synthesis. It has been known for some time that the opening of mitoK_(ATP) causes mitochondrial matrix swelling, and that this in turn activates the respiratory chain providing more ATP to support the recovering myocardium. (3) Changes in the levels of reactive oxygen species (ROS). Reactive oxygen species are a double-edge sword when it comes to cardioprotection. The ROS generated during the preconditioning period is thought to be protective. However, the ROS that is produced during reperfusion, is detrimental and causes cell death. It is thought that the opening of mitoK_(ATP) results in an increase in the protective ROS produced during preconditioning phase and a decrease in the levels of ROS generated during reperfusion phase.
A study of our lab suggested that the opening of mitoK_(ATP) followed by a depolarization of m in hypoxia might contribute to the alterations in the expression of cell membrane Kv1.5 mRNA and protein leading to the change in the cell membrane potential of hypoxic HPASMCs. This study continue to investige the detail mechanism about the role of mitochondrial ATP-sensitive K+ channel on the unbalance between proliferation and apoptosis of hypoxic rat and human pulmonary artery smooth muscle cells.
Part 1 The role of mitochondrial ATP-sensitive K+ channel on the balance between proliferation and apoptosis of hypoxic rat pulmonary artery smooth muscle cells
Subject 1 The effect of mitochondrial ATP-sensitive K+ channel on distributing of cytochrome C in rat pulmonary artery smooth muscle cells and on proliferation of hypoxic rat pulmonary artery smooth muscle cells
Objective : The objective of this paper is to investigate the contribution of mitochondrial ATP-sensitive K+ channel (MitoK_(ATP)) and mitochondrial membrane potential ( m) to distributing of cytochrome C in rat pulmonary artery smooth muscle cells and on proliferation of rat pulmonary artery smooth muscle cells induced by hypoxia.
Methods:Fresh normal rat lung tissues were obtained. Then rat PASMCs were isolated and cultured, which were divided into 6 groups, as follow:①control group: cultured under normoxia;②diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP);③5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP);④c hronic hypoxia group: cultured under hypoxia;⑤chronic hypoxia+diazoxide group;⑥chronic hypoxia +5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. Western blot technique was used to trace the expression of cytochrome C protein in cell plasma and mitochondria respectively. The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.
Results:After exposed to diazoxide for 24h, the intensity of R-123 fluorescence in normoxic rat PASMCs were significantly increased as compared with control group (P<0.05), but there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h (P>0.05); chronic hypoxia or chronic hypoxia+diazoxide could markedly increase the intensity of R-123 fluorescence in rat PASMC as compared with control group (P<0.05), the changes were more significant in chronic hypoxia +diazoxide group than those of chronic hypoxia group (P<0.05); 5-HD could partly weaken the effect of hypoxia on the intensity of R-123 fluorescence (P<0.05) too, After exposed to diazoxide for 24h, the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria were significantly decreased as compared with control group (P<0.05), The A value were significantly increased as compared with control group(P<0.05),and the apoptosis of rat PASMCs were significantly decreased as compared with control group(P<0.05) . But there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h; After exposed to hypoxia or hypoxia+diazoxide for 24h, the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria were significantly decreased as compared with control group (P<0.05), the A value were significantly increased as compared with control group (P<0.05), and the apoptosis of rat PASMCs were significantly decreased as compared with control group (P<0.05); The changes were more significant in hypoxia +diazoxide group than those of hypoxia group (P<0.05). 5-HD could partly weaken the effect of hypoxia on the changes of the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria in rat pulmonary artery smooth muscle cells and on the proliferation of rat PASMCs induced by hypoxia (P<0.05).
Conclusion:The results suggested that the opening of MitoK_(ATP) followed by a depolarization of m might contribute to the inhibition of the release of cytochrome C from cell mitochondria to cell plasma in rat PASMCs and proliferation of rat PASMCs induced by hypoxia. This might be a mechanism of the development of hypoxic pulmonary hypertension.
Subject 2 The effect of mitochondrial ATP-sensitive K+ channel on changes of reactive oxygen species in rat pulmonary artery smooth muscle cells and on proliferation of hypoxic rat pulmonary artery smooth muscle cells
Objective : The objective of this paper is to investigate the contribution of mitochondrial ATP-sensitive K+ channel (MitoK_(ATP)) and mitochondrial membrane potential ( m) to changes of H2O2 in rat pulmonary artery smooth muscle cells and on proliferation of hypoxic rat pulmonary artery smooth muscle cells.
Methods:Fresh normal rat lung tissues were obtained. Then rat PASMCs were isolated and cultured, which were divided into 6 groups, as follow:①control group: cultured under normoxia;②diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP);③5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP);④c hronic hypoxia group: cultured under hypoxia;⑤chronic hypoxia+diazoxide group;⑥chronic hypoxia +5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. The level of H2O2 in rat PASMCs were tested with chemiluminescence method. The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.
