Essential role of complex II of the respiratory chain in hypoxia-induced ROS generation in the pulmonary vasculature

In the pulmonary vasculature, the mechanisms responsible for oxygen sensing and the initiation of hypoxia-induced vasoconstriction and vascular remodeling are still unclear. Nitric oxide (NO) and reactive oxygen species (ROS) are discussed as early mediators of the hypoxic response. Here, we describe a quantitative analysis of NO- and ROS-producing cells within the vascular walls of murine lung sections cultured at normoxia or hypoxia. Whereas the number of NO-producing cells was not changed by hypoxia, the number of ROS-generating cells was significantly increased. Addition of specific inhibitors revealed that mitochondria were the source of ROS. The participation of the individual mitochondrial complexes differed in normoxic and hypoxic ROS generation. Whereas normoxic ROS production required complexes I and III, hypoxic ROS generation additionally demanded complex II. Histochemically demonstrable succinate dehydrogenase activity of complex II in the arterial wall decreased during hypoxia. Inhibition of the reversed enzymatic reaction, i.e., fumarate reductase, by application of succinate, specifically abolished hypoxic, but not normoxic, ROS generation. Thus complex II plays an essential role in hypoxic ROS production. Presumably, its catalytic activity switches from succinate dehydrogenase to fumarate reductase at reduced oxygen tension, thereby modulating the directionality of the electron flow.     

Hypoxia sensitizes cells to nitric oxide-induced apoptosis

Nitric oxide (NO) can induce apoptosis in a variety of cell types. A non-toxic concentration of nitric oxide under normal oxygen conditions triggered cell death under hypoxic conditions (1.5% O(2)) in fibroblasts. Nitric oxide administered during hypoxia induced the release of cytochrome c, caspase-9 activation, and the loss of mitochondrial membrane potential followed by DNA fragmentation and lactate dehydrogenase release (markers of cell death). Bcl-X(L) protected cells from nitric oxide-induced apoptosis during hypoxia by preventing the release of cytochrome c, caspase-9 activation, and by maintaining a mitochondrial membrane potential. Murine embryonic fibroblasts from bax(-/-) bak(-/-) mice exposed to nitric oxide during hypoxia did not die, indicating that pro-apoptotic Bcl-2 family members are required for NO-induced apoptosis during hypoxia. The nitric oxide-induced cell death during hypoxia was independent of cGMP and peroxynitrite. Cells devoid of mitochondrial DNA (rho secondary-cells) lack a functional electron transport chain and were resistant to nitric oxide-induced cell death during hypoxia, suggesting that a functional electron transport chain is required for nitric oxide-induced apoptosis during hypoxia.     

Hypoxia affects mitochondrial protein expression in rat skeletal muscle

Hypoxia affects mammalian mitochondrial function, as well as mitochondria-based energy metabolism. The detail mechanism has not been fully understood. In this study, we detected protein expression levels in mitochondrial fractions of Wistar rats exposed to hypobaric hypoxia by use of proteomic methods. Adult male Wistar rats were randomized into an hypoxic (4,500?m, 30 days) group and a normoxic control group (sea level). Gastrocnemius muscles mitochondria were extracted and purified. Mitochondrial oxygen consumption was measured with a Clark oxygen electrode; mitochondrial transmembrane potential was detected with Rhodamine 123 as a fluoresce probe. Using 2-DE and MALDI-TOF MS analysis, we identified eight mitochondrial protein spots that were differentially expressed in the hypoxic group compared with the normoxic control. These proteins included Chain A of F1-ATPase, voltage dependent anion channel 1 (VDAC), hydroxyacyl Coenzyme A dehydrogenase α-subunit, mitochondrial F1 complex γ-subunit, androgen-regulated protein and tripartite motif protein 50. Two of the spots, VDAC and ATP synthase α-subunit, were confirmed by Western blotting analysis. Oxygen consumption during State 3 respiration, as well as the respiratory control ratio (RCR) was significantly higher in the control than that in the hypoxic group; mitochondrial transmembrane potential was significantly higher in hypoxic group than that in the control. With successful use of multiple proteomic analysis techniques, we demonstrates that 30 days hypoxia exposure has effects on the expression of mitochondrial proteins involved in ATP production and lipid metabolism, decrease the stability of mitochondrial membrane, and affect the mitochondrial electron transport chain.     

