线粒体三磷酸腺苷敏感性钾通道在未成熟心肌缺血预处理中的作用

目的探讨线粒体三磷酸腺苷敏感性钾通道（mitoKATP）在未成熟心肌预处理保护中的作用,为未成熟心肌的保护提供依据。方法采用Langendorff离体心脏灌注模型,将24只新生（出生14～21d）日本长耳大白兔按随机数字表法分为4组：缺血／再灌注组（I／R组）,心脏缺血预处理组（E1组）,mitoKATP阻滞剂5-hydroxydecanoate（5-HD）＋心脏缺血预处理（E2组）,mitoKATP通道开放剂Diazoxide（Diaz）预处理组（E3组）;检测心脏功能恢复率、心肌含水量、血清肌酸激酶和乳酸脱氢酶漏出率、三磷酸腺苷（ATP）含量、超氧化物歧化酶活性、丙二醛含量、心肌细胞内Ca^2＋含量、心肌线粒体Ca^2＋含量、心肌线粒体Ca^2＋-三磷酸腺苷酶活性（Ca^2＋-ATPase）、心肌线粒体合成ATP的能力;电子显微镜观察心肌超微结构。结果E1组、E3组心功能恢复优于I／R组和E2组,心肌含水量低于I／R组和E2组（P〈0．05）;E1组、E3组三磷酸腺苷含量、超氧化物歧化酶活性、心肌线粒体Ca^2＋-ATPase活性、心肌线粒体合成ATP的能力均优于I／R组和E2组（P〈0．05）,丙二醛含量、血清肌酸激酶和乳酸脱氢酶漏出率、心肌细胞内Ca^2＋含量、心肌线粒体Ca^2＋含量低于I／R组、E2组（P〈0．05）;E1组、E3组心肌超微结构损伤较I／R组和E2组明显减轻。结论心肌缺血预处理对未成熟心肌具有明显的保护作用,其机制可能是通过mitoKATP通道的开放起作用。     

妊高征大鼠颈交感神经干离断对胎鼠脑的保护作用

目的探讨妊高征（PIH）大鼠颈交感神经干离断（TCST）对胎鼠脑的保护作用。方法妊娠Wistar大鼠32只，随机分为4组，每组8只，对照组（A组）、PIH组（B组）、TCST组（C组）和假手术组（D组）。B组自孕14d开始皮下注射工广硝基精氨酸甲酯（L-NAME）12．5mg／100g，每天平均分两次注射，至孕20d，A组给予等量生理盐水；C组孕14d行右TCST，余同B组；D组孕14d行右颈交感神经干分离，但不离断，余同B组。各组于孕21d剖宫取仔，观察胎鼠脑组织超微结构，测定脑组织ATP、ADP、AMP的含量、腺苷酸池（ATP＋ADP＋AMP）及神经元细胞膜Na^＋-K^＋-ATP限酶及Ca^2＋-ATP酶活性。结果A、C组脑组织超微结构基本正常，B、D组脑组织超微结构严重受损。与A组比较。B、D组脑组织ATP、ADP、AMP的含量、腺苷酸池及神经元细胞膜Na^＋-K^＋-ATP酶及Ca^2＋-ATP酶的活性均降低；与B组比较，C组脑组织ATP、ADP、AMP的含量、腺苷酸池及神经元细胞膜Na^＋-K^＋-ATP酶及Ca^2＋-ATP酶的活性均升高（P〈0．01）。结论PIH大鼠TCST对胎鼠脑有一定的保护作用，可能与改善脑细胞的能量代谢及相关的离子泵功能有关。     