Results:After exposed to diazoxide for 24h, the intensity of R-123 fluorescence in normoxic rat PASMCs were significantly increased as compared with control group (P<0.05), but there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h (P>0.05); chronic hypoxia or chronic hypoxia+diazoxide could markedly increase the intensity of R-123 fluorescence in rat PASMC as compared with control group (P<0.05), the changes were more significant in chronic hypoxia +diazoxide group than those of chronic hypoxia group (P<0.05); 5-HD could partly weaken the effect of hypoxia on the intensity of R-123 fluorescence (P<0.05) too, After exposed to diazoxide for 24h, the level of H2O2 in rat PASMCs were significantly increased as compared with control group (P<0.05), The A value were significantly increased as compared with control group(P<0.05),and the apoptosis of rat PASMCs were significantly decreased as compared with control group(P<0.05) . But there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24h; After exposed to hypoxia or hypoxia+diazoxide for 24h, the level of H2O2 in rat PASMCs were significantly increased as compared with control group (P<0.05), the A value were significantly increased as compared with control group (P<0.05), and the apoptosis of rat PASMCs were significantly decreased as compared with control group (P<0.05); The changes were more significant in hypoxia +diazoxide group than those of hypoxia group (P<0.05). 5-HD could partly weaken the effect of hypoxia on the changes of the level of H2O2 in rat pulmonary artery smooth muscle cells and on the proliferation of rat PASMCs induced by hypoxia (P<0.05).
Conclusion:The results suggested that the opening of MitoK_(ATP) followed by a depolarization of m might contribute to the the increasing of the level of H2O2 in rat PASMCs and proliferation of rat PASMCs induced by hypoxia. This might be a mechanism of the development of hypoxic pulmonary hypertension.
Part 2 The role of mitochondrial ATP-sensitive K+ channel on the balance between proliferation and apoptosis of hypoxic human pulmonary artery smooth muscle cells
Subject 1 The effect of mitochondrial ATP-sensitive K+ channel on distributing of cytochrome C in human pulmonary artery smooth muscle cells and on proliferation of hypoxic human pulmonary artery smooth muscle cells
Objective : The objective of this paper is to investigate the contribution of mitochondrial ATP-sensitive K+ channel (MitoK_(ATP)) and mitochondrial membrane potential (Δψ_m) to distributing of cytochrome C in human pulmonary arterial smooth muscle cells (HPASMCs) and to proliferation of HPASMCs induced by hypoxia.
Methods:HPASMCs were divided into several groups, as following:①control group: cultured under normoxia;②diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP);③5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP);④2 4-hours hypoxia group: cultured under hypoxia for 24 hours;⑤2 4-hours hypoxia+diazoxide group, cultured under hypoxia with diazoxide for 24 hours;⑥2 4-hours hypoxia+5-HD group, cultured under hypoxia with 5-HD for 24 hours. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. Western blot technique was used to trace the expression of cytochrome C protein in cell plasma and mitochondria respectively. The expression of cell caspase-9 protein was determined with western blot technique, too. The proliferation of HPASMCs was examined by cell cycle analysis and MTT colorimetric assay.
Results:After exposed to diazoxide for 24 h, the intensity of R-123 fluorescence in normoxic HPASMCs was significantly increased as compared with control group (P<0.05), but there was no significant change of the intensity of R-123 fluorescence after the HPASMCs had been exposed to 5-HD for 24 h; 24-hours hypoxia or 24-hours hypoxia+diazoxide could markedly increase the intensity of R-123 fluorescence in HPASMC as compared with control group (P<0.05), the change was more significant in 24-hours hypoxia +diazoxide group than that of 24-hours hypoxia group (P<0.05); 5-HD could weaken the effect of 24-hours hypoxia on the intensity of R-123 fluorescence. After exposed to diazoxide for 24 h, the ratio of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein was significantly decreased as compared with control group (P<0.05), the expression of caspase-9 protein was significantly decreased as compared with control group (P<0.05), the percentage of S phase and A value of MTT were significantly increased as compared with control group (P<0.05). But there were no significant changes of these tests after the HPASMCs had been exposed to 5-HD for 24 h (P>0.05). After exposed to hypoxia or hypoxia+diazoxide for 24 h, the rate of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein and the expression of caspase-9 protein were significantly decreased as compared with control group (P<0.05), the percentage of S phase and A value of MTT were significantly increased as compared with control group (P<0.05). These changes were more significant in 24-hours hypoxia +diazoxide group than those of 24-hours hypoxia group (P<0.05). 5-HD could weaken the effect of hypoxia on the changes of distributing of cytochrome C, the expression of caspase-9 in human pulmonary arterial smooth muscle cells and the proliferation of HPASMCs induced by hypoxia (P<0.05).
Conclusion:All these results suggested that the opening of MitoK_(ATP) followed by a depolarization ofΔψ_m induced by hypoxia might contribute to the inhibition of the release of cytochrome C from cell mitochondria to cell plasma in HPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension. The signal transduction pathway of mitochondria might play an important role in the relationship betweenΔψ_m and apoptosis of HPASMCs.
Subject 2 The effect of mitochondrial ATP-sensitive K+ channel on changes of reactive oxygen species in human pulmonary artery smooth muscle cells and on proliferation of hypoxic human pulmonary artery smooth muscle cells
Objective:The objective of this paper is to investigate the contribution of diazoxide, an opener of mitochondrial ATP-sensitive K+ channel (MitoK_(ATP)), and mitochondrial membrane potential(Δψ_m) to changes of reactive oxygen species in human pulmonary arterial smooth muscle cells (HPASMCs) and to proliferation of HPASMCs induced by hypoxia.