The Effect of Acute Hypoxia on Synaptosomes from Rat Brain

Abstract: Synaptosomes have been isolated from the brains of nonanesthetized and nembutal-anesthetized rats subjected to 30 min hypoxia induced by breathing 7% oxygen in nitrogen. The respiratory rate was depressed in synaptosomes from starved hypoxic animals but was not significantly different from the respective control values in preparations from fed hypoxic animals, anesthetized animals, and hypoxic nonanesthetized animals allowed to recover from the hypoxic episode by 60 min of normoxic conditions. Observations are also reported concerning the levels of various metabolites in the synaptosomes isolated from the brains of the same groups of animals. It is suggested that hypoxia results in damage to the synaptosomal and/or mitochondrial membrane, which modifies substrate oxidation in the mitochondria and decreases availability of reducing equivalents for the respiratory chain. Results obtained on afflicted and recovered animals indicate that synaptosomal preparations provide a useful model for the study of hypoxic damage.     

Hypoxia, reactive oxygen, and cell injury

Hypoxia usually decreases the formation of reactive oxygen species by oxidases and by autoxidation of components of cellular electron transfer pathways and of quinoid compounds such as menadione. In the case of menadione reactive oxygen species are liberated to a significant extent only at non-physiologically high oxygen partial pressures (PO2). At physiological and hypoxic PO2 values electron shuttling of menadione in the mitochondrial respiratory chain predominates. In contrast, lipid peroxidation induced by halogenated alkanes, such as carbon tetrachloride, in liver leads to an increase in the formation of reactive oxygen and thus in cell injury under hypoxic conditions. Reactive oxygen species may also be generated during reoxygenation of a previously hypoxic tissue. Based on experiments with isolated hepatocytes a three-zone-model of liver injury due to hypoxia and reoxygenation is presented; 1) a zone where the cells die by hypoxia; 2) a zone where the cells are destroyed upon reoxygenation, presumably mediated by an increase in the cellular ATP content; and 3) a zone where cell injury occurs upon reoxygenation, mediated by reactive oxygen species possibly liberated by xanthine oxidase.     

Human Systemic Reactions to a Dosed Exposure to Hypoxia: A Multiparameter Study

Human physiological reactions to acute hypoxic hypoxia were studied. Analysis of simultaneously recorded parameters of various physiological systems showed the following: activation of the general antihypoxic defense system is based on the formation of an intricate structure of intra- and intersystemic relations of specific and nonspecific elements of adaptation that support vital body functions during environmental oxygen deficit. These specific elements become more important in more severe hypoxia, which suppresses metabolism in some organs and tissues because of redistribution of blood flow. These factors allow the body to function at a lower oxygen tension in its tissues owing to an increased efficiency of mitochondria as a result of changes in the kinetics of enzymes of the mitochondrial respiratory chain. In acute hypoxia, the structure of intra- and intersystemic relations is rather intricate; its functional hierarchy is maintained by stronger individual amplitude-related controlling factors and by modulation of their phase- and time-related links. Advanced stages of hypoxia are associated with disintegration of central regulatory mechanisms, which is manifested by disturbances in amplitude-frequency and spatiotemporal parameters of the brain bioelectrical activity, changes in phasic interactions between elements of regulatory mechanisms, and signs of deregulation and decompensation of vital functions. The interpretation of the results is based on the general theory of adaptation, Medvedev's idea of adaptation as a successive involvement of genetically predetermined and newly-formed regulatory programs of the brain, Anokhin's theory of functional systems, and modern concepts of molecular and biochemical mechanisms of hypoxia. It was concluded that artificial normobaric hypoxia is a unique, biologically adequate model that makes it possible to study the rearrangements in systemic and autonomic regulatory mechanisms in response to strictly determined changes in the environmental concentration of oxygen as a principal factor supporting life.     

低氧与心肌细胞凋亡

细胞凋亡是心肌细胞低氧损伤的主要死亡形式之一。低氧引起心肌细胞凋亡可以通过外部的死亡受体通路以及内部的线粒体通路,两条通路之间又存在复杂的交互作用,其中,线粒体通路在低氧诱导的心肌细胞凋亡中起重要作用。另外,心肌细胞本身也具有多种内源性的凋亡抑制因子。因此,低氧时心肌细胞凋亡的产生是多种因素综合作用的结果,Bcl-2家族蛋白、线粒体通透性改变、细胞色素c的释放以及caspases的活化等参与了低氧引起的心肌细胞凋亡的调控。对低氧时心肌细胞凋亡的认识和深入研究,为人类在缺血性心脏病的防治中提供了一个新的治疗措施。     

Inborn errors of complex II--unusual human mitochondrial diseases.

The succinate dehydrogenase consists of only four subunits, all nuclearly encoded, and is part of both the respiratory chain and the Krebs cycle. Mutations in the four genes encoding the subunits of the mitochondrial respiratory chain succinate dehydrogenase have been recently reported in human and shown to be associated with a wide spectrum of clinical presentations. Although a comparatively rare deficiency in human, molecularly defined succinate dehydrogenase deficiency has already been found to cause encephalomyopathy in childhood, optic atrophy or tumor in adulthood. Because none of the typical housekeeping genes encoding this respiratory chain complex is known to present tissue-specific isoforms, the tissue-specific involvement represents a quite intriguing question, which is mostly addressed in this review. A differential impairment of electron flow through the respiratory chain, handling of oxygen, and/or metabolic blockade possibly associated with defects in the different subunits that can be advocated to account for tissue-specific involvement is discussed.     

Inborn errors of complex II – Unusual human mitochondrial diseases

The succinate dehydrogenase consists of only four subunits, all nuclearly encoded, and is part of both the respiratory chain and the Krebs cycle. Mutations in the four genes encoding the subunits of the mitochondrial respiratory chain succinate dehydrogenase have been recently reported in human and shown to be associated with a wide spectrum of clinical presentations. Although a comparatively rare deficiency in human, molecularly defined succinate dehydrogenase deficiency has already been found to cause encephalomyopathy in childhood, optic atrophy or tumor in adulthood. Because none of the typical housekeeping genes encoding this respiratory chain complex is known to present tissue-specific isoforms, the tissue-specific involvement represents a quite intriguing question, which is mostly addressed in this review. A differential impairment of electron flow through the respiratory chain, handling of oxygen, and/or metabolic blockade possibly associated with defects in the different subunits that can be advocated to account for tissue-specific involvement is discussed.     

Cytochrome b reducible by succinate in an isolated succinate dehydrogenase-cytochrome b complex from Bacillus subtilis membranes

In previous work with membranes of Bacillus subtilis, the succinate dehydrogenase complex was isolated by immunoprecipitation of Triton X-100-solubilized membranes. The complex included a polypeptide with an apparent molecular weight of 19,000, probably attributable to apocytochrome. This paper reports the further characterization of this cytochrome and its relation to the respiratory chain of B. subtilis. The cytochrome was identified as cytochrome b, and its difference absorption spectra showed maxima at 426, 529, and 558 nm at room temperature. The oxidized cytochrome had an absorption maximum at 413 nm. The cytochrome was reduced by succinate in the isolated succinate dehydrogenase complex and in Triton X-100-solubilized membranes. In whole membranes cytochromes b, c, and a were reduced by succinate. In membranes from a mutant containing normal cytochromes but lacking succinate dehydrogenase no reduction of cytochrome was seen with succinate. It was concluded that the isolated succinate dehydrogenase-cytochrome b complex is a functional unit in the intact B. subtilis membrane. An accompanying paper describes cytochrome b as a structural unit involved in the membrane binding of succinate dehydrogenase.     

