Interplay between ferritin metabolism, reactive oxygen species and nitric oxide

 The biological relevance of each of the three inorganic species – iron, oxygen, and nitric oxide (NO) – is crucial. Moreover, their metabolic pathways cross each other and thus create a complex network of connections responsible for the regulation of many essential biological processes. The iron storage protein ferritin, one of the main regulators of iron homeostasis, influences oxygen and NO metabolism. Here, examples are given of the biological interactions of the ferritin molecule (ferritin iron and ferritin shell) with reactive oxygen species (ROS) and NO. The focus is the regulation of ferritin expression by ROS and NO. From these data, ferritin emerges as an important cytoprotective component of the cellular response to ROS and NO. Also, by its ability to alter the amount of intracellular "free" iron, ferritin may affect the metabolism of ROS and NO. It is proposed that this putative activity of ferritin may constitute a missing link in the regulatory loop between iron, ROS, and NO. Received: 2 January 1997 / Accepted: 9 June 1997     

Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes

Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. Like chloroplasts and mitochondria, plant peroxisomes also produce superoxide radicals (O2*(-)) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29 and 32 kDa have been shown to generate radicals O2*(-). Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the ascorbate-glutathione cycle, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of peroxisomes. The four enzymes of the ascorbate-glutathione cycle (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) as well as the antioxidants glutathione and ascorbate have been found in plant peroxisomes. The recycling of NADPH from NADP(+) can be carried out in peroxisomes by three dehydrogenases: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase. In the last decade, different experimental evidence has suggested the existence of cellular functions for peroxisomes related to reactive oxygen species (ROS), but the recent demonstration of the presence of nitric oxide synthase (NOS) in plant peroxisomes implies that these organelles could also have a function in plant cells as a source of signal molecules like nitric oxide (NO*), superoxide radicals, hydrogen peroxide, and possibly S-nitrosoglutathione (GSNO).     

Mammalian peroxisomes and reactive oxygen species

The central role of peroxisomes in the generation and scavenging of hydrogen peroxide has been well known ever since their discovery almost four decades ago. Recent studies have revealed their involvement in metabolism of oxygen free radicals and nitric oxide that have important functions in intra- and intercellular signaling. The analysis of the role of mammalian peroxisomes in a variety of physiological and pathological processes involving reactive oxygen species (ROS) is the subject of this review. The general characteristics of peroxisomes and their enzymes involved in the metabolism of ROS are briefly reviewed. An expansion of the peroxisomal compartment with proliferation of tubular peroxisomes is observed in cells exposed to UV irradiation and various oxidants and is apparently accompanied by upregulation of PEX genes. Significant reduction of peroxisomes and their enzymes is observed in inflammatory processes including infections, ischemia-reperfusion injury, and allograft rejection and seems to be related to the suppressive effect of tumor necrosis factor- on peroxisome function and peroxisome proliferator activated receptor-. Xenobiotic-induced proliferation of peroxisomes in rodents is accompanied by the formation of hepatic tumors, and evidently the imbalance in generation and decomposition of ROS plays an important role in this process. In PEX5^–/– knockout mice lacking functional peroxisomes severe alterations of mitochondria in various organs are observed which seem to be due to a generalized increase in oxidative stress confirming the important role of peroxisomes in homeostasis of ROS and the implications of its disturbances for cell pathology.     

Interaction between nitric oxide,reactive oxygen intermediates,and peroxynitrite in the regulation of 5-lipoxygenase metabolism

