蒿甲醚引起线粒体跨膜电位下降在神经毒性中的作用

目的　用嗜铬细胞瘤细胞(PC12细胞)、原代培养的大鼠神经元细胞和大鼠脑线粒体作为体外模型观察线粒体跨膜电位(mitochondrialmembranepotential,MMP)和细胞膜通透性改变在蒿甲醚引起的神经毒性中的作用。　方法　用流式细胞仪测定蒿甲醚对线粒体跨膜电位和细胞膜通透性的影响,用分光光度法分析蒿甲醚对线粒体肿胀度的影响。　结果　蒿甲醚能够降低两种细胞的MMP,使其峰值左移,摄入的Rh123荧光强度降低,量效关系明显;能增加两种细胞的细胞膜通透性,使细胞内的PI摄入增加;能引起大鼠脑线粒体肿胀,有一定的时间效应和剂量效应关系。结论　MMP下降是蒿甲醚引起神经毒性的重要环节,可通过影响线粒体膜通透性转运孔,改变线粒体跨膜电位和线粒体肿胀度,并能增加细胞膜通透性,引起细胞能量代谢障碍,最终导致神经毒性     

磷酸肌酸对心肌细胞线粒体的保护作用

目的 :研究能量治疗对心力衰竭的治疗机制。观察磷酸肌酸 （phosphocreatine,CP）对心肌细胞线粒体膜电位的保护作用。方法 :采用胶原酶分离成年大鼠心肌细胞 ,0 .2 mmol/ L H2 O2 刺激细胞 ,于刺激前 10 min加入 10mm ol/ L CP,利用荧光探针 JC-1结合流式细胞仪检测线粒体膜电位的变化 ;Annexin-V/ PI及 TUNEL 染色法鉴定心肌细胞的凋亡。结果 :H2 O2 导致线粒体膜电位下降 ,细胞凋亡增加。CP有效地减低了线粒体膜电位的下降 ,减少了细胞凋亡。结论 :CP能够抗心肌细胞氧化损伤 ,其机制可能依赖于减少或抑制线粒体膜电位的下降 ,保护了线粒体的正常功能     

Utilization of intramitochondrial membrane cholesterol by cytochrome P-450-dependent cholesterol side-chain cleavage reaction in bovine adrenocortical mitochondria: steroidogenic and non-steroidogenic pools of cholesterol in the mitochondrial inner membranes

Inner and outer submitochondrial membranes were prepared after disruption of malate-treated bovine adrenocortical mitochondria. It was found that a part of the endogenous cholesterol in the inner membrane (approx. 50%) was rapidly utilized by the cholesterol side-chain cleavage reaction. The utilization of cholesterol in the outer membrane, on the other hand, was inefficient and slow in spite of the fact that cholesterol concentration is higher in the outer than in the inner membrane. When the inner membrane prepared from untreated mitochondria was incubated for 20 min in the presence of a reconstituted cytochrome P-450-reducing system, the inner membrane cholesterol was depleted by approximately 70%. The half-life of the depletion reaction was 2–3 min. In addition, when the outer membrane plus the soluble fraction from the untreated mitochondria were added as a source of cholesterol to the inner membrane fraction, a marginal increase in the production of steroids was observed. From these results it is concluded that a portion of the inner membrane cholesterol can be steroidogenic, whereas the rest of the cholesterol is non-steroidogenic.     

病毒性心肌炎小鼠心肌与骨骼肌细胞线粒体损伤及其相关性

目的探讨病毒性心肌炎（VMC）小鼠心肌与骨骼肌细胞线粒体损伤（线粒体膜磷脂脱失和线粒体DNA3867缺失）程度及二者的相关性。方法50只BALB/c小鼠随机分为2组,实验组（40只）腹腔注射内含柯萨奇B3病毒（Coxsackievirus B3,CVB3,TCID50=108）的Eagle液制备VMC小鼠模型,另10只为对照组。分别于病毒感染后3、11和24 d行心肌和骨骼肌细胞线粒体膜磷脂脱失程度和mtDNA3867缺失率的测定,并用Spearman法对其进行相关分析。结果实验组小鼠在病毒感染后3 d,可见心肌和骨骼肌细胞mtDNA3867缺失率显著高于对照组（P<0.05）,而线粒体膜磷脂脱失程度与对照组相比无显著性差异;病毒感染后11 d,心肌和骨骼肌细胞mtDNA3867缺失性损伤达高峰（P<0.05）,线粒体膜磷脂脱失程度亦显著高于对照组（P<0.05）;病毒感染后24 d,心肌和骨骼肌细胞线粒体膜磷脂脱失和mtDNA3867缺失程度与感染后11 d组比较无显著性差异,但与对照组和病毒感染后3 d组比较,仍有显著性差异（P<0.05）。线粒体的上述损伤性改变在心肌和骨骼肌细胞呈一致性同步变化,且具有良好相关性（P<0.05）。结论CVB3可显著损伤心肌和骨骼肌线粒体DNA和膜磷脂,两者损伤具有相关性,提示骨骼肌有望成为反映心肌细胞线粒体损伤的外周细胞“窗口”。     

