一氧化氮合酶激活参与烧伤后Ca~(2 )介导的心肌线粒体损伤

目的 :探讨线粒体一氧化氮合酶 (mtNOS)在严重烧伤早期心肌线粒体损害中的作用。方法 :复制30 %TBSAⅢ°烧伤大鼠模型 ,取正常及伤后 1、3、6、12、2 4h大鼠心肌分离线粒体 ,测其呼吸功能、Ca2 浓度 ([Ca2 ]m)及细胞色素c氧化酶、mtNOS活性。结果 :①伤后 1h心肌线粒体呼吸控制率 (RCR)显著高于正常组 ,但 3、6、12、2 4h明显低于正常组 ,Ⅲ态呼吸速率 (ST3 )变化与RCR平行 ,ST4 仅于伤后 3h明显升高 ;②伤后各时点 [Ca2 ]m 均明显高于正常组 ,尤以 3、6h为甚 ,而mtNOS活性于伤后 3、6、12、2 4h显著高于对照组 ,细胞色素c氧化酶活性于伤后 3、6、12、2 4h显著低于正常组 ;③伤后mtNOS活性与 [Ca2 ]m 呈明显正相关 ,相关系数为 0 8945 (P <0 0 5 ) ,RCR与mtNOS活性呈显著负相关 ,相关系数为 - 0 934 7(P <0 0 5 )。结论 :伤后 [Ca2 ]m 升高激活mtNOS ,可能参与严重烧伤早期心肌线粒体损害。     

严重烧伤早期心肌[Ca2+]m变化及其机制研究

目的:探讨严重烧伤早期心肌线粒体Ca²⁺浓度([Ca²⁺]_m)的动态变化规律及其发生机制。方法:复制30%Ⅲ°烫伤大鼠模型,测定伤后1、3、6、12、24h大鼠心肌[Ca²⁺]_m,同时检测影响[Ca²⁺]_m的相关指标—胞浆Ca^2＋浓度(_c)及线粒体Ca^2＋转运速率。结果:烧伤后1、3、6h[Ca²⁺]_m依次升高,12、24h较6h虽有所下降,但仍高于正常对照组;_c除伤后1h无明显变化外,其余各时相点变化趋势与[Ca²⁺]_m相同,且伤后[Ca²⁺]_m与_c呈显著正相关,相关系数为0.9177(P＜0.01)。伤后1h心肌线粒体Ca^2＋摄取速率明显升高,而Ca^2＋释放速率无明显改变,但3、6、12、24h心肌线粒体Ca^2＋摄取速率与Ca^2＋释放速率均显著降低,且烧伤后3、6、12、24h[Ca²⁺]_m分别与线粒体Ca^2＋释放速率呈明显负相关。结论:烧伤后心肌线粒体存在明显的Ca^2＋超载和转运紊乱。     

急、慢性缺氧对大鼠脑线粒体能量代谢的影响

目的:探讨缺氧大鼠脑线粒体能量代谢的特点。方法:雄性Wistar大鼠随机分为急性缺氧组(AH)、慢性缺氧组(CH)和对照组。急、慢性缺氧组动物分别连续暴露于模拟4000m高原3d(AH)和40d(CH)。分离脑线粒体,分别测定线粒体呼吸功能、线粒体内腺苷酸池含量、ATP生成能力和F₀F₁-ATP酶活性。结果:急性缺氧大鼠IV态呼吸(ST₄)显著升高,伴呼吸控制率(RCR)降低,同时线粒体内ATP含量、ATP生成率和F₀F₁-ATP酶活性均显著降低;慢性缺氧大鼠ST₄、RCR、线粒体ATP含量和F₀F₁-ATP酶活性部分恢复。结论:急性缺氧脑线粒体代谢是以功能受损为特点,而慢性缺氧时则表现为功能的部分代偿。     