δ阿片受体激动剂对脓毒症大鼠小肠能量代谢作用的研究

目的：探讨δ阿片受体激动剂DADLE（D—Ala^2-D—Leu^5-enkephali）对脓毒症大鼠小肠屏障功能的保护作用及其机制。方法：72只SD大鼠,分为假手术组、脓毒症组和DADLE（5mg／Kg）治疗组．每组24只。采用改良盲肠结扎穿孔方法（CLP）建立大鼠脓毒症模型,假手术组除不结扎刺穿盲肠外,其余操作同脓毒症组,DADLE治疗组模型建立后立即按5mL/kg剂量静脉注射浓度为0．5mg／mL的DALDE。于手术后4、8、12h处死大鼠,测定小肠黏膜组织中ATP、ADP、AMP含量;制备肠上皮细胞线粒体,测定各组大鼠线粒体呼吸控制率（RCR）、磷氧比（P／O）和肠道氧摄取率（Oext）;观察并比较各组小肠黏膜上皮组织病理改变。结果：DADLE治疗组的小肠黏膜ATP、ADP含量较脓毒症组均有明显升高（P〈0．05）,AMP含量明显下降（P〈0．05）;DADLE治疗组小肠上皮细胞中线粒体RCR、P／O和Oext较脓毒症组均明显升高（P〈0．05）;小肠黏膜上皮组织病理学提示DADLE组的组织损伤明显轻于脓毒症组。结论：δ阿片受体激动剂DADLE对脓毒症大鼠小肠氧代谢和能量代谢的抑制状态具有一定程度的改善作用。     

a-硫辛酸对阻塞性黄疸大鼠肝细胞线粒体内能量代谢保护作用及机制

目的 探讨α-硫辛酸对阻塞性黄疸大鼠肝细胞线粒体内能量代谢的保护作用及其机制. 方法 将72只SD雄性大鼠随机分为对照组（SO组）,胆总管结扎＋0.9%氯化钠溶液组（BDL＋NS组）,胆总管结扎α-硫辛酸组（BDL＋LA组）.分别于术后7 d、14 d和2l d检测肝细胞线粒体内MDA、SOD、ATP、ADP和AMP含量并计算总腺苷酸（TAN）和能荷（EC）. 结果 BDL＋NS组各时间点肝细胞线粒体内MDA、ADP、AMP含量明显升高,而SOD、ATP、EC含量却明显降低.胆总管结扎7 d、14 d时,肝细胞线粒体内MDA含量BDL＋LA组比BDL＋NS组低,差异有统计学意义（P＜0.01）;2l d时,AMP、MDA含量BDL＋NS组和BDL＋LA中都更进一步升高,但两组比较差异无统计学意义（P＞0.05）.胆总管结扎7 d、14 d时,肝细胞线粒体内SOD含量、ATP含量BDL＋LA组比BDL＋NS组下降程度有显著性差异（7 d,P＜0.01;14 d,P＜0.05）;21 d时,两组肝细胞线粒体内SOD含量都进一步下降,但差异无统计学意义（p＞0.05）. 结论 α-硫辛酸在阻塞性黄疸早、中期有保护线粒体能量代谢作用,减轻了阻塞性黄疸时肝的损伤.     

大鼠烧伤后心肌局部肾素—血管紧张素系统的改变

目的 观察烧伤早期心肌局部肾素－血管紧张素系统（RAS）变化及其对心肌肌浆网（SR）钙运转功能的影响,探讨烧伤早期心功能障碍的发病机制。方法 大鼠随机分为对照组（8只,不予致伤）,烧伤组（40只,致30％Ⅲ度烧伤）,治疗组（40只,Lisinopril灌胃3天后致30％Ⅲ度烧伤）,测定烧伤前（对照组）及烧伤后3,8,24h左心功能变化,心肌组织血管紧张素转换酶（ACE）活性,血管紧张素Ⅱ（AⅡ）及钙离子（Ca^2 ）含量变化,测定心肌SR Ca^2 -ATPase活性,及SR Ca^2 运转功能的变化。结果 烧伤组大鼠左心室内压变化最大速率（±dp/dtmax)明显降低,心肌组织ACE活性、AⅡ及Ca^2 含量显著增加,心肌SR Ca^2 -ATP活性降低,SR Ca^2 摄取减少。治疗组预防性给予ACE抑制剂Lisinopril可显著降低烧伤后心肌组织ACE活性,减少A Ⅱ产生,Ca^2 含量降低,增加SR Ca^2 -ATPase活性,SR Ca^2 摄取增加,左心室功能明显改善。结论 烧伤早期心肌局部RAS迅速激活,抑制SR Ca^2 运转,可能是心肌RAS促进烧伤早期心功能障碍的作用机制之一。     