Macromolecular organization of ATP synthase and complex I in whole mitochondria

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F(1) heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.     

Mitochondrial calcium uptake

Calcium (Ca²⁺) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca²⁺ levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca²⁺ ([Ca²⁺]_i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca²⁺ uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca²⁺ uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca²⁺ uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca²⁺ influx into the mitochondria via the MCU is small relative to other cytosolic Ca²⁺ extrusion pathways; (ii) single MCU conductance is ∼6–7 pS (105 mM [Ca²⁺]), and MCU flux appears to be modulated by [Ca²⁺]_i, suggesting Ca²⁺ regulation of MCU open probability (P_O); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca²⁺ critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca²⁺ under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca²⁺]_i, mitochondrial Ca²⁺ uptake can increase 10- to 1,000-fold and begin to shape [Ca²⁺]_i dynamics.     

线粒体TOM系统在心血管疾病中的研究现状

线粒体内稳态的维持与其内部蛋白密切相关,而绝大多数蛋白进入线粒体内部发挥作用均需通过线粒体的线粒体外膜转位酶(translocase of the outer mitochondrial membrane,TOM)系统的转运。研究表明,线粒体TOM系统相关组成亚基Tom70、Tom20和Tom40等参与了心血管疾病的发生发展,这为我们从线粒体蛋白水平研究心血管疾病的机制及开发新的治疗措施提供了思路。本文针对线粒体TOM系统在心血管相关疾病(如心肌缺血/再灌注、高血压及心功能衰竭)方面的研究现状进行了综述。     

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C–C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1—the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C–C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 Å away from the heme iron of CYP11A1. This structure suggests that after an initial protein–protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.     

Increased lactate production follows loss of mitochondrial membrane potential during apoptosis of human leukaemia cells

Acute tumour-lysis syndrome (ATLS) is a frequently fatal complication after cytoreductive leukaemia therapy. Lactic acidosis is associated with ATLS and its extent is correlated with the severity of ATLS. In the course of cytoreductive therapy, apoptosis is induced in tumour cells, which results in loss of mitochondrial function. We hypothesize that loss of mitochondrial function leads to compensatory glycolysis, which is the main cause of lactate accumulation and acidosis. We tested this hypothesis using the model of glucocorticoid-induced apoptosis in the human acute lymphoblastic leukaemia cell line CCRF-CEM. After induction of glucocorticoid-induced apoptosis, a biphasic course of lactate production was observed. Prior to the onset of apoptosis, i.e. prior to the loss of membrane potential, lactate production was reduced. However, subsequent to loss of mitochondrial membrane potential a massive increase in lactate production was observed (15.5 +/- 0.5 versus 10.17 +/- 0.09 mmol/10(6) cells, P = 0.001). We also demonstrated that inhibition of respiratory chain activity by antimycin A resulted in excess lactate production. In the model cell line used, conditional bcl-2 expression delayed glucocorticoid-induced apoptosis by protecting against loss of mitochondrial membrane potential; bcl-2 expression delayed the increase in lactate production and had no effect on the pre-apoptotic drop in lactate production. Apoptosis-induced lactate production was also observed in other cell lines (HL60, THP1 and OPM2) with various cytotoxic agents [doxorubicin, gemcitabine and vumon (VM26)]. Thus, the data suggest that lactate acidosis can be caused by apoptotic loss of mitochondrial function and massive apoptosis of a tumour mass via lactic acidosis may be the essential pathological event in ATLS.     

MICS-1 interacts with mitochondrial ATAD-3 and modulates lifespan in C. elegans

Caenorhabditis elegans open reading frame T21C9.1 encodes an uncharacterized protein, which is here named MICS-1 (mitochondrial scaffolding protein-1). It is predicted to be the homolog of human outer mitochondrial membrane protein 25 (OMP25 or synaptojanin-2-binding protein), which is a PDZ domain containing protein with a putative role in cellular stress response pathways. Here, we provide evidence that MICS-1 is an interacting partner of mitochondrial protein ATAD-3 (homologue of human ATAD3), which is essential for C. elegans development. We demonstrate that mics-1(RNAi) animals or mics-1 mutants display enhanced longevity with an increased mean lifespan of up to 54% compared to control animals. Of note, also atad-3(RNAi) promoted longevity, although to a lesser extend (29% compared to controls). In addition, thermal stress of mics-1 mutants induced low reactive oxygen species (ROS) production, whereas atad-3(RNAi) animals were highly sensitive to this assay, displaying drastically increased ROS levels. Further studies revealed that MICS-1 and ATAD-3 associated longevity was partially dependent on the presence of DAF-16. However, for both conditions, we also found a DAF-16 independent extension of lifespan. Finally, we observed an additional lifespan extension in mics-1 mutants when subjected to atad-3(RNAi) whereas heat induced ROS production was even aggravated under this condition. This suggests (partially) independent effects of MICS-1 and ATAD-3 on lifespan and ROS production in vivo.     

Platelets of type 2 diabetic patients are characterized by high ATP content and low mitochondrial membrane potential

Platelet dysfunction plays a critical role in vascular complications of type 2 diabetes mellitus (T2DM). But the relationship between platelet hyperactivity and its energy metabolic process remains unclear. This study was designed to explore alterations of platelet mitochondrial ATP production and the possible mechanism. A total of 39 T2DM patients without macrovascular and microvascular complications and 32 normal controls were fasting sampled. Platelet ATP content was measured by a high performance liquid chromatograph (HPLC). The flow cytometry technique was adopted to evaluate mitochondrial membrane potential (ΔΨm), the stored force for platelet ATP production. Consequently, T2DM patients exhibited obvious hyperglycemia, hyperlipidemia and hypertension, but normal platelet morphology. Platelet ATP content was significantly higher in T2DM (0.032 ± 0.010 µmol/10⁹ platelets versus 0.017 ± 0.006 µmol/10⁹ platelets, p < 0.001) than in the control group. Interestingly, ΔΨm was markedly decreased in T2DM patients (0.79 ± 0.18 versus 2.70 ± 1.03, p < 0.001) compared with normal controls. For whole subjects, a stepwise regression showed that plasma glycated hemoglobin A1c (HbA1c) level positively correlated to platelet ATP content (β = 0.552, 95% CI = 0.072–1.451), and fasting plasma glucose (FPG) level was negatively correlated to ΔΨm (β = ?0.372, 95% CI = ?0.471 to ?0.089). These data support that hyperglycemia of T2DM promotes platelet mitochondria to generate more ATP, but decreases platelet mitochondrial potential. The discordance between them requires further researches to elucidate.     

Bid-induced mitochondrial membrane permeabilization waves propagated by local reactive oxygen species (ROS) signaling

Bid-induced mitochondrial membrane permeabilization and cytochrome c release are central to apoptosis. It remains a mystery how tiny amounts of Bid synchronize the function of a large number of discrete organelles, particularly in mitochondria-rich cells. Looking at cell populations, the rate and lag time of the Bid-induced permeabilization are dose-dependent, but even very low doses lead eventually to complete cytochrome c release. By contrast, individual mitochondria display relatively rapid and uniform kinetics, indicating that the dose dependence seen in populations is due to a spreading of individual events in time. We report that Bid-induced permeabilization and cytochrome c release regularly demonstrate a wave-like pattern, propagating through a cell at a constant velocity without dissipation. Such waves do not depend on caspase activation or permeability transition pore opening. However, reactive oxygen species (ROS) scavengers suppressed the coordination of cytochrome c release and also inhibited Bid-induced cell death, whereas both superoxide and hydrogen peroxide sensitized mitochondria to Bid-induced permeabilization. Thus, Bid engages a ROS-dependent, local intermitochondrial potentiation mechanism that amplifies the apoptotic signal as a wave.     