We have shown that overnight lipopolysaccharide (LPS) suppresses alveolar macrophage (AM) leukotriene (LT) synthesis mediated in part by induction of inducible nitric oxide synthase (iNOS) and NO production. Here we examined the possibility that reactive oxygen intermediates (ROI) generated by LPS pretreatment contribute to the suppression of 5-lipoxygenase (5-LO) metabolism. Pretreatment of AM with xanthine/xanthine oxidase, which generates high concentrations of ROI, resulted in suppression of LT synthetic capacity. Since NO and ROI reactive species are known to react and form peroxynitrite (ONOO(-)), we examined the effect of ONOO(-) on 5-LO metabolism. Exogenous ONOO(-) caused a dose-dependent suppression of recombinant 5-LO cell-free activity. ONOO(-) also suppressed LT synthesis in intact AM, which was reversed by the ONOO(-) scavenger tetrakis(4-benzoic acid)porphyrin. ONOO(-) treatment also resulted in dose-dependent nitrotyrosination and S-nitrosylation of the recombinant 5-LO enzyme. Since the direct 5-LO inhibitor zileuton prevents the LPS-induced suppression of LT synthesis, we examined if 5-LO itself was the source of ROI. Zileuton reduced ROI generation in LPS-treated cells. These studies identify an important role for ROI and ONOO(-) in the suppression of 5-LO metabolism by LPS.     

Parameters of reactive oxygen species and nitric oxide metabolism in people with high arterial blood pressure

An exchange of active forms of oxygen and nitric oxide in normal conditions and under the development of oxidative stress in humans with high of arterial blood pressure was studied. The activity of NO-synthase was estimated in the human thrombocytes. The nitric oxide formations were determined by the quantity level of its final metabolites--nitrites and nitrates. The peroxynitrite formations were determined by the quantity level of 3-nitrotyrosine. An analysis of the investigation results has shown the increase of processes of oxidative stress, violation of nitric oxide formation in humans with high arterial blood pressure. Application of ascorbic acid allows to reduce the level of free radicals and to increase the formation of nitric oxide, but does not result in statistically reliable changes of the parameters describing formation of peroxynitrite and products of peroxide oxidation of lipids in humans with high arterial blood pressure. Application of ascorbic acid does not result in changes of researched parameters in the control group.     

Interaction between reactive oxygen metabolites and nitric oxide in oxidant tolerance

The excessive generation of reactive oxygen metabolites (ROM) leads to an oxidative stress in the microvasculature of a variety of tissues and has been implicated as a causative event in a number of pathologies. There are numerous reviews on this topic that have been published recently. Herein, we will focus on a beneficial effect of ROM generation that leads to the development of an adaptive response that protects tissue from a subsequent oxidative stress (oxidant tolerance). We will focus on reductionist approaches (studies in isolated cells) used by our laboratory and those of others to define the mechanisms involved in this adaptational response and potential interactions between different cells within the tissue. As our prototype organ system, we target the heart, which has received the greatest amount of attention in this area. We will summarize evidence from isolated endothelial cells and cardiac myocytes that supports (i) the role of ROM in the development of oxidant tolerance, (ii) the possibility of an interaction between cardiac myocytes and endothelial cells in this phenomenon, and (iii) the potential interactions between ROMs and nitric oxide.     

Ventilatory muscle activation and inflammation: cytokines, reactive oxygen species, and nitric oxide.

Strenuous diaphragmatic contractions that are induced by inspiratory resistive breathing initiate an inflammatory response that involves the elevation of pro- and anti-inflammatory cytokines within the diaphragm, which may then spill into the circulation. The production of reactive oxygen species within working respiratory muscles increases in response to these strenuous diaphragmatic contractions. At the same time, diaphragmatic nitric oxide (NO) production declines significantly, despite a time-dependent increase in NO synthase isoform protein expression. The increase in adhesion molecule expression and infiltration of granulocytes and macrophages that follows may contribute to the contraction-induced diaphragm injury. Enhanced generation of reactive oxygen species, oxidative stress augmentation, reduced NO production, and glycogen depletion are potential stimuli for the cytokine induction that is secondary to strenuous diaphragmatic contractions. This production of cytokines within the diaphragm may contribute to the diaphragmatic muscle fiber injury that occurs with strenuous contractions or to the expected repair process. TNF-alpha is a cytokine that compromises diaphragmatic contractility and may contribute to muscle wasting. IL-6 is a cytokine that may have beneficial systemic effects by mobilizing glucose from the liver and free fatty acids from the adipose tissue and providing them to the strenuously working respiratory muscles. Thus cytokine upregulation within the working diaphragm may be adaptive and maladaptive.     