L-精氨酸对兔缺血-再灌注心肌线粒体功能及结构的影响

目的:探讨L-精氨酸对心肌缺血-再灌注(MIR)时心肌细胞线粒体功能及结构的影响。方法:实验兔30只,随机分为正常对照组(control组)、心肌缺血-再灌注组(MIR组)和心肌缺血-再灌注+L-精氨酸治疗组(MRI+L-Arg组);分别观察心肌线粒体呼吸功能、Ca²⁺浓度([Ca²⁺]m)、丙二醛(MDA)浓度、超氧化物歧化酶(SOD)活性及超微结构的改变和心肌组织三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、一磷酸腺苷(AMP)含量、总腺苷酸量(TAN)、能荷(EC)的变化。结果:MIR+L-Arg组线粒体呼吸控制率(RCR)、Ⅲ态呼吸速率(V₃)、SOD、面密度(Sv)、比表面(δ)明显高于MIR组,Ⅳ态呼吸速率(V₄)、[Ca²⁺]m、MDA、体密度(Vv)、横径(Hd)显著低于MIR组,心肌组织ATP、ADP、TAN及EC均明显高于MIR组;且与control组比较,V₃、V₄、SOD、MDA、Vv、Sv、δ、数密度(Nv)、纵径(Vd)及AMP、TAN无明显差异。结论:L-精氨酸可通过降低氧自由基水平和减轻钙超载,而改善缺血-再灌注心肌的线粒体功能及结构。     

严重烧伤早期心肌[Ca~(2 )]_m变化及其机制研究

目的 :探讨严重烧伤早期心肌线粒体Ca2 浓度 ([Ca2 ]m)的动态变化规律及其发生机制。方法 :复制 30 %Ⅲ°烫伤大鼠模型 ,测定伤后 1、3、6、12、2 4h大鼠心肌 [Ca2 ]m,同时检测影响 [Ca2 ]m 的相关指标—胞浆Ca2 浓度 ([Ca2 ]c)及线粒体Ca2 转运速率。结果 :烧伤后 1、3、6h [Ca2 ]m 依次升高 ,12、2 4h较 6h虽有所下降 ,但仍高于正常对照组 ;[Ca2 ]c 除伤后 1h无明显变化外 ,其余各时相点变化趋势与 [Ca2 ]m 相同 ,且伤后 [Ca2 ]m 与 [Ca2 ]c呈显著正相关 ,相关系数为 0 .9177(P <0 .0 1)。伤后 1h心肌线粒体Ca2 摄取速率明显升高 ,而Ca2 释放速率无明显改变 ,但 3、6、12、2 4h心肌线粒体Ca2 摄取速率与Ca2 释放速率均显著降低 ,且烧伤后 3、6、12、2 4h [Ca2 ]m分别与线粒体Ca2 释放速率呈明显负相关。结论 :烧伤后心肌线粒体存在明显的Ca2 超载和转运紊乱     

盐霉素抑制人肺腺癌耐顺铂细胞株A549/DDP增殖及诱导凋亡的机制

目的: 探讨盐霉素对人肺腺癌耐药细胞株A549/DDP增殖的抑制作用及其可能机制。方法: 采用MTT法检测盐霉素对A549/DDP细胞生长的抑制作用;流式细胞术检测盐霉素对A549/DDP细胞凋亡及线粒体膜电位(ΔΨ_m)的影响;比色法检测caspase-3、8和9活性;Western blotting分析细胞色素C、Bcl-2、Bax、β-catenin和磷酸化低密度脂蛋白受体相关蛋白6(p-LRP6)蛋白水平。结果: 盐霉素对A549/DDP细胞生长具有剂量依赖性抑制作用。0.2 μmol/L盐霉素作用于A549/DDP细胞,ΔΨ_m显著下降,而细胞内活性氧和Ca²⁺浓度在短期显著升高,胞浆细胞色素C蛋白水平、caspase-3、8和9酶活性均显著增加,与对照组比较差异均有统计学意义(P<0.01);Bcl- 2 的表达下调,Bax 的表达明显增加,Bcl-2/Bax 比值显著降低。48 h时增殖抑制率为(34.61±1.97)%,细胞凋亡率为(18.74±2.08)%。盐霉素也减少A549/DDP细胞内β-catenin和p-LRP6蛋白水平。结论: 盐霉素通过抑制Wnt信号通路抑制A549/DDP细胞增殖,通过Bcl-2/Bax途径和线粒体凋亡途径诱导人肺腺癌耐药细胞株A549/DDP凋亡。     