Methods:HPASMCs were divided into several groups, as follow:①control group: cultured under normoxia;②diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP);③5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP);④chronic hypoxia group: cultured under hypoxia for 24 hours;⑤chronic hypoxia+diazoxide group;⑥chronic hypoxia+5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. The level of H2O2 in HPASMCs was tested with chemiluminescence method. The proliferation of HPASMCs was examined by examing the expression of PCNA, c-fos and c-jun proteins, and by MTT colorimetric assay.
Results:After exposed to diazoxide for 24h, the intensity of R-123 fluorescence in normoxic HPASMCs(105.45±4.38) was significantly increased as compared with control group (74.67±7.02) (P<0.05), but there was no significant change in this test after the HPASMCs had been exposed to 5-HD for 24h; chronic hypoxia (95.24±12.92)or chronic hypoxia +diazoxide (126.47±7.63)could markedly increase the intensity of R-123 fluorescence in HPASMC as compared with control group (P<0.05), the change was more significant in chronic hypoxia +diazoxide group than that of hypoxia group (P<0.05); CH+5-HD(70.69±3.73) could weaken the effect of hypoxia on the intensity of R-123 fluorescence. After exposed to diazoxide for 24h, the level of H2O2(3044.69±126.34)in HPASMCs was significantly increased as compared with control group (2771.69±48.66) (P<0.05), the expression of PCNA(53.52±2.28%), c-fos(164.47±53.47) and c-jun(130.25±10.39 ) proteins were significantly increased as compared with control group(PCNA 42.55±1.97%,c-fos 68.58±21.54 and c-jun 76.18±12.65 ) (P<0.05) too, The A value ( 0.3048±0.022 ) was significantly increased as compared with control group(0.2368±0.013) (P<0.05). But there were no significant changes in these tests after the HPASMCs had been exposed to 5-HD for 24h; After exposed to chronic hypoxia for 24h, the level of H2O2(3115.88±34.12) in HPASMCs was significantly increased as compared with control group (P<0.05), the expression of PCNA(54.55±2.14%), c-fos (160.95±47.49)and c-jun (127.87±17.44)proteins were significantly increased as compared with control group (P<0.05) too, The A value (0.3282±0.078)was significantly increased as compared with control group(P<0.05). These changes were more significant in chronic hypoxia +diazoxide group( the level of H2O2 3236.01±30.86,PCNA 66.13±2.59%, C-fos 370.65±90.85,C-jun 275.97±49.77 and A value 0.4398±0.023) than those of hypoxia group (P<0.05); 5-HD could weaken the effect of hypoxia on the changes of the level of H2O2 (2863.08±132.06) in human pulmonary arterial smooth muscle cells and on the proliferation of HPASMCs ( PCNA 43.48±1.23%,C-fos 70.87±21.46 , C-jun 79.87±12.75 and A value 0.2637±0.045) induced by hypoxia (P<0.05).
Conclusion:The results suggested that the opening of MitoK_(ATP) followed by a depolarization ofΔψ_m might contribute to the increasing of the level of ROS in HPASMCs. The ROS in HPASMCs play an important role in the prolifeation of HPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension.
引文
[1]. Akao M, Teshima Y, Marban E. Antiapoptotic effect of nicorandil mediated by mitochondrial atp-sensitive potassium channels in cultured cardiac myocytes. J Am Coll Cardiol 2002; 40:803-810.
[2]. Samavati L, Monick MM, Sanlioglu S, et al. Mitochdrial KATP channel openers activate the ERK kinase by an oxidant-dependent mechanism. Am J Physiol 2002; 283: C273-C281.
[3]. Liu X, Kim CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996 Jul 12; 86(1): 147-57.
[4]. Chen Q, Crosby M, Almasan A. Redox regulation of apoptosis before and after cytochrome C release. Korean J Biol Sci. 2003 Mar; 7(1): 1-9.
[5]. Das B, Sarkar C. Cardiomyocyte mitochondrial KATP channels participate in the antiarrhythmic and antiinfarct effets of KATP activators during ischemia and reperfusion in an intact anesthetized rabbit model. Pol J.Pharmacol, 2003, 55.771-786.
[1] Evangelos D M, Vaclav H, Ali N, et al. Diversity in mitochondrial function explains differences in vascular oxygen sensing. Cir Res. 2002, 90:1307-1315
[2] O’Rourke B. Evidence for Mitochondrial K + Channels and Their Role in Cardioprotection. Circ Res.; 2002, 94: 420-32
[3] Akao M, Teshima Y, Marban E. Antiapoptotic effect of nicorandil mediated by mitochondrial atp-sensitive potassium channels in cultured cardiac myocytes. J Am Coll Cardiol. 2002, 40:803-810.
[4] Teshima Y, Akao M, Li RA et al. Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by oxidative stress. Stroke. 2003 Jul; 349(7): 1796-802
[5] Das B, Sarkar C. Cardiomyocyte mitochondrial KATP channels participate in the antiarrhythmic and antiinfarct effets of KATP activators during ischemia and reperfusion in an intact anesthetized rabbit model. Pol J. Pharmacol. 2003, 55:771-786.
[6] Turrens J, Freeman B, Levitt J et al. The effect of hyperoxia on superoxide production by lung submitochondrial particles. Arch Biochem Biophys. 1982, 217(2): 401-10.
[7] Ardehali H. Role of the mitochondrial ATP-sensitive K channels in cardioprotection. Acta Biochim Pol. 2004; 51(2): 379-90. Review
[8] Kietzmann T,Gorlach A. Reactive oxygen species in the control of hypoxia-inducible factor-mediated gene expression. Semin Cell Dev Biol. 2005; 16(4-5): 474-86. Review
[9] Ryan K. A, Smith M. F. Jr, Sanders M.K et al. Reactive oxygen and nitrogen species differentially regulate toll-like receptor 4-mediated activation of and interleukin-8 expression. Infect Immun. 2004 Apr; 72 (4): 2123-30
[10] Mendes A.F., Caramona M.M., Carvalho AP, et al. Hydrogen peroxide mediates interleukin-1 beta-induced AP-1 activation in articular chondrocytes: implications for the regulation of iNOS expression.Cell Biol Toxicol. 2003 Aug; 19(4): 203-14.
[11] Greene EL, Velarde V, Jaffa AA. Role of reactive oxygen species in bradykinin-induced mitogen-activated protein kinase and c-fos induction in vascular cells.Hypertension. 2000 Apr; 35 (4): 942-7.
[1] Akao M, Teshima Y, Marban E. Antiapoptotic effect of nicorandil mediated by mitochondrial ATP-sensitive potassium channels in cultured cardiac myocytes. J Am Coll Cardiol 2002; 40:803-810.
[2] Samavati L, Monick MM, Sanlioglu S, Buettner GR, Oberley LW, Hunninghake GW. Mitochondrial KATP channel openers activate the ERK kinase by an oxidant-dependent mechanism. Am J Physiol 2002; 283: C273-C281.
[3] Suzuki A, Tsutomi Y, Yamamoto N, Shibutani T, Akahane K. Mitochondrial regulation of cell death: mitochondria are essential for procase - 3 p21 complex formation to resist Fas mediated cell death [J]. Mol Cell Biol, 1999, 19(9): 3842
[4] Wang T, Zhang ZX, XuYJ. Effect of mitochondrial KATP channel on voltage-gated K+ channel in 24 hour-hypoxic human pulmonary artery smooth muscle cells. Chin Med J (Engl). 2005 Jan 5; 118(1): 12-9.
[5] Krick S, Platoshyn O, Mcdaniel SS, Rubin LJ, Yuan JX. Augmented K+ currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis.Am J Physiol Lung Cell Mol Physiol 2001; 281:L887-L894.
[6] Das B, Sarkar C. Cardiomyocyte mitochondrial KATP channels participate in the antiarrhythmic and antiinfarct effets of KATP activators during ischemia and reperfusion in an intact anesthetized rabbit model. Pol J.Pharmacol, 2003, 55. 771-786.
[7] Kroemer G, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunology Today, 1997; 18 (1): 44.
[8] Gurney AM, Osipenko ON, MacMillan D, Kempsill FE. Potassium
[9] channels underlying the resting potential of pulmonary artery smooth muscle cells. Clin Exp Pharmacol Physiol 2002; 29: 33023331
[10] Platoshyn O, Zhang S, McDaniel SS, Yuan JX. Cytochrome C activates K+ channels before inducing apoptosis. Am J Physiol Cell Physiol. 2002 Oct; 283(4): C1298-305.
[1] Michelakis ED , Hampl V, Nsair A. et al. Diversity in mitochondrial function explains differences in vascular oxygen sensing. Cir Res. 2002, 90:1307-1315
[2] Akao M, Teshima Y, Marban E. Antiapoptotic effect of nicorandil mediated by mitochondrial ATP-sensitive potassium channels in cultured cardiac myocytes. J Am Coll Cardiol. 2002, 40:803-810.
[3] Teshima Y, Akao M, Li RA et al. Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by oxidative stress. Stroke. 2003 Jul; 349(7): 1796-802
[4] Das B, Sarkar C. Cardiomyocyte mitochondrial KATP channels participate in the antiarrhythmic and antiinfarct effets of KATP activators during ischemia and reperfusion in an intact anesthetized rabbit model. J. Pharmacol. 2003, 55:771-786.
[5] Turrens J, Freeman B, Levitt J et al. The effect of hyperoxia on superoxide production by lung submitochondrial particles. Arch Biochem Biophys. 1982, 217(2): 401-10.
[6] Boveris A, Oshino N, Chance B et al. The cellar production of hydrogen peroxide. Biochem J. 1972, 128:617-630
[7] Shibanuma M, Kuroki T, Nose K. Stimulation by hydrogen peroxide of DNA synthesis, competence family gene expression and phosphorylation of a specific protein in quiescent Balb/3T3 cells. Oncogene. 1990, Jul; 5(7): 1025-32.
[8] Maki A, Berezesky I K, Fargnoli J et al. Role of [Ca2+]i in induction of c-fos, c-jun and c-myc mRNA in rat PTE after oxidative stress. FASEB J. 1992 Feb 1; 6(3): 919-24.
[9] Huang R P, Peng A, Hossain M Z et al. Tumor promotion by hydrogen peroxide in rat liver epithelial cells. Carcinogenesis. 1999, 20(3): 485-492
[10] Rao GN, Katki KA, Madamanchi NR, Wu Y, Birrer MJ. JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells the tourenal or biolocical chemistry 1999,274(9): 6003-6010
[1] Inoue I, Nagase H, Kishi K, Higuti T. ATP-sensitive K+ channel in the mitochondrial inner membrane. Nature; 1991,352: 244–7.
[2] Paucek P, Mironova G, Mahdi F, Beavis AD, Woldegiorgis G, Garlid KD. Reconstitution and partial purification of the glibenclamide-sensitive ATP-dependent K+ channel from rat liver and beef heart mitochondria. J Biol Chem. 1992; 267: 26062–9.
[3] Bajgar R, Seetharaman S, Kowaltowski AJ, Garlid KD, Paucek P. Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain. J Biol Chem. 2001; 276: 33369–74.
[4] Jezek P, Orosz DE, Garlid KD. Reconstitution of the uncoupling protein of brown adipose tissue mitochondria. Demonstration of GDP-sensitive halide anion uniport. J Biol Chem. 1990; 265: 19296–302.
[5] Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D’Alonzo AJ, Lodge NJ, Smith MA, Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection. Circ Res. 1997; 81: 1072–82.
[6] Jaburek M, Yarov-Yarovoy V, Paucek P, Garlid KD. State-dependent inhibition of the mitochondrial KATP channel by glyburide and 5-hydroxydecanoate. J Biol Chem. 1998; 273:13578–82.
[7] Kowaltowski AJ, Seetharaman S, Paucek P,Garlid KD. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria. Am J Physiol Heart Circ Physiol. 2001; 280: H649–57.
[8] Liu Y, Sato T, O’Rourke B, Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection?
[9] Circulation. 1998; 97: 2463–9.
[10] Hu H, Sato T, Seharaseyon J, Liu Y, Johns DC, O’Rourke B, Marban E. Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels. Mol Pharmacol. 1999; 55: 1000–5.
[11] Sato T, Sasaki N, O’Rourke B, Marban E. Adenosine primes the opening of mitochondrial ATP-sensitive potassium channels:a key step in ischemic preconditioning?Circulation. 2000; 102: 800–5.
[12] Sato T, Sasaki N, Seharaseyon J, O’Rourke B,Marban E. Selective pharmacological agents implicate mitochondrial but not sarcolemmal KATP channels in ischemic cardioprotection. Circulation. 2000; 101: 2418–23.
[13] Seharaseyon J, Ohler A, Sasaki N, Fraser H,Sato T, Johns DC, O’Rourke B, Marban E. Molecular composition of mitochondrial
[14] ATP-sensitive potassium channels probed by viral Kir gene transfer. J Mol Cell Cardiol. 2000; 32: 1923–30.
[15] Sasaki N, Sato T, Ohler A, O’Rourke B, Marban E. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation.2000; 101: 439–45.
[16] Sasaki N, Murata M, Guo Y, Jo SH, Ohler A, Akao M, O’Rourke B, Xiao RP, Bolli R, Marban E. MCC-134 a single pharmacophore opens surface ATP-sensitive potassium channels blocks mitochondrial
[17] ATP-sensitive potassium channels and suppresses preconditioning. Circulation.2003; 107:1183–8.
[18] Grigoriev SM, Skarga YY, Mironova GD,Marinov BS. Regulation of mitochondrial KATP channel by redox agents. Biochim
[19] Biophys Acta.1999; 1410: 91–6.
[20] Mironova GD, Skarga YY, Grigoriev SM,Negoda AE, Kolomytkin OV, Marinov BS. Reconstitution of the mitochondrial ATP-dependent potassium channel into bilayer lipid membrane. J Bioenerg Biomembr. 1999; 31: 159–63.
[21] Marinov BS, Grigoriev SM, Skarga Y, Olovjanishnikova GD, Mironova GD. Effects of pelargonidine and a benzocaine analogue p-diethylaminoethyl benzoate on mitochondrial KATP channel. Membr Cell Biol. 2001; 14: 663–71.
[22] Zhang DX, Chen YF, Campbell WB, Zou AP,Gross GJ, Li PL. Characteristics and superoxide-induced activation of reconstituted myocardial mitochondrial ATP-sensitive potassium channels. Circ Res. 2001; 89: 1177–83.
[23] Minners J, van den Bos EJ, Yellon DM, Schwalb H, Opie LH, Sack MN. Dinitrophenol cyclosporin A and trimetazidine modulate preconditioning in theisolated rat heart: support for a mitochondrial role in cardioprotection. Cardiovasc Res. 2000; 47: 68–73.
[24] Debska G, Kicinska A, Skalska J, Szewczyk A,May R, Elger CE, Kunz WS. Opening of potassium channels modulates mitochondrial function in rat skeletal muscle. Biochim Biophys Acta. 2002; 1556: 97–105.
[25] Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria. J Physiol (Lond). 1999; 519 (Pt 2): 347–60.
[26] O’Rourke B. Myocardial KATP channels in preconditioning. Circ Res. 2000; 87:845–55.
[27] Lim KH, Javadov SA, Das M, Clarke SJ, Suleiman MS, Halestrap AP. The effects of ischaemic preconditioning diazoxide and 5-hydroxydecanoate on rat heart mitochondrial volume and respiration. J Physiol. 2002; 545: 961–74.
[28] Forbes RA, Steenbergen C, Murphy E. Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism. Circ Res. 2001; 88: 802–9.
[29] Ozcan C, Bienengraeber M, Dzeja PP, Terzic A. Potassium channel openers protect cardiac mitochondria by attenuating oxidant stress at reoxygenation. Am J Physiol Heart Circ Physiol. 2002; 282: H531–9.
[30] O’Rourke B. Evidence for mitochondrial K+ channels and their role in cardioprotection. Circ Res. 2004; 94: 420–32.
[31] Suzuki M, Kotake K, Fujikura K, Inagaki N, Suzuki T, Gonoi T, Seino S, Takata K. Kir6.1: a possible subunit of ATP-sensitive K+ channels in mitochondria. Biochem Biophys Res Commun. 1997; 241: 693–7.
[32] Lacza Z, Snipes JA, Kis B, Szabo C, Grover G, Busija DW. Investigation of the subunit composition and the pharmacology of the mitochondrial TP-dependent K+ channel in the brain. Brain Res. 2003; 994: 27–36.
[33] Miki T, Suzuki M, Shibasaki T, Uemura H, Sato T, Yamaguchi K, Koseki H, Iwanaga T, Nakaya H, Seino S. Mouse model of
[34] Prinzmetal angina by disruption of the inward rectifier Kir6.1. Nat Med. 2002; 8: 466–72.
[35] Hanley PJ, Mickel M, Loffler M, Brandt U, Daut J. KATP channel -independent targets of diazoxide and 5-hydroxydecanoate in the heart. J Physiol. 2002; 542: 735–41.
[36] Ardehali H, Chen Z, Ko Y, Mejia-Alvarez R, Marban E. Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity. Circulation. 2003; 108 (suppl I): I–1004.
[37] Takashi E, Wang Y, Ashraf M. Activation of mitochondrial KATP channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res. 1999; 85: 1146–53.
[38] Akao M, Ohler A, O’Rourke B, Marban E. Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by
[39] oxidative stress in cardiac cells. Circ Res. 2001; 88: 1267–75.
[2]. Samavati L, Monick MM, Sanlioglu S, et al. Mitochdrial KATP channel openers activate the ERK kinase by an oxidant-dependent mechanism. Am J Physiol 2002; 283: C273-C281.
[3]. Liu X, Kim CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996 Jul 12; 86(1): 147-57.
[4]. Chen Q, Crosby M, Almasan A. Redox regulation of apoptosis before and after cytochrome C release. Korean J Biol Sci. 2003 Mar; 7(1): 1-9.
[5]. Das B, Sarkar C. Cardiomyocyte mitochondrial KATP channels participate in the antiarrhythmic and antiinfarct effets of KATP activators during ischemia and reperfusion in an intact anesthetized rabbit model. Pol J.Pharmacol, 2003, 55.771-786.
[1] Evangelos D M, Vaclav H, Ali N, et al. Diversity in mitochondrial function explains differences in vascular oxygen sensing. Cir Res. 2002, 90:1307-1315
[2] O’Rourke B. Evidence for Mitochondrial K + Channels and Their Role in Cardioprotection. Circ Res.; 2002, 94: 420-32
[3] Akao M, Teshima Y, Marban E. Antiapoptotic effect of nicorandil mediated by mitochondrial atp-sensitive potassium channels in cultured cardiac myocytes. J Am Coll Cardiol. 2002, 40:803-810.
[4] Teshima Y, Akao M, Li RA et al. Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by oxidative stress. Stroke. 2003 Jul; 349(7): 1796-802
[5] Das B, Sarkar C. Cardiomyocyte mitochondrial KATP channels participate in the antiarrhythmic and antiinfarct effets of KATP activators during ischemia and reperfusion in an intact anesthetized rabbit model. Pol J. Pharmacol. 2003, 55:771-786.
[6] Turrens J, Freeman B, Levitt J et al. The effect of hyperoxia on superoxide production by lung submitochondrial particles. Arch Biochem Biophys. 1982, 217(2): 401-10.
[7] Ardehali H. Role of the mitochondrial ATP-sensitive K channels in cardioprotection. Acta Biochim Pol. 2004; 51(2): 379-90. Review
[8] Kietzmann T,Gorlach A. Reactive oxygen species in the control of hypoxia-inducible factor-mediated gene expression. Semin Cell Dev Biol. 2005; 16(4-5): 474-86. Review
[9] Ryan K. A, Smith M. F. Jr, Sanders M.K et al. Reactive oxygen and nitrogen species differentially regulate toll-like receptor 4-mediated activation of and interleukin-8 expression. Infect Immun. 2004 Apr; 72 (4): 2123-30
[10] Mendes A.F., Caramona M.M., Carvalho AP, et al. Hydrogen peroxide mediates interleukin-1 beta-induced AP-1 activation in articular chondrocytes: implications for the regulation of iNOS expression.Cell Biol Toxicol. 2003 Aug; 19(4): 203-14.
[11] Greene EL, Velarde V, Jaffa AA. Role of reactive oxygen species in bradykinin-induced mitogen-activated protein kinase and c-fos induction in vascular cells.Hypertension. 2000 Apr; 35 (4): 942-7.
[1] Akao M, Teshima Y, Marban E. Antiapoptotic effect of nicorandil mediated by mitochondrial ATP-sensitive potassium channels in cultured cardiac myocytes. J Am Coll Cardiol 2002; 40:803-810.
[2] Samavati L, Monick MM, Sanlioglu S, Buettner GR, Oberley LW, Hunninghake GW. Mitochondrial KATP channel openers activate the ERK kinase by an oxidant-dependent mechanism. Am J Physiol 2002; 283: C273-C281.
