急性缺氧肺动脉平滑肌细胞线粒体活性氧变化的观察

 目的:观察急性缺氧时大鼠肺动脉平滑肌细胞（PASMCs）线粒体活性氧（ROS）的变化。方法:分离并培养大鼠PASMCs,常氧（35 ℃、5% CO2、21% O2、74% N2）和急性缺氧(35 ℃、5% CO2、1% O2、94% N2)条件下,利用分子探针chloromethyl dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) 和RedoxSensor Red CC-1通过激光共聚焦显微镜来检测细胞内ROS的生成量;直接分离线粒体,用线粒体电子传递链（ETC）复合物抑制剂通过荧光分光光度计检测线粒体ROS及其生成的具体位点。结果:急性缺氧细胞内ROS生成明显增加,其中缺氧组H2O2较常氧组增加3.35倍 (P<0.01),而H2O2 及O-·2较常氧组增加1.61倍（P<0.01）。与缺氧组比,用线粒体ETC复合物I抑制剂MPP、复合物II抑制剂NPA和TTFA及复合物III前泛半醌位点抑制剂myxothiazol都能显著降低缺氧时PASMCs胞内ROS的生成量（分别降低60%、73%、75%和61%,P<0.01）;而复合物III后泛半醌位点抑制剂antimycin A及复合物IV 抑制剂NaN3对ROS的生成无明显影响（升高13%和9.1%,P>0.05）。 直接检测线粒体ROS与测定细胞内ROS结果一致。结论: 急性缺氧PASMCs线粒体ROS（主要是H2O2）的生成量明显增加;其生成位点主要是线粒体ETC复合物Ⅰ、Ⅱ及III前泛半醌位点,而与复合物III后泛半醌位点和IV位点无明显关系。     

Chemoreception in the context of the general biology of ROS

Superoxide anion is the most important reactive oxygen species (ROS) primarily generated in cells. The main cellular constituents with capabilities to generate superoxide anion are NADPH oxidases and mitochondrial respiratory chain. The emphasis of our article is centered in critically examining hypotheses proposing that ROS generated by NADPH oxidase and mitochondria are key elements in O(2)-sensing and hypoxic responses generation in carotid body chemoreceptor cells. Available data indicate that chemoreceptor cells express a specific isoform of NADPH oxidase that is activated by hypoxia; generated ROS acting as negative modulators of the carotid body (CB) hypoxic responses. Literature is also consistent in supporting that poisoned respiratory chain can produce high amounts of ROS, making mitochondrial ROS potential triggers-modulators of the CB activation elicited by mitochondrial venoms. However, most data favour the notion that levels of hypoxia, capable of strongly activating chemoreceptor cells, would not increase the rate of ROS production in mitochondria, making mitochondrial ROS unlikely triggers of hypoxic responses in the CB. Finally, we review recent literature on heme oxygenases from two perspectives, as potential O(2)-sensors in chemoreceptor cells and as generators of bilirubin which is considered to be a ROS scavenger of major quantitative importance in mammalian cells.     

Oxygen sensing in hypoxic pulmonary vasoconstriction: using new tools to answer an age-old question

Hypoxic pulmonary vasoconstriction (HPV) becomes activated in response to alveolar hypoxia and, although the characteristics of HPV have been well described, the underlying mechanism of O(2) sensing which initiates the HPV response has not been fully established. Mitochondria have long been considered as a putative site of oxygen sensing because they consume O(2) and therefore represent the intracellular site with the lowest oxygen tension. However, two opposing theories have emerged regarding mitochondria-dependent O(2) sensing during hypoxia. One model suggests that there is a decrease in mitochondrial reactive oxygen species (ROS) levels during the transition from normoxia to hypoxia, resulting in the shift in cytosolic redox to a more reduced state. An alternative model proposes that hypoxia paradoxically increases mitochondrial ROS signalling in pulmonary arterial smooth muscle. Experimental resolution of the question of whether the mitochondrial ROS levels increase or decrease during hypoxia has been problematic owing to the technical limitations of the tools used to assess oxidant stress as well as the pharmacological agents used to inhibit the mitochondrial electron transport chain. However, recent developments in genetic techniques and redox-sensitive probes may allow us eventually to reach a consensus concerning the O(2) sensing mechanism underlying HPV.     