甲基强的松龙对脊髓损伤后伤段脊髓线粒体功能的影响

目的：探讨脊髓损伤后伤段脊髓线粒体呼吸功能和线粒体内游离钙的变化和早期使用甲基强的松龙（MP）对其的影响。方法：54只SD大鼠，随机分组为假手术组（对照组）、脊髓损伤组（SCI组，采用Allen’s打击法造成大鼠脊髓损伤模型）和脊髓损伤后应用MP治疗组（MP组），每组又分为处理后6h、12h、24h三个时间相，每个时间相6只。在各时间相处死动物后提取伤段脊髓线粒体，测定线粒体呼吸Ⅲ态（R3）、呼吸Ⅳ态（R4）、呼吸控制率（RCR）、磷氧比（P／0）和线粒体内游离Ca^2＋浓度。结果：SCI组在伤后6h、12h和24hR3、RCR和P／0显著低于对照组，R4和线粒体内游离Ca^2＋浓度显著高于对照组。差异有显著性（P〈0．01）；伤后6h和12h MP组R3、RCR和P／0高于SCI组，R4和线粒体内游离Ca^2＋浓度低于SCI组，差异有显著性（P〈0．01）；MP组R3、R4和RCR在6h和12h时与对照组之间无显著性差异，24h时R3、RCR和P／0低于正常对照组，有显著性差异（P〈0．05）。结论：脊髓损伤后伤段脊髓线粒体呼吸功能和线粒体内游离Ca^2＋浓度明显受到影响，线粒体内膜通透性增加，线粒体氧化磷酸化的偶联程度明显受到抑制。早期使用甲基强的松龙可明显改善线粒体的呼吸功能，抑制Ca^2＋内流，保护伤段脊髓线粒体的稳定性。     

肾缺血预处理对未成熟心肌的保护作用

目的探讨肾缺血预处理对未成熟心肌保护的影响，为未成熟心肌的保护提供新的方法。方法建立兔Langendorff灌注模型，将18只幼兔随机分为3组，缺血／再灌注组（I／R组）：灌注15min转为工作心15min，停灌45min，恢复灌注15min改为工作心30min；心脏缺血预处理组（CIP组）：灌注15min转为工作心15min，反复2次缺血5min再灌注5min，重复I／R组的方法；肾缺血预处理组（RIP组）：反复3次阻断左肾动脉血流5min再灌注5min，取离体心脏，灌注15min转为工作心15min，重复I／R组的方法。观察血流动力学、生化等指标。结果CIP组和RIP组的冠状动脉流量（CF）、心排血量（CO）、左心室收缩压（LVSP）恢复百分率均较I／R组升高，左心室舒张期末压（LVEDP）恢复率则较I／R组降低，差异有统计学意义（P〈0.01）；三组间比较，HR、AF恢复率差异无统计学意义（P〉0.05）；RIP组与CIP组比较各指标恢复率差异无统计学意义（P〉0．05）。RIP组与I／R组比较：心肌含水量（MWC）、血清肌酸激酶（cK）和乳酸脱氢酶（LDH）漏出率、ATP含量、丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性、心肌细胞内Ca^2＋含量、心肌线粒体Ca^2＋-ATPase活性、心肌线粒体Ca^2＋含量、心肌线粒体合成ATP能力差异有统计学意义（P〈0.01），RIP组和CIP组比较各项指标差异无统计学意义（P〉0.05）。结论肾缺血预处理对未成熟心肌具有心肌保护作用。     

创面脓毒症大鼠组织ATP含量及合成酶活性的变化

目的：探讨烧伤后创面脓毒症对组织能量合成的影响。方法：60只背部30%Ⅲ度烫伤大鼠随机分为单纯烫伤组和刨面脓毒症组,创面脓毒症组大鼠创面涂以1×10~9 cfu/ml的铜绿假单胞菌观察伤后96h内心、肝、骨骼肌的ATP的含量及合成酶活性变化,同时电镜观察组织超微结构的相应改变。结果：伤后早期心肌、肝脏、骨骼肌细胞内ATP含量及合成酶活性均明显下降,此后单纯烫伤组逐渐恢复,至伤后96h脓毒症组大鼠ATP含量及合成酶活性明显低于对照组,并有与此相对应的线粒体数量和形态结构的改变。结论：烧伤后发生创面脓毒症时机体重要器官的线粒体结构损伤严重,组织ATP含量及合成酶活性明显下降,ATP合成不足加重了能量代谢的紊乱状况,线粒体的早期损伤可能是导致代谢紊乱的病理基础。     