汉黄芩素对流感病毒感染肺泡巨噬细胞炎症相关因子的影响

目的: 研究汉黄芩素对甲型流感病毒鼠肺适应株A/FM/1/47(H1N1)感染的大鼠肺泡巨噬细胞(NR8383)产生促炎症细胞因子、炎症介质及氧自由基的影响。 方法: 流感病毒感染 NR8383细胞1 h后,加入含汉黄芩素的培养基(终浓度16 mg/L),药物作用后6 h、12 h和24 h,ELISA法检测细胞上清中肿瘤坏死因子α(TNF-α)和单核细胞趋化蛋白 1(MCP-1)的含量,放射免疫测定法检测细胞上清中前列腺素E₂(PGE₂)、磷脂酸A₂(PLA₂)和白三烯B₄(LTB₄)的含量;药物作用后8 h、24 h、36 h和48 h,生化法检测细胞内一氧化氮(NO)含量和诱导型一氧化氮合酶(iNOS)活性,4 h、8 h、18 h和24 h,生化法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;药物作用后24 h,real-time PCR检测细胞内TNF-α和MCP-1的mRNA水平。 结果: 汉黄芩素抑制了流感病毒感染NR8383细胞后TNF-α、MCP-1的转录和表达(P<0.01),降低了PGE₂、PLA₂、LTB₄和MDA的含量(P<0.05);减少了NO和iNOS的产生(P<0.05),增强了SOD的活性(P<0.05)。 结论: 汉黄芩素明显抑制了流感病毒感染后肺泡巨噬细胞内各种炎症相关因子的产生,具有抗炎作用。     

14-3-3γ对烧伤及LPS诱导所致大鼠心肌损伤的保护作用

目的: 研究14-3-3γ在烧伤及脂多糖(LPS)引起的心肌损伤中的保护作用。方法: 体内实验建立大鼠烧伤及LPS损伤模型,检测3、6、12、24 h各时点心功能及心肌细胞14-3-3γ蛋白表达水平的改变;体外实验采用新生大鼠心肌细胞,构建pFLAG-14-3-3γ质粒,于LPS损伤前24 h转染至心肌细胞,实验结束后检测培养液中乳酸脱氢酶(LDH) 活性,MTT法测定细胞存活率,流式细胞术检测细胞凋亡,线粒体肿胀实验检测线粒体通透性转换孔(mPTP)开放。结果: 烧伤及LPS损伤模型大鼠心电图ST段均有明显改变,心肌细胞14-3-3γ蛋白表达水平均显著升高。转染了pFLAG-14-3-3γ质粒的心肌细胞能明显对抗随后的LPS损伤,与未转染组比较,14-3-3γ能明显提高细胞存活率,降低LDH活性,减少细胞凋亡,抑制mPTP的开放。结论: 14-3-3γ可能通过抑制mPTP的开放对烧伤及LPS所致的心肌损伤起保护作用。     

盐霉素抑制耐格列卫的人慢性粒细胞白血病细胞株K562/Glv增殖并诱导其凋亡

目的: 探讨盐霉素对耐格列卫的人慢性粒细胞白血病细胞株K562/Glv抑制增殖和诱导凋亡的作用及其机制。方法: 采用CCK-8的方法检测盐霉素对K562/Glv细胞生长的抑制作用;流式细胞术检测细胞凋亡、活性氧、细胞内Ca²⁺浓度([Ca²⁺]_i)和线粒体膜电位(ΔΨ_m);比色法检测caspase-3、-8和-9活性;Western blotting 分析细胞色素C、Bcl-2、Bax、β-catenin和磷酸化低密度脂蛋白受体相关蛋白6(p-LRP6)蛋白水平。结果: 盐霉素对K562/Glv细胞生长具有剂量依赖性抑制作用,0.2 μmol/L时细胞增殖抑制率为(36.70±2.31)%,细胞凋亡率为(19.66±2.43)%;0.2 μmol/L盐霉素作用于K562/Glv细胞,ΔΨ_m显著下降,24 h时下降至对照组的(19.8±2.4)%,细胞内活性氧和[Ca²⁺]_i在短期显著升高。Caspase-3、-8和-9活性均显著增加,与对照组比较,差异有统计学意义(P<0.01)。Bcl-2 的表达下调,Bax 的表达明显增加,Bcl-2/Bax 比值显著降低。同时,盐霉素也减少K562/Glv细胞内β-catenin和p-LRP6蛋白水平。结论: 盐霉素不仅通过Bcl-2/Bax途径和线粒体凋亡途径诱导耐格列卫人慢性粒细胞白血病细胞K562/Glv的凋亡,而且通过抑制Wnt信号途径抑制K562/Glv细胞增殖。     