Respiratory Responses to Hypoxia in Fish

Hypoxia is discussed in the widest sense, i.e., as the conditionsunder which cells suffer a lack of oxygen. From a considerationof the transfer of oxygen from the water to the cellular sitesas a series of resistances, it is suggested that hypoxia canresult from an increase in resistance anywhere along the chain,and possible types of hypoxia in fish are defined from thispoint of view. More detailed discussion is given in relation to (1) interferencewith gas transfer at the gill surface; (2) reduction in theblood O₂ carrying capacity; (3) modifications in the cardiacand ventilatory frequencies and in the coupling between thesetwo rhythms, and (4) the time course of the hypoxic stimulus. It is concluded that most of the increase in knowledge of hypoxiaduring the past ten years can be fitted into known theoreticalframeworks, but there is still a great need for measurementsof oxygen tension at all levels in the respiratory chain; attentionneeds to be directed to both diurnal and seasonal variationsand especially to long-term changes in those environmental conditionswhich tend to result in hypoxia.     

Nature of the activation of succinate dehydrogenase by various effectors and in hypobaria and hypoxia

1. Hepatic mitochondrial succinate dehydrogenase (succinate:(acceptor)oxidoreductase, EC 1.3.99.1) was activated by preincubation of mitochondria with four diverse classes of compounds, the dicarboxylic acids, nitrophenols, quinols (and ubiquinols) and pyrophosphates. Of the various compounds tested malonate, oxaloacetate and pyrophosphate, well-known competitive inhibitors of the enzyme, and also hydroquinone and ubiquinols were effective even at low concentrations and showed maximal stimulation in 2 min.2. Activation of succinate dehydrogenase by ubiquinol-9 and ubiquinol-10 was comparable to succinate activation in fresh mitochondria, and was much higher in the aged samples.3. Preincubation of mitochondria with succinate, 2,4-dinitrophenol, pyrophosphate and ATP also stimulated the succinate-2,2′,5,5′-tetraphenyl-3,3′-(4,4′-biphenylene) ditetrazolium chloride (NT) reductase activity, whereas malonate, hydroquinone and ubiquinol-9 were ineffective. A differential activation of the flavoprotein by the oxidized and reduced forms of ubiquinone-9 was observed, the former stimulating the reduction of NT and the latter of phenazine methosulphate-2,6-dichlorophenolindophenol.4. Repeated washing of the activated mitochondrial samples with the sucrose homogenizing medium, partially reversed the activation by effectors other than succinate. Further washing of the activated preparations after a second preincubation with succinate reverted the enzyme activity to the basal level in the case of malonate, ATP and pyrophosphate but not that of hydroquinone and ubiquinol-9.5. Increase in the activity of hepatic mitochondrial succinate dehydrogenase, but not of succinate-NT reductase, known to occur in rats exposed to hypobaria was also observed in hypoxia indicating that it is an effect of lowered O₂ tension. The enzyme activity in these “partially activated” preparations was stable to washing with the sucrose homogenizing medium and could be fully activated to the same level as in the controls showing thereby the qualitative nature of the change. On washing these succinate-activated preparations further with the medium, the “hypobaric activation” was not reversed to the basal level, whereas the “hypoxic activation” was reversed. These results suggest that the effectors responsible for the activation of succinate dehydrogenase under hypobaric and hypoxic conditions are probably different; the former may be of the ubiquinol type and the latter of the malonate type.     