肺泡巨噬细胞凋亡在老年多器官功能衰竭肺启动中作用的研究

目的探讨肺泡巨噬细胞(AM)凋亡在老年多器官功能衰竭(MOF)肺启动中的可能机制。方法选择SD大鼠40只,随机分为油酸+脂多糖组20只和对照组20只。应用油酸-内毒素序贯打击,复制以肺功能障碍为首发的MOF模型,分别在造模6、12、24 h后处死2组大鼠各5只,通过支气管肺泡灌洗和细胞贴壁的方法获取AM,用碘化丙啶染色流式细胞仪测定AM凋亡率。结果造模后6、12、24 h,油酸+脂多糖组大鼠AM的多器官功能障碍综合征发生率、凋亡率和游离钙含量逐渐升高,线粒体膜电位逐渐变小(P<0.01);而对照组各时间点AM的凋亡率、游离钙含量及线粒体膜电位无明显变化(P>0.05)。与对照组比较,油酸+脂多糖组造模后各时间点多器官功能障碍综合征发生率、AM的凋亡率和游离钙含量均明显升高(P<0.01),线粒体膜电位明显降低(P<0.01)。结论 AM凋亡率逐渐增加严重影响了其功能的发挥,可能是MOF发生的重要原因。AM细胞内游离钙含量逐渐升高、线粒体膜电位逐渐变小,是诱导老年大鼠AM凋亡增加的重要机制。     

Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.Commitment of cells to apoptosis is determined primarily by interactions within the B-cell lymphoma-2 (Bcl-2) protein family on the mitochondrial outer membrane (MOM) (1–4). The proapoptotic members Bcl-2 antagonist/killer (Bak) and Bcl-2–associated X protein (Bax) mediate the pivotal step of MOM permeabilization, which releases proteins, such as cytochrome c, that promote the proteolytic demolition by caspases. Two other Bcl-2 subfamilies tightly control Bak and Bax activation. Their activation is promoted by the Bcl-2 homology domain 3 (BH3)-only proteins, such as BH3-interacting domain death agonist (Bid), the truncated form of which (tBid) can directly bind both. Conversely, prosurvival family members can bind and inhibit activated Bak and Bax, as well as the BH3-only proteins.Like their prosurvival relatives, Bak and Bax in healthy cells are globular monomers, comprising similar helical bundles with a hydrophobic α-helix (α5) surrounded by amphipathic helices (5, 6). Their C-terminal helix (α9) is a hydrophobic transmembrane (TM) domain that anchors them in the MOM. In healthy cells Bak is already anchored there, presumably solely by α9, whereas Bax is primarily cytosolic (5), accumulating at the MOM after an apoptotic signal and inserting its α9. Other major conformational changes in both Bak and Bax, reviewed in ref 4, include exposure of their BH3 (α2) and its reburial within the surface groove of another activated Bak or Bax molecule (7–10). These novel “symmetric” homo-dimers can multimerize via association of α6 helices (8, 11, 12).Although oligomeric Bak and Bax are highly implicated in MOM permeabilization, how they interact with the membrane to form pores remains a mystery. The first structure of a Bcl-2 family member, the prosurvival protein Bcl-x_L (13), and later those of Bax (5) and Bak (6), provided a tantalizing clue: similarities with the pore-forming domains of bacterial toxins, such as diphtheria toxin or colicin A. To form pores, these toxins are thought to insert their two hydrophobic core helices as a hairpin across the membrane (14), suggesting that the central helices of Bak and Bax (α5 and α6) might penetrate the MOM similarly (reviewed in refs 3, 15, and 16). Consistent with this hairpin insertion model, α5 and α6 peptides can permeabilize membranes (17–19). More pertinently, Bax α5 and α6 were reported to insert into and span the MOM as a hairpin before oligomerization (20).This longstanding model, however, does not fit well with recent evidence on the structure of Bak and Bax oligomers, as recently reviewed (2, 4). Analysis of Bak oligomers in liposomes by electron paramagnetic resonance (EPR) suggests that α6 inserts only shallowly in the lipid bilayer (21). Additionaly, the first 3D structures of activated forms of Bax (10) suggest that, early in its activation, α5 and α6 separate. Moreover, a Bax core domain containing only helices α2 to α5 generated a BH3:groove symmetric dimer in which two α4 and two α5 helices form an aromatic face that might sit on the bilayer (10). These findings fit better with an “in-plane model” in which only α9 is a TM domain and other helices (including α5 and α6) insert only shallowly into the bilayer.The nature of the apoptotic pores remains uncertain. Some findings favor a proteinaceous pore (22), but studies with model membranes suggest that Bax oligomers can perturb the bilayer and produce lipidic pores (i.e., pores not bounded entirely by protein) (23–26).These important unresolved questions about the pivotal event in apoptosis prompted us to explore the membrane topology of Bak, before, during, and after an apoptotic signal, and to reinvestigate that of Bax. In accord with recent Bax structures (10) and recent EPR studies on Bak (21, 27), the results show that neither oligomeric Bak nor Bax inserts an α5–α6 hairpin across the MOM. We propose instead that the α5 and α6 helices lie in the bilayer plane and disrupt membrane integrity by imposing tension and curvature to the membrane that provoke its permeabilization.     