内毒素血症时大鼠肝细胞线粒体损伤及其机制的研究

目的:观察急性感染性内毒素血症大鼠肝细胞线粒体呼吸链的损伤及其机制。方法:将体重在250-280g健康SD大鼠随机分为空白对照组、内毒素组。提取内毒素血症大鼠肝细胞线粒体,测定其超氧阴离子O₂生成量,同时测定线粒体呼吸链功能:复合体Ⅱ+Ⅲ的电子传递与质子转移定量关系(H⁺/2e^-)、ADP/O、呼吸控制率(RCR)。结果:内毒素血症大鼠肝细胞线粒体电子漏显著增加;大鼠肝细胞线粒体以琥珀酸为底物的态4和态3呼吸速率增加,ADP/O、RCR及复合体Ⅱ+Ⅲ的H⁺/2e^-显著降低。结论:在内毒素血症的发病机制中存在由于肝细胞线粒体内源性氧自由基生成增加对线粒体呼吸功能所造成的氧化损伤。     

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca²⁺ plays important roles in the regulation of energy metabolism and cellular Ca²⁺ homeostasis. In this study, we characterized mitochondrial Ca²⁺ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 μM Ca²⁺ ([Ca²⁺]_o) caused a concentration-dependent increase in intramitochondrial Ca²⁺ concentrations ([Ca²⁺]_m). The [Ca²⁺]_m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca²⁺]_o was higher than 1 μM and a decrease of about 52% was detected at [Ca²⁺]_o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca²⁺ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ _m). Using a tetraphenylphosphonium (TPP⁺) electrode, the measured Δψ _m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Δψ _m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca²⁺ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.     

Cytoplasmic and mitochondrial Ca2+ levels in brown adipocytes

Aim: We elucidated the mitochondrial functions of brown adipocytes in intracellular signalling, paying attention to mitochondrial activity and noradrenaline‐ and forskolin‐induced Ca²⁺ mobilizations in cold‐acclimated rats. Methods: A confocal laser‐scanning microscope of brown adipocytes from warm‐ or cold‐acclimated rats was employed using probes rhodamine 123 which is a mitochondria‐specific cationic dye, and the cytoplasmic and mitochondrial Ca²⁺ probes fluo‐3 and rhod‐2. X‐ray microanalysis was also studied. Results: The signal of rhodamine 123 in the cells was decreased by antimycin A which effect was less in cold‐acclimated cells than warm‐acclimated cells. Cytoplasmic and mitochondrial Ca²⁺ in cold‐acclimated brown adipocytes double‐loaded with fluo‐3 and rhod‐2 were measured. Noradrenaline induced the rise in cytoplasmic Ca²⁺ ([Ca²⁺]_cyto) followed by mitochondrial Ca²⁺ ([Ca²⁺]_mito), the effect being transformed into an increase in [Ca²⁺]_cyto whereas a decrease in [Ca²⁺]_mito by antimycin A or carbonyl cyanide m‐chlorophenylhydrazone (CCCP). Antimycin A induced small Ca²⁺ release from mitochondria. CCCP induced Ca²⁺ release from mitochondria only after the cells were stimulated with noradrenaline. Further, forskolin also elicited an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito in the cells. The Ca measured by X‐ray microanalysis was higher both in the cytoplasm and mitochondria whereas K was higher in the mitochondria of cold‐acclimated cells in comparison to warm‐acclimated cells. Conclusions: These results suggest that noradrenaline and forskolin evoked an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito, in which H⁺ gradient across the inner membrane is responsible for the accumulation of calcium on mitochondria. Moreover, cAMP also plays a role in intracellular and mitochondrial Ca²⁺ signalling in cold‐acclimated brown adipocytes.     