异丙酚对脓毒症大鼠肝线粒体呼吸功能的保护作用

目的探讨异丙酚对脓毒症大鼠肝线粒体呼吸功能的保护作用。方法 健康SD大鼠 18只,随机分为3组(n=6):假手术组(C组)、生理盐水组(NS组)、异丙酚组(P组)。NS、P组采用盲 肠结扎穿孔法(CLP)制备脓毒症模型,C组仅做开腹、分离盲肠远端、关腹手术。CLP后12 hP组颈外 静脉输注异丙酚5 mg／kg负荷量,继以10 mg·kg-1·h-1维持,C组、NS组输注生理盐水0.5 ml／h。持续 静脉输注异丙酚4 h时处死大鼠,取肝脏,采用Clark氧电极技术测定线粒体呼吸功能,分光光度法测 定丙二醛(MDA)、超氧化物歧化酶(SOD)、NO2／NO3-(NO)水平,采用定磷法测定ATP酶活性。结果 与C组比较,NS组、P组SOD及ATP的酶活性、呼吸控制率(RCR)、磷／氧比(ADP／O)降低,MDA水平、 态3及态4的呼吸速率升高(P<0．01);与NS组比较,P组SOD及ATP酶的活性、RCR及其ADP／O水 平升高,MDA、NO水平、态3及态4的呼吸速率降低(P<0．01)。结论 异丙酚通过中和自由基,对 脓毒症大鼠肝线粒体呼吸功能产生一定的保护作用。     

成体大鼠心肌细胞微管解聚对线粒体分布及能量代谢的影响

目的 了解成体大鼠心肌细胞微管解聚对线粒体分布、线粒体活性及细胞能量代谢的影响. 方法 分离培养成体SD大鼠及SD大鼠乳鼠心肌细胞,按随机数字表法分为:大(乳)鼠对照组(常规培养,不加任何刺激因素)、大(乳)鼠微管解聚剂组(用含终浓度8μmol/L秋水仙碱的培养液培养,作用30 min).(1)用蛋白质印迹法检测各组大鼠和乳鼠心肌细胞聚合态β微管蛋白表达量.(2)取2组大鼠心肌细胞,用蛋白质印迹法检测细胞色素c表达量;免疫荧光染色法观察细胞聚合态β微管蛋白、电压依赖型阴离子通道(VDAC)分布情况;免疫细胞化学法检测细胞线粒体内膜电位;噻唑蓝法测量细胞活性;采用高效液相色谱法,检测细胞ATP、腺苷二磷酸(ADP)、腺苷一磷酸(AMP)含量及能荷. 结果 (1)聚合态β微管蛋白表达量:大鼠微管解聚剂组为0.52±0.07,较大鼠对照组1.25±0.12明显减少(F=31.002,P=0.000);乳鼠微管解聚剂组为0.76±0.12,较乳鼠对照组1.11±0.24显著减少(F=31.002,P=0.000),但明显高于大鼠微管解聚剂组(F=31.002,P=0.009).(2)细胞色素c表达量:大鼠对照组为0.26±0.03,明显低于大鼠微管解聚剂组(1.55±0.13,t=-24.056,P=0.000).(3)免疫荧光染色:大鼠对照组心肌细胞微管多呈线性管状、与心肌纤维平行分布;VDAC着色显示线粒体呈颗粒状与微管同向分布.大鼠微管解聚剂组微管正常排列规律被破坏,表现为免疫荧光强度减弱,微管结构不清晰、连续性丧失、粗糙;线粒体分布散乱.(4)线粒体内膜电位:大鼠对照组荧光强度为1288±84,明显高于大鼠微管解聚剂组(331±27,t=26.508,P=0.000).(5)细胞活性:大鼠对照组吸光度值为1.75±0.11;大鼠微管解聚剂组为0.81±0.07,较前者明显降低(t=17.348,P=0.000).(6)能量代谢:与大鼠对照组比较,大鼠微管解聚剂组心肌细胞ATP含量下降,ADP、AMP含量上升,ATP/ADP值与能荷均降低. 结论 在正常成体大鼠心肌细胞内,微管与线粒体分布方向一致.微管解聚后心肌细胞线粒体排列紊乱,细胞色素c从线粒体漏出,线粒体内膜电位下降、能量供应降低,细胞活性下降.     