[3] Suzuki A, Tsutomi Y, Yamamoto N, Shibutani T, Akahane K. Mitochondrial regulation of cell death: mitochondria are essential for procase - 3 p21 complex formation to resist Fas mediated cell death [J]. Mol Cell Biol, 1999, 19(9): 3842
[4] Wang T, Zhang ZX, XuYJ. Effect of mitochondrial KATP channel on voltage-gated K+ channel in 24 hour-hypoxic human pulmonary artery smooth muscle cells. Chin Med J (Engl). 2005 Jan 5; 118(1): 12-9.
[5] Krick S, Platoshyn O, Mcdaniel SS, Rubin LJ, Yuan JX. Augmented K+ currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis.Am J Physiol Lung Cell Mol Physiol 2001; 281:L887-L894.
[6] Das B, Sarkar C. Cardiomyocyte mitochondrial KATP channels participate in the antiarrhythmic and antiinfarct effets of KATP activators during ischemia and reperfusion in an intact anesthetized rabbit model. Pol J.Pharmacol, 2003, 55. 771-786.
[7] Kroemer G, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunology Today, 1997; 18 (1): 44.
[8] Gurney AM, Osipenko ON, MacMillan D, Kempsill FE. Potassium
[9] channels underlying the resting potential of pulmonary artery smooth muscle cells. Clin Exp Pharmacol Physiol 2002; 29: 33023331
[10] Platoshyn O, Zhang S, McDaniel SS, Yuan JX. Cytochrome C activates K+ channels before inducing apoptosis. Am J Physiol Cell Physiol. 2002 Oct; 283(4): C1298-305.
[1] Michelakis ED , Hampl V, Nsair A. et al. Diversity in mitochondrial function explains differences in vascular oxygen sensing. Cir Res. 2002, 90:1307-1315
[2] Akao M, Teshima Y, Marban E. Antiapoptotic effect of nicorandil mediated by mitochondrial ATP-sensitive potassium channels in cultured cardiac myocytes. J Am Coll Cardiol. 2002, 40:803-810.
[3] Teshima Y, Akao M, Li RA et al. Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by oxidative stress. Stroke. 2003 Jul; 349(7): 1796-802
[4] Das B, Sarkar C. Cardiomyocyte mitochondrial KATP channels participate in the antiarrhythmic and antiinfarct effets of KATP activators during ischemia and reperfusion in an intact anesthetized rabbit model. J. Pharmacol. 2003, 55:771-786.
[5] Turrens J, Freeman B, Levitt J et al. The effect of hyperoxia on superoxide production by lung submitochondrial particles. Arch Biochem Biophys. 1982, 217(2): 401-10.
[6] Boveris A, Oshino N, Chance B et al. The cellar production of hydrogen peroxide. Biochem J. 1972, 128:617-630
[7] Shibanuma M, Kuroki T, Nose K. Stimulation by hydrogen peroxide of DNA synthesis, competence family gene expression and phosphorylation of a specific protein in quiescent Balb/3T3 cells. Oncogene. 1990, Jul; 5(7): 1025-32.
[8] Maki A, Berezesky I K, Fargnoli J et al. Role of [Ca2+]i in induction of c-fos, c-jun and c-myc mRNA in rat PTE after oxidative stress. FASEB J. 1992 Feb 1; 6(3): 919-24.
[9] Huang R P, Peng A, Hossain M Z et al. Tumor promotion by hydrogen peroxide in rat liver epithelial cells. Carcinogenesis. 1999, 20(3): 485-492
[10] Rao GN, Katki KA, Madamanchi NR, Wu Y, Birrer MJ. JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells the tourenal or biolocical chemistry 1999,274(9): 6003-6010
[1] Inoue I, Nagase H, Kishi K, Higuti T. ATP-sensitive K+ channel in the mitochondrial inner membrane. Nature; 1991,352: 244–7.
[2] Paucek P, Mironova G, Mahdi F, Beavis AD, Woldegiorgis G, Garlid KD. Reconstitution and partial purification of the glibenclamide-sensitive ATP-dependent K+ channel from rat liver and beef heart mitochondria. J Biol Chem. 1992; 267: 26062–9.
[3] Bajgar R, Seetharaman S, Kowaltowski AJ, Garlid KD, Paucek P. Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain. J Biol Chem. 2001; 276: 33369–74.
[4] Jezek P, Orosz DE, Garlid KD. Reconstitution of the uncoupling protein of brown adipose tissue mitochondria. Demonstration of GDP-sensitive halide anion uniport. J Biol Chem. 1990; 265: 19296–302.
[5] Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D’Alonzo AJ, Lodge NJ, Smith MA, Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection. Circ Res. 1997; 81: 1072–82.
[6] Jaburek M, Yarov-Yarovoy V, Paucek P, Garlid KD. State-dependent inhibition of the mitochondrial KATP channel by glyburide and 5-hydroxydecanoate. J Biol Chem. 1998; 273:13578–82.
[7] Kowaltowski AJ, Seetharaman S, Paucek P,Garlid KD. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria. Am J Physiol Heart Circ Physiol. 2001; 280: H649–57.
[8] Liu Y, Sato T, O’Rourke B, Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection?
[9] Circulation. 1998; 97: 2463–9.
[10] Hu H, Sato T, Seharaseyon J, Liu Y, Johns DC, O’Rourke B, Marban E. Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels. Mol Pharmacol. 1999; 55: 1000–5.