Inheritance of mitochondrial DNA in humans: implications for rare and common diseases

The first draft human mitochondrial DNA (mtDNA) sequence was published in 1981, paving the way for two decades of discovery linking mtDNA variation with human disease. Severe pathogenic mutations cause sporadic and inherited rare disorders that often involve the nervous system. However, some mutations cause mild organ‐specific phenotypes that have a reduced clinical penetrance, and polymorphic variation of mtDNA is associated with an altered risk of developing several late‐onset common human diseases including Parkinson’s disease. mtDNA mutations also accumulate during human life and are enriched in affected organs in a number of age‐related diseases. Thus, mtDNA contributes to a wide range of human pathologies. For many decades, it has generally been accepted that mtDNA is inherited exclusively down the maternal line in humans. Although recent evidence has challenged this dogma, whole‐genome sequencing has identified nuclear‐encoded mitochondrial sequences (NUMTs) that can give the false impression of paternally inherited mtDNA. This provides a more likely explanation for recent reports of ‘bi‐parental inheritance’, where the paternal alleles are actually transmitted through the nuclear genome. The presence of both mutated and wild‐type variant alleles within the same individual (heteroplasmy) and rapid shifts in allele frequency can lead to offspring with variable severity of disease. In addition, there is emerging evidence that selection can act for and against specific mtDNA variants within the developing germ line, and possibly within developing tissues. Thus, understanding how mtDNA is inherited has far‐reaching implications across medicine. There is emerging evidence that this highly dynamic system is amenable to therapeutic manipulation, raising the possibility that we can harness new understanding to prevent and treat rare and common human diseases where mtDNA mutations play a key role.     

Increased apoptosis in acquired sideroblastic anaemia

Idiopathic acquired sideroblastic anaemias (IASAs) form a subgroup of the myelodysplastic syndromes and are characterized by mitochondrial iron accumulation, bone marrow erythroid hyperplasia and decreased peripheral red blood cell counts. Increased intramedullary apoptosis of erythroid precursors is presumed to constitute the pathophysiological mechanism explaining this ineffective erythropoiesis, but if and how mitochondrial dysfunction is implicated in this process is currently unknown. We therefore studied bone marrow precursor cells obtained from nine patients with IASA for (i) caspase 3 activity, (ii) numbers of Annexin V- and 7-amino-actinomycin-positive cells, (iii) numbers of cells with diminished mitochondrial membrane potential, Delta Psi(m), and (iv) numbers of cells producing reactive oxygen species (ROS), and we compared the results with those of five normal bone marrow samples. Compared with controls, we found increased caspase 3 activity in all IASA samples, which correlated with increased numbers of Annexin-V-positive cells (r = 0.7). Analysis of different subpopulations showed increased apoptosis in erythroid populations compared with myeloid and/or lymphoid populations in five out of nine cases, and increased apoptosis in the last two populations in four out of nine cases. As evidence of mitochondrial dysfunction, Delta Psi(m) was found to be diminished in the erythroid subpopulations of all cases of IASA (66.6 +/- 17% vs. 34.6 +/- 12% in normals). Delta Psi(m) decrease was correlated to Annexin V positivity (r = 0.7). Astonishingly, no difference was found between IASA and normal bone marrows with regard to the number of ROS-producing cells. In fact, both groups exhibited a similar low proportion of ROS production (10.3 +/- 7% in normals vs. 6.8 +/- 5% in IASA). Taken together, our results show that mitochondria are clearly implicated in the apoptotic process in IASA patients. Whether this is a result of an intramitochondrial defect (e.g. Fe accumulation, secondary to mitochondrial or nuclear DNA mutations) or is secondary to an extracellular stimulus [e.g. tumour necrosis factor (TNF), Fas ligand (FasL)] remains to be determined.     