Ruthenium red attenuated cardiomyocyte and mitochondrial damage during the early stage after severe burn

OBJECTIVE: To investigate the effects of ruthenium red on cardiomyocyte and mitochondrial damage during the early stage after severe burn. METHODS: Adult Wistar rats were randomized into normal control group (control), burn group (burns) and ruthenium red-treated group (RR) (n=8). Rats in the burn and RR groups were scalded to 30% TBSA III degree, and 30 min later, resuscitated with Ringer's solution. Ruthenium red was injected into rats of RR group at a dose of 2mg kg(-1), and again 3h later. Rats were sacrificed at the 6th hour post-scald, myocardial mitochondrial respiratory function and Ca(2+) concentration ([Ca(2+)](m)), serum creatine kinase (CK) and lactate dehydrogenase (LDH), myocardial ATP, ADP, AMP and lactate contents were determined. RESULTS: [Ca(2+)](m) in the RR group was significantly lower than that of the burn group. Mitochondrial respiratory control rate (RCR) and ST(3) were higher than those of burns, but ST(4) was high compared to the control group. Serum CK and LDH of the RR group decreased significantly, and were 65.0 and 45.5% of the burn group, myocardial ATP content in the ruthenium red-treated group increased by 100.4% and lactate level was decreased by 53.5% compared to the burn group. CONCLUSIONS: Ruthenium red attenuated myocyte and mitochondrial damage during the early stage after severe burns.     

Stimulation of mitochondrial Ca2+ efflux by NADP+ with maintenance of respiratory control

Experiments in this paper demonstrate that mitochondrial damage associated to NAD(P)+-induced Ca2+ efflux, is the consequence of inappropriate reaction conditions. The major findings are (i) Added oxaloacetate and acetoacetate readily oxidize NAD(P)H in intact rat liver mitochondria without causing swelling or membrane damage. (ii) Ca2+ efflux can be induced by the oxidized state of mitochondrial NADP in the presence of ATP, ADP, Pi and Mg2+, without mitochondrial swelling. (iii) Ca2+ efflux induced by NADP+ in the presence of ruthenium red causes no significant impairment of respiratory control, a sensitive measure of membrane potential.     

Changes of myocardial mitochondrial Ca(2+) transport and mechanism in the early stage after severe burns

OBJECTIVE: To investigate the change of myocardial mitochondrial Ca(2+) transport and its mechanism in the early stage after burns. METHODS: Forty-eight Wistar rats were randomized into a normal control group (n=8) and a burns group (n=40). The rats of the burns group were given a 30%TBSA full-thickness. Myocardial mitochondria were isolated from normal and scalded rats which were sacrificed at the 1st, 3rd, 6th, 12th and 24th hour post-burn. Mitochondrial Ca(2+) transport velocity, membrane potential (MP), ATP content and cytosolic Ca(2+) concentration [Ca(2+)](c) were determined. The effects of exogenous ATP on mitochondrial Ca(2+) transport velocity were also investigated. RESULTS: Mitochondrial Ca(2+) uptake velocity of the 1st hour post-burn was higher than that of the control, and Ca(2+) release velocity did not change significantly, but mitochondrial Ca(2+) transport velocity, MP and ATP content were all decreased at the 3rd, 6th, 12th and 24th hour post-burn. Mitochondrial Ca(2+) uptake velocity was positively correlated with MP after burn, and Ca(2+) release velocity with mitochondrial ATP content. [Ca(2+)](c) was increased at the 3rd, 6th, 12th and 24th hour post-burn. Exogenous ATP increased myocardial mitochondrial Ca(2+) uptake velocity of rats at the 3rd and 6th hour post-burn and Ca(2+) release velocity at the 3rd, 6th and 12th hour post-burn. CONCLUSIONS: Increase of [Ca(2+)](c) led to reinforcement of mitochondrial Ca(2+) uptake at the beginning of the post-burns period. ATP depletion and MP collapse cause myocardial mitochondrial Ca(2+) transport disorder in the following stages.     