Stress Reaction and Biochemical Shock as Interrelated and Unavoidable Components in the Formation of High Radioresistance of the Body in Acute Hypoxia

Stress reactions with activation of the sympathetic-adrenal system due to acute hypoxia reflects the degree of sensitivity of the body to this extreme factor. Succinate dehydrogenase (SDH) activation in cells as an adaptive response to acute hypoxia is closely associated with the degree of disturbance of tissue respiration through a lack of oxygen in the tissues, including the manifestation of “biochemical shock,” which is an unavoidable component of implementation of the protective effect of radioprotectors. In experiments on mice, rats, and dogs, the correlation between the manifestation of the radioprotective effect of acute hypoxia and SDH activation in blood lymphocytes, caused primarily by adrenergic stimulation during stress reactions, is confirmed. The degree of SDH activation in blood lymphocytes by hypoxia of different origins including that induced by radioprotectors may indicate its radioprotective potential irrespective of the differences in the oxygen consumption intensity and the resistance to acute hypoxia in animals and humans.     

Hypoxia induced metabolism dysfunction of rat astrocytes in primary cell cultures

In order to study the astroglial contribution to hypoxic injury on brain tissue metabolism, modifications of glutamine synthetase (GS) lactate dehydrogenase (LDH) enolase and malate dehydrogenase activity produced by reduced oxygen supply have been determined in primary cultures of astrocytes prepared from newborn rat cerebral cortex. Enzymatic activities were measured immediately after the hypoxic treatment (9 h) and during post injury recovery. GS level is significantly decreased in response to low oxygen pressure and increased above control value during the post hypoxic recovery period. The magnitude of GS reduction by hypoxia depends on the age of the cells in culture. Lactate dehydrogenase and enolase levels were significantly enhanced during the two periods considered. No modification of the MDH level was observed. The synthesis of LDH isoenzymes containing mainly M subunits is specifically induced by hypoxia. Our results suggest that astroglial cells may represent a particularly sensitive target toward hypoxia injury in brain tissue. Low oxygen pressure available may modify some fundamental metabolical functions of these cells such as glutamate turnover and lactic acid accumulation.     

Hypoxia-adaptation involves mitochondrial metabolic depression and decreased ROS leakage

Through long-term laboratory selection, we have generated a Drosophila melanogaster population that tolerates severe, normally lethal, level of hypoxia. This strain lives perpetually under severe hypoxic conditions (4% O(2)). In order to shed light on the mechanisms involved in this adaptation, we studied the respiratory function of isolated mitochondria from the thorax of hypoxia-adapted flies (AF) using polarographic oxygen consumption while monitoring superoxide generation by electron paramagnetic resonance (EPR) techniques. AF mitochondria exhibited a significant 30% decrease in respiratory rate during state 3, while enhancing the resting respiratory rate during State 4-oligo by 220%. The activity of individual electron transport complexes I, II and III were 107%, 65%, and 120% in AF mitochondria as compared to those isolated from control flies. The sharp decrease in complex II activity and modest increase in complexes I and III resulted in >60% reduction in superoxide leakage from AF mitochondria during both NAD(+)-linked state 3 and State 4-oligo respirations. These results provide evidence that flies with mitochondria exhibiting decreased succinate dehydrogenase activity and reduced superoxide leakage give flies an advantage for survival in long-term hypoxia.     

Purification and characterisation of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile Sulfolobus acidocaldarius