A rotenone-sensitive site and H2O2 are key components of hypoxia-sensing in neonatal rat adrenomedullary chromaffin cells

In the perinatal period, adrenomedullary chromaffin cells (AMC) directly sense PO2 and secrete catecholamines during hypoxic stress, and this response is lost in juvenile ( approximately 2 week-old) chromaffin cells following postnatal innervation. Here we tested the hypothesis that a rotenone-sensitive O2-sensor and ROS are involved in the hypoxic response of AMC cultured from neonatal and juvenile rats. In whole-cell recordings, hypoxia (PO2=5-15 mm Hg) inhibited outward current in neonatal AMC; this response was reversed by exogenous H2O2 and mimicked and occluded by intracellular catalase (1000 units/ml), as well as the antioxidants, N-acetyl-L-cysteine (NAC; 50 microM) and Trolox (200 microM). Acute hypoxia decreased ROS levels and stimulated ATP secretion in these cells, as measured by luminol and luciferin-luciferase chemiluminescence, respectively. Of several mitochondrial electron transport chain (ETC) inhibitors tested, only rotenone, a complex I blocker, mimicked and occluded the effects of hypoxia on outward current, cellular ROS, and ATP secretion. Succinate donors, which act as complex II substrates, reversed the effects of hypoxia and rotenone in neonatal AMC. In contrast, in hypoxia-insensitive juvenile AMC, neither NAC nor rotenone stimulated ATP secretion though they both caused a decrease in ROS levels. We propose that O2-sensing by neonatal AMC is mediated by decreased ROS generation via a rotenone-sensitive site that is coupled to outward current inhibition and secretion. Interestingly, juvenile AMC display at least two modifications, i.e. an uncoupling of the O2-sensor from ROS regulation, and an apparent insensitivity of outward current to decreased ROS.     

Frataxin, iron-sulfur clusters, heme, ROS, and aging

A deficiency in mitochondrial frataxin causes an increased generation of mitochondrial reactive oxygen species (ROS), which may contribute to the cell degenerative features of Friedreich's ataxia. In this work the authors demonstrate mitochondrial iron-sulfur cluster (ISC) defects and mitochondrial heme defects, and suggest how both may contribute to increased mitochondrial ROS in lymphoblasts from human patients. Mutant cells are deficient in the ISC-requiring mitochondrial enzymes aconitase and succinate dehydrogenase, but not in the non-ISC mitochondrial enzyme citrate synthase; also, the mitochondrial iron-sulfur scaffold protein IscU2 co-immunoprecipitates with frataxin in vivo. Presumably as a consequence of the iron-sulfur cluster defect, cytochrome c heme is deficient in mutants, as well as heme-dependent Complex IV. Mitochondrial superoxide is elevated in mutants, which may be a consequence of cytochrome c deficiency. Hydrogen peroxide, glutathione peroxidase activity, and oxidized glutathione (GSSG) are each elevated in mutants, consistent with activation of the glutathione peroxidase pathway. Mutant status blunted the effects of Complex III and IV inhibitors, but not a Complex I inhibitor, on superoxide production. This suggests that heme defects late in the electron transport chain of mutants are responsible for increased mutant superoxide. The impact of ISC and heme defects on ROS production with age are discussed.     