New insights into reactive oxygen species and nitric oxide signalling under low oxygen in plants

Plants produce reactive oxygen species (ROS) when exposed to low oxygen (O₂). Much experimental evidence has demonstrated the existence of an oxidative burst when there is an O₂ shortage. This originates at various subcellular sites. The activation of NADPH oxidase(s), in complex with other proteins, is responsible for ROS production at the plasma membrane. Another source of low O₂‐dependent ROS is the mitochondrial electron transport chain, which misfunctions when low O₂ limits its activity. Arabidopsis mutants impaired in proteins playing a role in ROS production display an intolerant phenotype to anoxia and submergence, suggesting a role in acclimation to stress. In rice, the presence of the submergence 1A (SUB1A) gene for submergence tolerance is associated with a higher capacity to scavenge ROS. Additionally, the destabilization of group VII ethylene responsive factors, which are involved in the direct O₂ sensing mechanism, requires nitric oxide (NO). All this evidence suggests the existence of a ROS and NO – low O₂ mechanism interplay which likely includes sensing, anaerobic metabolism and acclimation to stress. In this review, we summarize the most recent findings on this topic, formulating hypotheses on the basis of the latest advances.     

Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria

Chloroplasts and mitochondria are the powerhouses of photosynthetic cells. The oxidation-reduction (redox) cascades of the photosynthetic and respiratory electron transport chains not only provide the driving forces for metabolism but also generate redox signals, which participate in and regulate every aspect of plant biology from gene expression and translation to enzyme chemistry. Plastoquinone, thioredoxin and reactive oxygen have all been shown to have signalling functions. Moreover, the intrinsic involvement of molecular oxygen in electron transport processes with the inherent generation of superoxide, hydrogen peroxide and singlet oxygen provides a repertoire of additional extremely powerful signals. Accumulating evidence implicates the major redox buffers of plant cells, ascorbate and glutathione, in redox signal transduction. The network of redox signals from energy-generating organelles orchestrates metabolism to adjust energy production to utilization, interfacing with hormone signalling to respond to environmental change at every stage of plant development.     

Enhanced nitric oxide and reactive oxygen species production and damage after inhalation of silica

In previous reports from this study, measurements of pulmonary inflammation, bronchoalveolar lavage cell cytokine production and nuclear factor-kappa B activation, cytotoxic damage, and fibrosis were detailed. In this study, we investigated the temporal relationship between silica inhalation, nitric oxide (NO), and reactive oxygen species (ROS) production, and damage mediated by these radicals in the rat. Rats were exposed to a silica aerosol (15 mg/m(3) silica, 6 h/day, 5 days/wk) for 116 days. We report time-dependent changes in 1) activation of alveolar macrophages and concomitant production of NO and ROS, 2) immunohistochemical localization of inducible NO synthase and the NO-induced damage product nitrotyrosine, 3) bronchoalveolar lavage fluid NO(x) and superoxide dismutase concentrations, and 4) lung lipid peroxidation levels. The major observations made in this study are as follows: 1) NO and ROS production and resultant damage increased during silica exposure, and 2) the sites of inducible NO synthase activation and NO-mediated damage are associated anatomically with pathological lesions in the lungs.     

Age-dependent change in reactive oxygen species and nitric oxide generation by rat alveolar macrophages