钙和氧自由基在急性心肌梗死时线粒体膜介导的细胞死亡途径中的作用

在急性心肌梗死时,过量产生的活性氧和细胞内钙积聚对启动程序性细胞死亡起重要作用。细胞死亡包括坏死、凋亡、自噬及其共同作用。在缺血过程中,肌浆网、肌丝之间的钙处理被中断,同时钙转移至线粒体导致其肿胀,再灌注激活能量传导和心肌收缩导致氧自由基释放及其他离子失衡,在急性缺血一再灌注过程中,主要的死亡途径是线粒体通透性转换孔开放和线粒体外膜通透性增加启动内源性程序性坏死和凋亡。尽管国内外学者做了深入的研究,但调节线粒体膜通透性的作用和机制尚未完全了解。外源性凋亡、坏死性凋亡和自噬也可能加重缺血一再灌注所致的损伤。在这篇综述中,我们将讨论心肌梗死时钙失调和氧自由基、Bcl一2蛋白、线粒体膜通透性改变在心肌细胞死亡途径中的作用。     

Role of cellular defense against hydrogen peroxide-induced inhibition of myocyte respiration

Hydrogen peroxide (H₂O₂) serves as a precursor for highly reactive oxygen intermediates. However, the respiratory function of myocytes is relatively resistant to exogenously administered H₂O₂. In this study, we examined whether or not the reduction of cellular defense increases the toxicity of H₂O₂. Rat heart myocytes were isolated by collagenase digestion. Respiratory rates of myocytes, suspended in a medium containing sucrose, 3-N-morpholino-propanesulfonic acid, EGTA and bovine serum albumin, were determined polarographically in the presence of pyruvate and malate with or without 2,4-dinitrophenol (DNP). Mitochondrial membrane potentials were measured by using [³H]triphenylmethylphosphonium⁺. Cellular defense was attenuated by i) inhibiting the catalase activity by 3-amino-1,2,4-triazole (AT), ii) reducing the glutathione concentration by diethyl maleate (DEM) or ethacrinic acid (EA), and iii) permeabilizing the sarcolemmal membrane by saponin. The dose-response relationship between H₂O₂ (0.1–5 mM) and mitochondrial membrane potential was not greatly affected by these experimental conditions. Myocyte respiration was inhibited by 5 mM H₂O₂, particularly that measured in the presence of DNP (48% of control). DEM treatment did not significantly affect the respiratory inhibition by H₂O₂, whereas the degree of inhibition was somewhat greater following EA or AT treatment. By contrast, the sensitivity of cellular respiration to H₂O₂ was potentiated approximately two orders of magnitude by the permeabilization of sarcolemmal membrane; thus, 100 M H₂O₂ inhibited both DNP-stimulated and unstimulated respiration to 17% and 35% of control, respectively. The results indicate that factors existing in the sarcolemma and/or in the cytosol, which become ineffective and/or are diluted, respectively, following permeabilization with saponin, are important cellular defense mechanisms in alleviating the toxic effect of exogenous H₂O₂ on the respiration of mitochondria in situ in myocytes.     

BH3-domain mimetic compound BH3I-2' induces rapid damage to the inner mitochondrial membrane prior to the cytochrome c release from mitochondria

The Bcl-2 family proteins are major regulators of cell survival and death in human leukaemia. BH3-containing peptides induce apoptosis by binding to the hydrophobic pocket of the anti-apoptotic proteins, such as Bcl-2 or Bcl-XL. A small cell-permeable compound, BH3I-2' (3-iodo-5-chloro-N-[2-chloro-5-((4-chlorophenyl)sulphonyl)phenyl]-2-hydroxybenzamide), has been recently reported to have a function similar to Bak BH3 peptide. BH3I-2' induces apoptosis by disrupting interactions mediated by the BH3 domain, between pro-apoptotic and anti-apoptotic members of the Bcl-2 family. This study found that BH3I-2' induced cytochrome c release from the mitochondrial outer membrane in a Bax-dependent manner and that this correlated with the sensitivity of leukaemic cells to apoptosis. Moreover, it also induced rapid damage to the inner mitochondrial membrane, represented by a rapid collapse of mitochondrial membrane potential (DeltaPsim), prior to the cytochrome c release. This occurred both in whole cells and isolated mitochondria, and was not associated with the sensitivity of cells to BH3I-2'-induced apoptosis. Exogenous Bcl-2 or Bcl-XL neutralized BH3I-2'in vitro and diminished its effect on the inner mitochondrial membrane. Our results indicate that BH3I-2' not only induces cytochrome c release from the outer mitochondrial membrane but also damages the inner mitochondrial membrane, probably by interacting with Bcl-2 family proteins.     

Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.