Ischemic preconditioning improves mitochondrial tolerance to experimental calcium overload

BACKGROUND: Ca(2+) overload leads to mitochondrial uncoupling, decreased ATP synthesis, and myocardial dysfunction. Pharmacologically opening of mitochondrial K(ATP) channels decreases mitochondrial Ca(2+) uptake, improving mitochondrial function during Ca(2+) overload. Ischemic preconditioning (IPC), by activating mitochondrial K(ATP) channels, may attenuate mitochondrial Ca(2+) overload and improve mitochondrial function during reperfusion. The purpose of these experiments was to study the effect of IPC (1) on mitochondrial function and (2) on mitochondrial tolerance to experimental Ca(2+) overload. METHODS: Rat hearts (n = 6/group) were subjected to (a) 30 min of equilibration, 25 min of ischemia, and 30 min of reperfusion (Control) or (b) two 5-min episodes of ischemic preconditioning, 25 min of ischemia, and 30 min of reperfusion (IPC). Developed pressure (DP) was measured. Heart mitochondria were isolated at end-Equilibration (end-EQ) and at end-Reperfusion (end-RP). Mitochondrial respiratory function (state 2, oxygen consumption with substrate only; state 3, oxygen consumption stimulated by ADP; state 4, oxygen consumption after cessation of ADP phosphorylation; respiratory control index (RCI, state 3/state 4); rate of oxidative phosphorylation (ADP/Deltat), and ADP:O ratio) was measured with polarography using alpha-ketoglutarate as a substrate in the presence of different Ca(2+) concentrations (0 to 5 x 10(-7) M) to simulate Ca(2+) overload. RESULTS: IPC improved DP at end-RP. IPC did not improve preischemic mitochondrial respiratory function or preischemic mitochondrial response to Ca(2+) loading. IPC improved state 3, ADP/Deltat, and RCI during RP. Low Ca(2+) levels (0.5 and 1 x 10(-7) M) stimulated mitochondrial function in both groups predominantly in IPC. The Control group showed evidence of mitochondrial uncoupling at lower Ca(2+) concentrations (1 x 10(-7) M). IPC preserved state 3 at high Ca(2+) concentrations. CONCLUSIONS: The cardioprotective effect of IPC results, in part, from preserving mitochondrial function during reperfusion and increasing mitochondrial tolerance to Ca(2+) loading at end-RP. Activation of mitochondrial K(ATP) channels by IPC and their improvement in Ca(2+) homeostasis during RP may be the mechanism underlying this protection.     

Hepatic energy charge and adenine nucleotide status in rats anesthetized with halothane, isoflurane or enflurane

Background: Volatile anesthetics are known to have varying effects on hepatic oxygen supply in vivo and have been shown to depress hepatic mitochondrial respiration and so energy charge in vitro. However, the effect of halothane, isoflurane and enflurane on hepatic adenine nucleotide status in viuo has not been evaluated.
Methods: Ninety male rats were exposed to 40% oxygen (n=22) or 40% oxygen in equipotent (1 MAC) concentrations of halothane (1%) (n=23), isoflurane (1.4%) (n=22) or enflurane (2%) (n=23) for 2 hours. All animals were then administered intraperitoneal pentobarbital and anesthesia continued and laparotomy was performed. A liver biopsy was taken for determination of hepatocellular adenosine-5-triphosphate (ATP), adenosine-5-diphosphate (ADP) and adenosine-5-monophosphate (AMP) and computation of energy charge (EC) from ((ATP+1/2 ADP)+(ATP+ADP+AMP)) and total ade nine nucleotides (TAN) from (ATP+ADP+AMP). After the biopsy the aorta was cannulated for blood sampling.
Results: Rats in each group were similar in weight, as well as acid base and blood gas status just after liver biopsy. Hepatic energy charge, ATP, ADP, AMP, and TAN levels were not different in animals receiving either halothane, isoflurane or enflurane when compared with those receiving only oxygen.
Conclusions: One MAC of anesthesia for a period of 2 hours with the described volatile anesthetic agents did not affect adenine nucleotide status in viuo in rats.     