Immunosenescence is an age-associated dysregulation of the immune function, which contributes to increased susceptibility to disease in the elderly. Alveolar macrophages (AM) are known phagocytes that generate reactive oxygen species (ROS) and nitric oxide (NO), essential mediators for host defence. We studied phagocytosis, ROS and NO production in AM obtained from young, adult and senescent rats (1-2, 9-12 and 18-24 months old, respectively) after exposure to lipopolysaccharide (LPS, 0.1-10 microg mL(-1)), 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 microg mL(-1)) or LPS + TPA in culture. Phagocytosis was significantly lower in control AM from adult rats than in AM from young animals. Nevertheless, AM from adult animals pretreated with LPS exhibited higher phagocytic capacity than AM from younger animals. ROS was identified by the NBT test at single cell level and quantified by automated image analysis. When TPA was added to all three populations, AM from adult and senescent animals responded more than AM from young animals. All LPS-stimulated AM produce more NO than controls. However, NO production increased three-, four- and two-fold in young, adult and senescent animals, respectively. Our results demonstrate that AM from young, adult and senescent animals display differential responsiveness to inflammatory mediators. Therefore, aging processes markedly affect AM metabolic functions and may further compromise the lung immune defence response, increasing adverse long-term health effects.     

植物线粒体、活性氧与信号转导

活性氧(ROS)的产生是需氧代谢不可避免的结果。在植物细胞中,线粒体电子传递链(ETC)的复合物Ⅰ和Ⅱ是ROS产生的主要的部位。交替氧化酶和可能的内源鱼藤酮不敏感的NADH脱氢酶通过保持ETc的相对氧化状态限制线粒体产生ROS。线粒体基质中的抗氧化酶系统与小分子量的抗氧化剂一道起ROS的解毒作用。ROS除了引起细胞的伤害外,在植物中还能够作为一种普遍存在的信号分子起作用。在低浓度时,ROS能诱导防御基因的表达和引起适应反应;在高浓度时,引起细胞死亡。一氧化氮是植物合成和释放的一种气体,也可作为信号分子调节植物的生长和发育。     

Plant hemoglobins: important players at the crossroads between oxygen and nitric oxide

Plant hemoglobins constitute a diverse group of hemeproteins and evolutionarily belong to three different classes. Class 1 hemoglobins possess an extremely high affinity to oxygen and their main function consists in scavenging of nitric oxide (NO) at very low oxygen levels. Class 2 hemoglobins have a lower oxygen affinity and they facilitate oxygen supply to developing tissues. Symbiotic hemoglobins in nodules have mostly evolved from class 2 hemoglobins. Class 3 hemoglobins are truncated and represent a clade with a very low similarity to class 1 and 2 hemoglobins. They may regulate oxygen delivery at high O₂ concentrations. Depending on their physical properties, hemoglobins belong either to hexacoordinate non-symbiotic or pentacoordinate symbiotic groups. Plant hemoglobins are plausible targets for improving resistance to multiple stresses.     

Levodopa modulating effects of inducible nitric oxide synthase and reactive oxygen species in glioma cells

Neurological injury and Parkinson disease (PD) are often associated with the increase of nitric oxide (NO) and free radicals from resident glial cells in the brain. In vitro, exposure to L-3-4-dihydroxyphenylalanine (L-DOPA), one of the main therapeutic agents for the treatment of PD, can lead to neurotoxicity. In this study, lipopolysaccharide (LPS) and interferon-gamma (IFN-g) were used to stimulate C6 glioma cells in the presence of varying concentrations of L-DOPA (1 microM-1 mM). The results indicated a slight augmentation of NO(2)(-) production at low concentrations of L-DOPA (<100 microM) and complete inhibition of NO(2)(-) at higher concentrations (500 microM, 1 mM), (p < 0.001). Western blot analysis corroborated that L-DOPA effects on iNOS was at the level of its protein expression. Total reactive oxygen species (ROS) were detected using 2', 7'-dichlorofluorescein diacetate fluorescence dye (2', 7'-DCFC) and there was an increase of intensity with the increasing concentrations of L-DOPA. Furthermore, large amounts of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) were generated from the autoxidation of L-DOPA. C6 cells contain high levels of catalase, with inadequate levels of superoxide dismutase (SOD); therefore, there was an accumulation of O(2)(-), tantamount to elevation in 2'7'-DCFC intensity. Simultaneous accumulation of O(2)(-) and NO(2)(-) would propel formation of peroxynitrite (ONOO-). SOD completely attenuated the autoxidation of L-DOPA and significantly reversed the inhibitory effects on iNOS at high concentrations. The data obtained confirmed that the observed effects on iNOS were not due to the activation of the D(1) or beta1 adrenergic receptors by L-DOPA. It was concluded from this study that L-DOPA contributed to the modulation of iNOS and to the increase of O(2)(-) production in the stimulated glioma cells in vitro.     