A succinate dehydrogenase complex was isolated in a three-step purification from plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. It consists of four subunits: a, 66 kDa; b, 31 kDa; c, 28 kDa and d, 12.8 kDa. In the 141-kDa native protein, the four subunits are present in an equimolar stoichiometry. The complex contains acid-non-extractable flavin, iron and acid-labile sulphide. Maximal succinate dehydrogenase activities were recorded at pH 6.5, which coincides with the internal pH of Sulfolobus cells. The temperature optimum of 81 degrees C defines the Sulfolobus succinate dehydrogenase as a thermophilic enzyme complex. The Km for succinate was found to be 1.42 mM (55 degrees C). Similar to the mitochondrial soluble succinate dehydrogenase, this enzyme is capable of transferring electrons to artificial electron acceptors, for instance phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine and ferricyanide. In contrast to the mitochondrial succinate dehydrogenase, the archaebacterial enzyme reduces 1,4-dichloroindophenol also in the absence of phenazine methosulfate. Caldariella quinone, the physiological electron mediator in the Sulfolobus respiratory chain, was only slowly reduced under adjusted conditions. The succinate--phenazine methosulfate-(1,4-dichloroindophenol) oxidoreductase of the isolated complex was strongly inhibited by tetrachlorobenzoquinone. In plasma membranes the complex reduces molecular oxygen in a cyanide-sensitive reaction. Polyclonal Sulfolobus anti-a antibodies crossreacted with 66-67-kDa polypeptides from membranes of Thermoplasma acidophilium, Sulfolobus solfataricus and beef heart submitochondrial particles.     

Anaerobic formation of succinate and facilitation of its oxidation -- possible mechanisms of cell adaptation to oxygen deficiency

An important role of anaerobic formation of succinate in anoxic and hypoxic states and the activation of succinate oxidation under hypoxia were shown. It was concluded that, for maintaining the energetics of animal cells under conditions of oxygen deficiency, it is advisable to use substrates capable of participating in the anaerobic formation of succinate, whereas under hypoxia it is reasonable to use succinate itself.     

Intermittent hypoxic training and L-arginine as corrective agents for myocardial energy supply under acute hypoxia

It NO has been shown play to the primary role in several mitochondrial functions. Our aim for this study was to investigate whether exogenous NO (L-arginine) or NO blocker L-NNA modulated the adaptive reactions of rat myocardial tissue respiration on intermittent hypoxic training (IHT). In the control rats an acute hypoxic test (inhalation of 7% O2, 30 min) provoked sharp augmentation of ADP-stimulated tissue respiration with the increase of respiratory coefficient and phosphorylation rate, the decrease of O2 uptake efficacy and switching the energy supply to succinate oxidation pathway. The same hypoxic test but following 14 days of IHT (11% O2, 15-min sessions with 15 min rest intervals, 5 times daily) produced a stimulation of oxidative phosphorylation with primary activation of NAD-dependent pathway, the marked increase of ADP/O ratio. The combination of IHT with L-arginine treatment (600 mg/kg intraperitoneally, daily before IHT sessions) provoked the decrease of tissue oxygen consumption in comparison with untrained animals. L-arginine effects abolished by the NO-synthase blocker L-NNA. Its effects on mitochondrial function deals with succinic acid inhibition utilizatin (increasing level ADP/O) and activation NADH-dependent oxidation. We conclude that the combination of IHT with NO-precursor treatment was capable to increase significantly the tolerance to episodes of acute hypoxia.     

Hypoxia up-regulates telomerase activity via mitogen-activated protein kinase signaling in human solid tumor cells.

Solid tumor cells are often exposed to hypoxia in vivo, which has been suggested to promote genetic instability in those cells. Telomere elongation by telomerase is implicated in chromosome stabilization in immortal cells. Here we found that hypoxia enhanced telomerase activity in the solid tumor A2780 and HT-29 cells but not in the leukemia U937 cells. The telomerase activation correlated with activation of mitogen-activated protein kinase (MAPK) and c-fos expression. The MEK1 inhibitor PD98059 repressed telomerase activation in the hypoxic cells. Consistently, a dominant negative MEK1 inhibited telomerase activation by hypoxia. Finally, we found a good correlation between telomerase activation and resistance to apoptotic cell death under hypoxic conditions. These findings indicate that hypoxia up-regulates telomerase activity via MAPK cascade signaling especially in solid tumor cells and suggest that solid tumor cells might enhance the telomerase activity as a stress response against genotoxicity induced by hypoxia.