Opening of potassium channels protects mitochondrial function from calcium overload

Ischemic preconditioning (IPC) protects myocardium from ischemia reperfusion injury by activating mitochondrial K(ATP) channels. However, the mechanism underlying the protective effect of K(ATP) channel activation has not been elucidated. It has been suggested that activation of mitochondrial K(ATP) channels may prevent mitochondrial dysfunction associated with Ca(2+) overload during reperfusion. The purpose of this experiment was to study, in an isolated mitochondrial preparation, the effects of mitochondrial K(ATP) channel opening on mitochondrial function and to determine whether it protects mitochondria form Ca(2+) overload. Mitochondria (mito) were isolated from rat hearts by differential centrifugation (n = 5/group). Mito respiratory function was measured by polarography without (CONTROL) or with a potassium channel opener (PINACIDIL, 100 microM). Different Ca(2+) concentrations (0 to 5 x 10(-7) M) were used to simulate the effect of Ca(2+) overload; state 2, mito oxygen consumption with substrate only; state 3, oxygen consumption stimulated by ADP; state 4, oxygen consumption after cessation of ADP phosphorylation; respiratory control index (RCI: ratio of state 3 to state 4); rate of oxidative phosphorylation (ADP/Deltat); and ADP:O ratio were measured. PINACIDIL increased state 2 respiration and decreased RCI compared to CONTROL. Low Ca(2+) concentrations stimulated state 2 and state 4 respiration and decreased RCI and ADP:O ratios. High Ca(2+) concentrations increased state 2 and state 4 respiration and further decreased RCI, state 3, and ADP/Deltat. PINACIDIL improved state 3, ADP/Deltat, and RCI at high Ca(2+) concentrations compared to CONTROL. Pinacidil depolarized inner mitochondrial membrane, as evidenced by decreased RCI and increased state 2 at baseline. Depolarization may decrease Ca(2+) influx into mito, protecting mito from Ca(2+) overload, as evidenced by improved state 3 and RCI at high Ca(2+) concentrations. The myocardial protective effects resulting from activating K(ATP) channels either pharmacologically or by IPC may be the result of protecting mito from Ca(2+) overload.     

烧伤血清导致大鼠离体心室肌细胞钙超载的研究

目的观察烫伤血清对分离的成年大鼠心室肌细胞游离钙浓度(［Ca     

Altered ATP-dependent mitochondrial Ca2+ uptake in cold ischemia is attenuated by ruthenium red

BACKGROUND: Graft dysfunction as a result of preservation injury remains a major clinical problem in liver transplantation. This is related in part to accumulation of mitochondrial calcium (Ca(2+)), which has been linked to activation of proapoptotic factors. We hypothesized that cold ischemia increases mitochondrial Ca(2+) uptake in a concentration dependent fashion and that ruthenium red (RR) will attenuate these changes by inhibiting the mitochondrial Ca(2+) uniporter. METHODS: Rat livers perfused with cold University of Wisconsin (UW) solution (4 degrees C) with or without RR (10 microM) via the portal vein (n = 3 per group) were processed immediately (no ischemia) or after 24 h cold-storage (24 h cold ischemia). Mitochondria were separated by differential centrifugation, and adenosine triphosphate (ATP)-dependent (45)Ca(2+) uptake was determined in the presence of ATP (5 mM), adenosine diphosphate (ADP), or adenosine 5'-beta,gamma-imidotriphosphate (AMP-PNP); variable concentrations of extramitochondrial (45)Ca(2+) were used. All measurements were performed in triplicate. Student's t test with P < 0.05 was taken as significant. RESULTS: Our data demonstrate the following: 1) ATP-dependent (45)Ca(2+) uptake in mitochondria separated from livers following 24 h of cold ischemia in UW alone was higher than in mitochondria isolated from non-ischemic livers; the increased uptake was dependent on the concentration of (45)Ca(2+) in the incubation buffer. 2) There was no difference in ATP-dependent (45)Ca(2+) uptake between nonischemic mitochondria and those separated from livers stored in UW-RR for 24 h. 3) (45)Ca(2+) uptake in mitochondria from livers subjected to 24 h of cold ischemia in UW-RR was significantly lower compared to those from livers stored in UW alone when (45)Ca(2+) concentrations were greater than 1 microM. CONCLUSION: 1) Cold ischemia affects mitochondrial Ca(2+) handling, especially when it is challenged by high extramitochondrial Ca(2+) concentrations. 2) The addition of RR in preservation solution attenuates the effects of cold ischemia on mitochondrial Ca(2+) handling. 3) Inhibition of mitochondrial Ca(2+) uniporter with RR protects mitochondria from Ca(2+) overload at high Ca(2+) concentrations. These findings may offer a potentially effective strategy for prevention of ischemia-reperfusion injury in liver transplantation.     