Adrenergic response of splanchnic arteries from cirrhotic patients: role of nitric oxide, prostanoids, and reactive oxygen species

Peripheral and splanchnic vasodilatation in cirrhotic patients has been related to hyporesponsiveness to vasoconstrictors, but studies to examine the vascular adrenergic response provide contradictory results. Hepatic arteries from cirrhotic patients undergoing liver transplantation and mesenteric arteries from liver donors were obtained. Segments 3 mm long from these arteries were mounted in organ baths for testing isometric adrenergic response. The concentration-dependent contraction to noradrenaline (10(-8) to 10(-4) M) was similar in hepatic and mesenteric arteries, and prazosin (alpha 1-adrenergic antagonist, 10(-6) M), but not yohimbine (alpha 2-adrenergic antagonist, 10(-6) M), produced a rightward parallel displacement of this contraction in both types of arteries. Phenylephrine (alpha 1-adrenergic agonist, 10(-8) to 10(-4) M) and clonidine (alpha 2-adrenergic agonist, 10(-8) to 10(-4) M) also produced concentration-dependent contractions that were comparable in hepatic and mesenteric arteries. The inhibitor of cyclooxygenase meclofenamate (10(-5) M), but not the inhibitor of nitric oxide synthesis N(w)-nitro-l-arginine methyl ester (l-NAME, 10(-4) M), potentiated the response to noradrenaline in hepatic arteries; neither inhibitor affected the response to noradrenaline in mesenteric arteries. Diphenyleneiodonium (DPI; 5 x 10(-6) M), but neither catalase (1000 U/ml) nor tiron (10(-4) M), decreased the maximal contraction for noradrenaline similarly in hepatic and mesenteric arteries. Therefore, it is suggested that, in splanchnic arteries from cirrhotic patients, the adrenergic response and the relative contribution of alpha 1- and alpha 2-adrenoceptors in this response is preserved, and prostanoids, but not nitric oxide, may blunt that response. Products dependent on NAD(P)H oxidase might contribute to the adrenergic response in splanchnic arteries from control and cirrhotic patients.     

Ambient oxygen regulates epithelial metabolism and nitric oxide production in the human nose.

The effects of ambient O(2) tension on epithelial metabolism and nitric oxide (NO) production (VNO) in the nasal airway were examined in nine healthy volunteers. Nasal VNO, O(2) consumption (VO(2)), and CO(2) production (VCO(2)) were measured during normoxia followed by gradual hypoxia from 21 to 0% O(2) concentration. Nasal VO(2), VCO(2), and respiratory quotient during normoxia were determined to be 1.19 +/- 0.04 ml/min, 1.60 +/- 0.04 ml/min, and 1.35 +/- 0.04, respectively. Hypoxia exposure to the nasal cavity significantly decreased both VCO(2) and VNO [VCO(2): 1.60 +/- 0.04 to 0.96 +/- 0.03 ml/min (P < 0.01), VNO: 530 +/- 15 to 336 +/- 9 nl/min (P < 0.01)]. VNO was reduced commensurately with gradual decline in O(2) tension, and the apparent K(m) value for O(2) was determined to be 23.0 microM. These results indicate that the nasal epithelial cells exchange O(2) and CO(2) with ambient air in the course of their metabolism and that nasal epithelial cells can synthesize NO by using ambient O(2) as a substrate. We conclude that air-borne O(2) diffuses into the epithelium where it may be utilized for either cell metabolism or NO synthesis.     