Response of skeletal muscle mitochondria to hypoxia

This review explores the current concepts relating the structural and functional modifications of skeletal muscle mitochondria to the molecular mechanisms activated when organisms are exposed to a hypoxic environment. In contrast to earlier assumptions it is now established that permanent or long-term exposure to severe environmental hypoxia decreases the mitochondrial content of muscle fibres. Oxidative muscle metabolism is shifted towards a higher reliance on carbohydrates as a fuel, and intramyocellular lipid substrate stores are reduced. Moreover, in muscle cells of mountaineers returning from the Himalayas, we find accumulations of lipofuscin, believed to be a mitochondrial degradation product. Low mitochondrial contents are also observed in high-altitude natives such as Sherpas. In these subjects high-altitude performance seems to be improved by better coupling between ATP demand and supply pathways as well as better metabolite homeostasis. The hypoxia-inducible factor 1 (HIF-1) has been identified as a master regulator for the expression of genes involved in the hypoxia response, such as genes coding for glucose transporters, glycolytic enzymes and vascular endothelial growth factor (VEGF). HIF-1 achieves this by binding to hypoxia response elements in the promoter regions of these genes, whereby the increase of HIF-1 in hypoxia is the consequence of a reduced degradation of its dominant subunit HIF-1a. A further mechanism that seems implicated in the hypoxia response of muscle mitochondria is related to the formation of reactive oxygen species (ROS) in mitochondria during oxidative phosphorylation. How exactly ROS interfere with HIF-1a as well as MAP kinase and other signalling pathways is debated. The current evidence suggests that mitochondria themselves could be important players in oxygen sensing.     

O(2) sensing in hypoxic pulmonary vasoconstriction: the mitochondrial door re-opens

The identity of the O(2) sensor underlying the hypoxic pulmonary vasoconstriction (HPV) response has been sought for more than 50 years. Recently, the mitochondria have again come into sharp focus as the cellular organelle responsible for triggering the events that culminate in pulmonary artery constriction. Studies from different laboratories propose two disparate models to explain how mitochondria react to a decrease in P(O(2)). One model proposes that hypoxia slows or inhibits mitochondrial electron transport resulting in the accumulation of reducing equivalents and a decrease in the generation of reactive oxygen species (ROS). This is proposed to activate a redox-sensitive pathway leading to pulmonary vasoconstriction. A second and opposing model suggests that hypoxia triggers a paradoxical increase in mitochondrial ROS generation. This increase would then lead to the activation of an oxidant-sensitive signaling transduction pathway leading to HPV. This article summarizes the potential involvement of mitochondria in these two very different models.     

Functional analysis of neurotransmission at β2-laminin deficient terminals

Carotid body glomus cells release transmitters in response to hypoxia due to the increase of excitability resulting from inhibition of O₂ -regulated K⁺ channels. However, the mechanisms involved in the detection of changes of O₂ tension are unknown. We have studied the interaction between glomus cell O₂ sensitivity and inhibition of the mitochondrial electron transport chain (ETC) in a carotid body thin slice preparation in which catecholamine release from intact single glomus cells can be monitored by amperometry. Inhibition of the mitochondrial ETC at proximal and distal complexes induces external Ca²⁺-dependent catecholamine secretion. At saturating concentration of the ETC inhibitors, the cellular response to hypoxia is maintained. However, rotenone, a complex I blocker, selectively occludes the responsiveness to hypoxia of glomus cells in a dose-dependent manner. The effect of rotenone is mimicked by 1-methyl-4-phenylpyridinium ion (MPP⁺), an agent that binds to the same site as rotenone, but not by complex I inhibitors acting on different sites. In addition, the effect of rotenone is not prevented by incubation of the cells with succinate, a substrate of complex II. These data strongly suggest that sensitivity to hypoxia of carotid body glomus cells is not linked in a simple way to mitochondrial electron flow and that a rotenone (and MPP⁺)-sensitive molecule critically participates in acute oxygen sensing in the carotid body.     

硫化氢对缺氧诱导的大鼠皮层神经元损伤的保护作用

 目的:探讨硫化氢(H 2S)对缺氧诱导的皮层神经元损伤的影响及作用机制。方法:将SD大鼠皮层神经元在2% O 2、5% CO 2、93 % N 2、37 ℃培养箱培养24 h,建立细胞缺氧模型。以硫氢化钠（NaHS）作为H 2S的供体,应用CCK-8分析细胞活性;采用荧光探针DCFH-DA检测神经元活性氧(ROS)含量;用Rh123染色测定线粒体膜电位(MMP);采用乳酸脱氢酶(LDH)试剂盒分析神经元LDH释放率,反映神经元的损伤情况。结果:(1)缺氧引起神经元ROS含量和LDH释放率升高,NaHS预处理可抑制缺氧所致神经元ROS含量和LDH释放率的升高;(2)缺氧降低神经元MMP和细胞活性,NaHS和活性氧清除剂NAC预处理均显著抑制缺氧所致神经元MMP和细胞活性的降低。结论:缺氧增加神经元ROS含量,降低神经元MMP和细胞活性,而H 2S通过其抗氧化作用,减轻缺氧所致神经元的损伤。     

Lessons from chronic intermittent and sustained hypoxia at high altitudes

Recurrent sleep apnea (RSA), mimicking chronic intermittent hypoxia (CIH), may trigger unique adaptations in oxygen sensing in the carotid body, and consequent cellular functions unlike the effects of sustained hypoxia (SH). As a mechanism, an augmented generation of reactive oxygen species (ROS) in CIH has been invoked at the exclusion of SH effects. The ROS might act at hypoxia inducible factors (HIF-1s), giving rise to various genes whose function is to restore the tissue P(O(2)) close to the original. In a spate, review articles on the CIH effects at sea level have appeared but little on high altitude (HA). Their views have been reexamined with the primary focus on the peripheral chemoreception. At HA, RSA is more common in the lowlanders because of a high ventilatory sensitivity to hypoxia (with the consequent effects) unlike the high altitude natives (HAN). Undoubtedly, the HIF-1s play a central role at HA, the mechanisms of which are unknown and explorable.     

不同氧体积分数对人胚胎干细胞基因表达谱的影响

背景：低氧培养人胚胎干细胞的相关研究主要集中在干细胞多能性的维持及分化方面,而低氧对人胚胎干细胞基因表达水平的影响研究甚少。 目的：观察不同氧体积分数对人胚胎干细胞基因表达谱的影响。 方法：分别在低氧(体积分数5%O2)和常氧(体积分数21%O2)的条件下持续培养FY-hES-7细胞,收集晚期(第52代)细胞,运用全基因组表达谱芯片技术检测不同氧体积分数组FY-hES-7细胞的基因表达谱,并进行差异基因的功能富集分析及通路分析。 结果与结论：与常氧组相比,低氧组表达上调(>2倍)的基因1 840个,表达下调(>2倍)的基因1 676个。利用基因本体分析发现低氧组表达上调基因与细胞表面受体信号转导、免疫反应、离子转运、生物代谢及细胞活动等功能相关,而表达下调基因则与转录调控,依赖DNA的转录调控、神经分化、细胞形态、胚胎形态、胚胎器官及各系统发育等功能相关。通路分析发现低氧组表达上调的基因与细胞因子受体的相互作用、免疫反应以及造血系统等通路的改变相关,而表达下调的基因则多与胚胎发育及肿瘤通路改变相关。不同氧体积分数下长期培养的人胚胎干细胞基因表达谱存在明显的差异,低氧组出现差异表达基因的功能分析表明低氧有助于人胚胎干细胞的自我更新,防止分化及降低成瘤性。     

Downregulation of the Expression of Mitochondrial Electron Transport Complex Genes in Autism Brains

Mitochondrial dysfunction (MtD) and abnormal brain bioenergetics have been implicated in autism, suggesting possible candidate genes in the electron transport chain (ETC). We compared the expression of 84 ETC genes in the post‐mortem brains of autism patients and controls. Brain tissues from the anterior cingulate gyrus, motor cortex, and thalamus of autism patients (n = 8) and controls (n = 10) were obtained from Autism Tissue Program, USA. Quantitative real‐time PCR arrays were used to quantify gene expression. We observed reduced expression of several ETC genes in autism brains compared to controls. Eleven genes of Complex I, five genes each of Complex III and Complex IV, and seven genes of Complex V showed brain region‐specific reduced expression in autism. ATP5A1 (Complex V), ATP5G3 (Complex V) and NDUFA5 (Complex I) showed consistently reduced expression in all the brain regions of autism patients. Upon silencing ATP5A1, the expression of mitogen‐activated protein kinase 13 (MAPK13), a p38 MAPK responsive to stress stimuli, was upregulated in HEK 293 cells. This could have been induced by oxidative stress due to impaired ATP synthesis. We report new candidate genes involved in abnormal brain bioenergetics in autism, supporting the hypothesis that mitochondria, critical for neurodevelopment, may play a role in autism.     

Is antioxidant potential of the mitochondrial targeted ubiquinone derivative MitoQ conserved in cells lacking mtDNA?

MitoQ has been developed as a mitochondrial targeted antioxidant for diseases associated with oxidative stress. Here we show that MitoQ blocks the generation of reactive oxygen species (ROS) and mitochondrial protein thiol oxidation, and preserves mitochondrial function and ultrastructure after glutathione (GSH) depletion. Furthermore, the antioxidant effect of MitoQ is conserved in cells lacking mitochondrial DNA, indicating that its antioxidant properties do not depend on a functional electron transport chain (ETC). Our results elucidate the antioxidant mechanism of MitoQ and suggest that it may be a useful therapeutic for disorders associated with a dysfunctional ETC and increased ROS production.     

Hyperglucagonemia and alpha-adrenergic receptor in acute hypoxia.

The plasma immunoreactive glucagon (IRG) response to hypoxia was studied in puppies. Three groups of paired experiments were performed. In group I, 8% O2:92% N2 ventilation (PaO2 20--30 torr) produced a rise in plasma IRG and glucose as well as hypotension and bradycardia. However, when group I was air ventilated (PaO2 greater than 70 torr) and given glucose infusions producing hyperglycemia of similar degree, plasma IRG was unchanged. Group II received alpha-adrenergic blockade (phenoxybenzamine). When made hypoxic, group II developed no significant IRG rise and less hyperglycemia than with hypoxia alone. Hypotension was more severe with hypoxia plus alpha-blockade. Phenoxybenzamine itself did not change plasma IRG or glucose during air breathing. Group III receivi developed hyperglucagonemia and hyperglycemia not significantly different from that with hypoxia alone. However, hypoxia-caused hypotension and bradycardia was more pronounced with beta-blockade. No change in plasma IRG or glucose occurred in group III animals breathing air. These data suggest that a) glucagon release is caused by acute oxygen deficiency, and b) the hypoxic response is largely adrenergically mediated with the major role played by the alpha-receptor.     

Significance of ROS in oxygen sensing in cell systems with sensitivity to physiological hypoxia

Reactive oxygen species (ROS) are oxygen-containing molecular entities which are more potent and effective oxidizing agents than is molecular oxygen itself. With the exception of phagocytic cells, where ROS play an important physiological role in defense reactions, ROS have classically been considered undesirable byproducts of cell metabolism, existing several cellular mechanisms aimed to dispose them. Recently, however, ROS have been considered important intracellular signaling molecules, which may act as mediators or second messengers in many cell functions. This is the proposed role for ROS in oxygen sensing in systems, such as carotid body chemoreceptor cells, pulmonary artery smooth muscle cells, and erythropoietin-producing cells. These unique cells comprise essential parts of homeostatic loops directed to maintain oxygen levels in multicellular organisms in situations of hypoxia. The present article examines the possible significance of ROS in these three cell systems, and proposes a set of criteria that ROS should satisfy for their consideration as mediators in hypoxic transduction cascades. In none of the three cell types do ROS satisfy these criteria, and thus it appears that alternative mechanisms are responsible for the transduction cascades linking hypoxia to the release of neurotransmitters in chemoreceptor cells, contraction in pulmonary artery smooth muscle cells and erythropoietin secretion in erythropoietin producing cells.