[11] Sato T, Sasaki N, O’Rourke B, Marban E. Adenosine primes the opening of mitochondrial ATP-sensitive potassium channels:a key step in ischemic preconditioning?Circulation. 2000; 102: 800–5.
[12] Sato T, Sasaki N, Seharaseyon J, O’Rourke B,Marban E. Selective pharmacological agents implicate mitochondrial but not sarcolemmal KATP channels in ischemic cardioprotection. Circulation. 2000; 101: 2418–23.
[13] Seharaseyon J, Ohler A, Sasaki N, Fraser H,Sato T, Johns DC, O’Rourke B, Marban E. Molecular composition of mitochondrial
[14] ATP-sensitive potassium channels probed by viral Kir gene transfer. J Mol Cell Cardiol. 2000; 32: 1923–30.
[15] Sasaki N, Sato T, Ohler A, O’Rourke B, Marban E. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation.2000; 101: 439–45.
[16] Sasaki N, Murata M, Guo Y, Jo SH, Ohler A, Akao M, O’Rourke B, Xiao RP, Bolli R, Marban E. MCC-134 a single pharmacophore opens surface ATP-sensitive potassium channels blocks mitochondrial
[17] ATP-sensitive potassium channels and suppresses preconditioning. Circulation.2003; 107:1183–8.
[18] Grigoriev SM, Skarga YY, Mironova GD,Marinov BS. Regulation of mitochondrial KATP channel by redox agents. Biochim
[19] Biophys Acta.1999; 1410: 91–6.
[20] Mironova GD, Skarga YY, Grigoriev SM,Negoda AE, Kolomytkin OV, Marinov BS. Reconstitution of the mitochondrial ATP-dependent potassium channel into bilayer lipid membrane. J Bioenerg Biomembr. 1999; 31: 159–63.
[21] Marinov BS, Grigoriev SM, Skarga Y, Olovjanishnikova GD, Mironova GD. Effects of pelargonidine and a benzocaine analogue p-diethylaminoethyl benzoate on mitochondrial KATP channel. Membr Cell Biol. 2001; 14: 663–71.
[22] Zhang DX, Chen YF, Campbell WB, Zou AP,Gross GJ, Li PL. Characteristics and superoxide-induced activation of reconstituted myocardial mitochondrial ATP-sensitive potassium channels. Circ Res. 2001; 89: 1177–83.
[23] Minners J, van den Bos EJ, Yellon DM, Schwalb H, Opie LH, Sack MN. Dinitrophenol cyclosporin A and trimetazidine modulate preconditioning in theisolated rat heart: support for a mitochondrial role in cardioprotection. Cardiovasc Res. 2000; 47: 68–73.
[24] Debska G, Kicinska A, Skalska J, Szewczyk A,May R, Elger CE, Kunz WS. Opening of potassium channels modulates mitochondrial function in rat skeletal muscle. Biochim Biophys Acta. 2002; 1556: 97–105.
[25] Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria. J Physiol (Lond). 1999; 519 (Pt 2): 347–60.
[26] O’Rourke B. Myocardial KATP channels in preconditioning. Circ Res. 2000; 87:845–55.
[27] Lim KH, Javadov SA, Das M, Clarke SJ, Suleiman MS, Halestrap AP. The effects of ischaemic preconditioning diazoxide and 5-hydroxydecanoate on rat heart mitochondrial volume and respiration. J Physiol. 2002; 545: 961–74.
[28] Forbes RA, Steenbergen C, Murphy E. Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism. Circ Res. 2001; 88: 802–9.
[29] Ozcan C, Bienengraeber M, Dzeja PP, Terzic A. Potassium channel openers protect cardiac mitochondria by attenuating oxidant stress at reoxygenation. Am J Physiol Heart Circ Physiol. 2002; 282: H531–9.
[30] O’Rourke B. Evidence for mitochondrial K+ channels and their role in cardioprotection. Circ Res. 2004; 94: 420–32.
[31] Suzuki M, Kotake K, Fujikura K, Inagaki N, Suzuki T, Gonoi T, Seino S, Takata K. Kir6.1: a possible subunit of ATP-sensitive K+ channels in mitochondria. Biochem Biophys Res Commun. 1997; 241: 693–7.
[32] Lacza Z, Snipes JA, Kis B, Szabo C, Grover G, Busija DW. Investigation of the subunit composition and the pharmacology of the mitochondrial TP-dependent K+ channel in the brain. Brain Res. 2003; 994: 27–36.
[33] Miki T, Suzuki M, Shibasaki T, Uemura H, Sato T, Yamaguchi K, Koseki H, Iwanaga T, Nakaya H, Seino S. Mouse model of
[34] Prinzmetal angina by disruption of the inward rectifier Kir6.1. Nat Med. 2002; 8: 466–72.
[35] Hanley PJ, Mickel M, Loffler M, Brandt U, Daut J. KATP channel -independent targets of diazoxide and 5-hydroxydecanoate in the heart. J Physiol. 2002; 542: 735–41.
[36] Ardehali H, Chen Z, Ko Y, Mejia-Alvarez R, Marban E. Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity. Circulation. 2003; 108 (suppl I): I–1004.
[37] Takashi E, Wang Y, Ashraf M. Activation of mitochondrial KATP channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res. 1999; 85: 1146–53.
[38] Akao M, Ohler A, O’Rourke B, Marban E. Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by
[39] oxidative stress in cardiac cells. Circ Res. 2001; 88: 1267–75.