活性氧、NO和线粒体ATP敏感钾通道在TNF-α预处理对缺血/再灌注心肌保护中的作用

目的: 观察TNF-α预处理对缺血/再灌注心脏功能和酶学指标的影响及其可能机制.方法: 采用心脏Langendorff灌流模型.结果:与单独缺血/再灌注组相比,TNF-α(104U/L)预处理明显减弱缺血/再灌注对左室发展压、左室舒张末压、最大收缩/舒张速率和左室发展压与心率乘积的抑制作用(P<0.05),并显著降低复灌后冠脉流出液中乳酸脱氢酶(LDH)含量,增加线粒体中锰超氧化物歧化酶(Mn-SOD)活性(P<0.05);分别使用抗氧化剂2-MPG(0.3 mmol/L)、一氧化氮合酶抑制剂L-NAME(0.5 mmol/L)或线粒体ATP敏感钾通道抑制剂5-HD(100 μmol/L)预处理,减弱了TNF-α改善缺血/再灌注后心功能、抑制心肌LDH释放和诱导Mn-SOD活性增高的作用.结论: TNF-α预处理具有减轻心脏缺血/再灌注损伤的作用,这一作用可能与其诱导Mn-SOD活性增高有关,活性氧、一氧化氮和线粒体ATP敏感钾通道参与介导TNF-α的心肌保护作用.     

Rodent and human mast cells produce functionally significant intracellular reactive oxygen species but not nitric oxide

In immunity, reactive oxygen species (ROS) and nitric oxide (NO) are important antimicrobial agents and regulators of cell signaling and activation pathways. However, the cellular sources of ROS and NO are much debated. Particularly, there is contention over whether mast cells, key secretory cells in allergy and immunity, can generate these chemical species, and if so, whether they are of functional significance. We therefore examined directly by flow cytometry the capacity of mast cells to generate intracellular ROS and NO using the respective cell-permeable fluorescent probes dichlorodihydrofluorescein and diaminofluorescein and evaluated the effects of inhibitors of ROS and NO synthesis on cell degranulation. For each of three mast cell types (rat peritoneal mast cells, mouse bone marrow-derived mast cells, and human blood-derived mast cells), degranulation stimulated by IgE/antigen was accompanied by production of intracellular ROS but not NO. Inhibition of ROS production led to reduced degranulation, indicating a facilitatory role for ROS, whereas NO synthase inhibitors were without effect. Likewise, bacterial lipopolysaccharide and interferon-gamma over a wide range of conditions failed to generate intracellular NO in mast cells, whereas these agents readily induced intracellular NO in macrophages. NO synthase protein, as assessed by Western blotting, was readily induced in macrophages but not mast cells. We conclude that rodent and human mast cells generate intracellular ROS but not NO and that intracellular ROS but not intracellular NO are functionally linked to mast cell degranulation.     

Interaction between reactive oxygen species and nitric oxide in the microvascular response to systemic hypoxia.

Systemic hypoxia results in oxidative stress due to a change in the reactive oxygen species (ROS)-nitric oxide (NO) balance. These experiments explored two mechanisms for the altered ROS-NO balance: 1) decreased NO synthesis by NO synthase due to limited O(2) substrate availability and 2) increased superoxide generation. ROS levels and leukocyte adherence in mesenteric venules of rats during hypoxia were studied in the absence and presence of an NO donor [spermine NONOate (SNO)] and of the NO precursor L-arginine. We hypothesized that if the lower NO levels during hypoxia were due to O(2) substrate limitation, L-arginine would not prevent hypoxia-induced microvascular responses. Graded hypoxia (produced by breathing 15, 10, and 7.5% O(2)) increased both ROS (123 +/- 6, 148 +/- 11, and 167 +/- 3% of control) and leukocyte adherence. ROS levels during breathing of 10 and 7.5% O(2) were significantly attenuated by SNO (105 +/- 6 and 108 +/- 3%, respectively) and L-arginine (117 +/- 5 and 115 +/- 2%, respectively). Both interventions reduced leukocyte adherence by similar degrees. The fact that the effects of L-arginine were similar to those of SNO does not support the idea that NO generation is impaired in hypoxia and suggests that tissue NO levels are depleted by the increased ROS during hypoxia.