Changes in hepatic energy metabolism in rats with acute necrotizing pancreatitis

In this study, changes of hepatic cellular ATP, ADP, and AMP, concentrations and mitochondrial oxidative phosphorylation were investigated in rats with experimental acute necrotizing pancreatitis (ANP). It was found that energy change (ATP + 1/2 ADP)/(ATP + ADP + AMP) of the liver decreased from 0.866 to 0.806 (P less than 0.05) 24 h after ANP, and to 0.769 (P less than 0.01) at 48 h. On the other hand, mitochondrial phosphorylative activity increased to 130% and 157% over the control at 12 h and 24 h respectively, and then rapidly dropped to 62% of normal value at 48 h. Blood ketone body ratio was positively correlated with hepatic energy charge level in ANP. The authors came to the following conclusions that: (1) In ANP, mitochondrial function damage resulted in decreased hepatic energy charge, which, in turn, led to hepatocellular impairment; (2) the measurement of blood ketone body ratio was a reliable indicator by which to assess the energy status of the liver in ANP.     

Calcium induced the damage of myocardial mitochondrial respiratory function in the early stage after severe burns

OBJECTIVE: To explore the role of Ca(2+) in the damage to myocardial mitochondrial respiratory function in the early stage after severe burns. METHODS: An experimental model of 30%TBSA full-thickness skin scalding was reproduced in rats. Myocardial mitochondria were isolated from control and burned rats in the 1st, 3rd, 6th, 12th and 24th hour post-burn. The mitochondrial respiratory function, contents of mitochondrial calcium ([Ca(2+)](m)), activities of mtPLA(2), mtNOS, F(0)F(1)-ATPase and cytochrome c oxidase were determined. RESULTS: (1) At the 1st hour post-burn, [Ca(2+)](m) was increased significantly and the myocardial mitochondrial respiratory function was significantly reinforced. At the same time, mitochondrial respiratory control rate (RCR) was elevated and positively correlated with [Ca(2+)](m) (r=0.8415, P<0.01). At the 3rd, 6th, 12th and 24th hour post-burn, [Ca(2+)](m) increased further to a higher level, however, the mitochondrial respiratory function was decreased from the peak value at 6h, and RCR was negatively correlated with [Ca(2+)](m). (2) The activities of mtNOS and mtPLA(2) were higher significantly at the 3rd, 6th, 12th and 24th hour post-burn than that of the control. After severe burns, mtNOS and mtPLA(2) activities were both positively correlated with [Ca(2+)](m) (r=0.8945, P<0.05; r=0.9271, P<0.01, respectively). (3) The F(0)F(1)-ATPase synthetic activity increased at the 1st hour post-burn, but it decreased to 51.4, 44.9, 77.6 and 87.4% of that of the control at the 3rd, 6th, 12th and 24th hour post-burn respectively. The F(0)F(1)-ATPase hydrolytic activity decreased at the 1st hour post-burn and increased at the 3rd, however, it decreased again at the 6th, 12th and 24th hour post-burn. The activity of cytochrome c oxidase at the 3rd, 6th, 12th and 24th hour was low compared to the control. CONCLUSIONS: The changes of [Ca(2+)](m) were involved in damage to or regulation of mitochondrial respiratory function after severe burns. Appropriate increase of [Ca(2+)](m) reinforced the mitochondrial respiration at 1st hour after of burn injury, but Ca(2+) severe overload impairing F(0)F(1)-ATPase and cytochrome c oxidase directly, or, indirectly by activation of mtPLA(2) and mtNOS, might play an important role in damage to myocardial mitochondrial respiratory function at later stages after severe burns.