The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers

We report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca²⁺ marker in enzymatically dissociated mouse FBD muscle fibers. Using laser scanning confocal microscopy and the dyes Mitotracker Green (MTG), di-8-ANEPPS and endoplasmic reticulum tracker green (ERTG), we determined the relative position of mitochondria, transverse tubules and sarcoplasmic reticulum in the sarcomere. Comparison with electron micrographies showed that mitochondria are mostly present at both sides of Z lines and near the triads located at the A-I band border. CaOr-5N fluorescence was mainly distributed in mitochondria, highly co-localised with MTG and basically excluded from the A band space. ERTG localised mostly between the two t-tubules present in each sarcomere. We studied the effect of the protonophore FCCP using CaOr-5N to measure mitochondrial Ca²⁺ and JC-1 dye to measure mitochondria inner membrane potential (ΔΨ _m). After FCCP treatment, the CaOr-5N fluorescence diminished by about 33% in 80 s, while JC-1 fluorescence diminished by 36% in 200 s. Our results show the loss of Ca²⁺ from mitochondria when ΔΨ_m is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial [Ca²⁺]_m. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Frequency-dependent mitochondrial Ca2+ accumulation regulates ATP synthesis in pancreatic β cells

Pancreatic β cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K⁺ channels and electrical spiking. The latter results in oscillatory Ca²⁺ influx through voltage-gated Ca²⁺ channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca²⁺]_cyt and [Ca²⁺]_mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca²⁺-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca²⁺ pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary β cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca²⁺ and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca²⁺ uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca²⁺ oscillation frequency as stable increases in [Ca²⁺]_mit and cytosolic ATP/ADP; (3) mitochondrial Ca²⁺ uptake rates remained constant between individual spikes, arguing against activity-dependent regulation (“plasticity”) and (4) the relationship between [Ca²⁺]_cyt and [Ca²⁺]_mit is essentially unaffected by changes in endoplasmic reticulum Ca²⁺ ([Ca²⁺]_ER). Our findings thus highlight new aspects of Ca²⁺ signalling in β cells of relevance to the actions of both glucose and sulphonylureas.     

Regulation of the mitochondrial proton gradient by cytosolic Ca2+ signals

Mitochondria convert the energy stored in carbohydrate and fat into ATP molecules that power enzymatic reactions within cells, and this process influences cellular calcium signals in several ways. By providing ATP to calcium pumps at the plasma and intracellular membranes, mitochondria power the calcium gradients that drive the release of Ca²⁺ from stores and the entry of Ca²⁺ across plasma membrane channels. By taking up and subsequently releasing calcium ions, mitochondria determine the spatiotemporal profile of cellular Ca²⁺ signals and the activity of Ca²⁺-regulated proteins, including Ca²⁺ entry channels that are themselves part of the Ca²⁺ circuitry. Ca²⁺ elevations in the mitochondrial matrix, in turn, activate Ca²⁺-dependent enzymes that boost the respiratory chain, increasing the ability of mitochondria to buffer calcium ions. Mitochondria are able to encode and decode Ca²⁺ signals because the respiratory chain generates an electrochemical gradient for protons across the inner mitochondrial membrane. This proton motive force (??p) drives the activity of the ATP synthase and has both an electrical component, the mitochondrial membrane potential (???? _m ), and a chemical component, the mitochondrial proton gradient (??pH _m ). ???? _m contributes about 190?mV to ??p and drives the entry of Ca²⁺ across a recently identified Ca²⁺-selective channel known as the mitochondrial Ca²⁺ uniporter. ??pH _m contributes ~30?mV to ??p and is usually ignored or considered a minor component of mitochondria respiratory state. However, the mitochondrial proton gradient is an essential component of the chemiosmotic theory formulated by Peter Mitchell in 1961 as ??pH _m sustains the entry of substrates and metabolites required for the activity of the respiratory chain and drives the activity of electroneutral ion exchangers that allow mitochondria to maintain their osmolarity and volume. In this review, we summarize the mechanisms that regulate the mitochondrial proton gradient and discuss how thermodynamic concepts derived from measurements in purified mitochondria can be reconciled with our recent findings that mitochondria have high proton permeability in situ and that ??pH _m decreases during mitochondrial Ca²⁺ elevations.     

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca²⁺]_i) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca²⁺]_i). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca²⁺]_i increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca²⁺]_i. Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca²⁺]_i, demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca²⁺]_i and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca²⁺]_i and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival.