Interplay Among Nitric Oxide and Reactive Oxygen Species: A Complex Network Determining Cell Survival or Death

Programmed cell death (PCD) is an integrated cellular process occurring in plant growth, development, and defense responses to facilitate normal growth and development and better survival against various stresses as a whole. As universal toxic chemicals in plant and animal cells, reactive oxygen or nitrogen species (ROS or RNS), mainly superoxide anion (O₂^−•), hydrogen peroxide (H₂O₂) or nitric oxide (^•NO), have been studied extensively for their roles in PCD induction. Physiological and genetic studies have convincingly shown their essential roles. However, the details and mechanisms by which ROS and ^•NO interplay and induce PCD are not well understood. Our recent study on Cupressus lusitanica culture cell death revealed the elicitor-induced co-accumulation of ROS and ^•NO and interactions between ^•NO and H₂O₂ or O₂-^• in different ways to regulate cell death. ^•NO and H₂O₂ reciprocally enhanced the production of each other whereas ^•NO and O₂^−• showed reciprocal suppression on each other''s production. It was the interaction between ^•NO and O₂-^• but not between ^•NO and H₂O₂ that induced PCD, probably through peroxynitrite (ONOO⁻). In this addendum, some unsolved issues in the study were discussed based on recent studies on the complex network of ROS and ^•NO leading to PCD in animals and plants.Key Words: cell death, nitric oxide, reactive oxygen species, interaction, posttranslational modification     

Hydrogen peroxide-independent generation of superoxide catalyzed by soybean peroxidase in response to ferrous ion

It is well documented that extracellular alkalization occurs in plants under the challenges by pathogenic microbes. This may eventually induce the pH-dependent extracellular peroxidase-mediated oxidative burst at the site of microbial challenges. By employing the purified proteins of horseradish peroxidase as a model, we have recently proposed a likely role for free Fe²⁺ in reduction of ferric enzyme of plant peroxidases into ferrous intermediate and oxygen-bound form of enzyme known as Compound III which may eventually releases superoxide anion radical (O₂^•−), especially under alkaline condition, possibly contributing to the plant defense mechanism. In the present study, we employed the purified protein of soybean peroxidase (SBP) as an additional model, and examined the changes in the redox status of enzyme accompanying the generation of O₂^•− in response to Fe²⁺ under alkaline condition.     

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O₂^•-, H₂O₂, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death^1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response⁷. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O₂^•- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells⁸. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others^9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O₂^•- that are important in the host defense mechanism^11,12. Although paracrine-derived O₂^•- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca²⁺ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O₂^•- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O₂^•- lead to calcium signaling in adjacent endothelial cells.     

Production of Superoxide Anions by Keratinocytes Initiates P. acnes-Induced Inflammation of the Skin

Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes), a gram-positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at determining whether reactive oxygen species (ROS) are produced by keratinocytes upon P. acnes infection, dissecting the mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response induced by P. acnes. In our hands, ROS, and especially superoxide anions (O₂ ^•−), were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In P. acnes-stimulated keratinocytes, O₂ ^•− was produced by NAD(P)H oxidase through activation of the scavenger receptor CD36. O₂ ^•− was dismuted by superoxide dismutase to form hydrogen peroxide which was further detoxified into water by the GSH/GPx system. In addition, P. acnes-induced O₂ ^•− abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O₂ ^•− with nitric oxide to form peroxynitrites. Finally, retinoic acid derivates, the most efficient anti-acneic drugs, prevent O₂ ^•− production, IL-8 release and keratinocyte apoptosis, suggesting the relevance of this pathway in humans.     

Rac-dependent feedforward autoactivation of NOX2 leads to oxidative burst

NADPH oxidase 2 (NOX2) produces the superoxide anion radical (O₂⁻), which has functions in both cell signaling and immune defense. NOX2 is a multimeric-protein complex consisting of several protein subunits including the GTPase Rac. NOX2 uniquely facilitates an oxidative burst, which is described by initially slow O₂⁻ production, which increases over time. The NOX2 oxidative burst is considered critical to immune defense because it enables expedited O₂⁻ production in response to infections. However, the mechanism of the initiation and progression of this oxidative burst and its implications for regulation of NOX2 have not been clarified. In this study, we show that the NOX2 oxidative burst is a result of autoactivation of NOX2 coupled with the redox function of Rac. NOX2 autoactivation begins when active Rac triggers NOX2 activation and the subsequent production of O₂⁻, which in turn activates redox-sensitive Rac. This activated Rac further activates NOX2, amplifying the feedforward cycle and resulting in a NOX2-mediated oxidative burst. Using mutagenesis-based kinetic and cell analyses, we show that enzymatic activation of Rac is exclusively responsible for production of the active Rac trigger that initiates NOX2 autoactivation, whereas redox-mediated Rac activation is the main driving force of NOX2 autoactivation and contributes to generation of ∼98% of the active NOX2 in cells. The results of this study provide insight into the regulation of NOX2 function, which could be used to develop therapeutics to control immune responses associated with dysregulated NOX2 oxidative bursts.     

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability^{1, 2}. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation³, and ultimately to oxidative stress leading to cell injury or death⁴. Superoxide radical anion (O₂•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O₂•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O₂•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O₂•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase⁵. Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO⁶. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O₂•- in a process called uncoupling, which is believed to be caused by oxidation of heme⁷ or the co-factor, tetrahydrobiopterin (BH₄)⁸.There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production⁹.The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO¹⁰, the phosphoryl-substituted DEPMPO¹¹, and the ester-substituted, EMPO¹² and BMPO¹³, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O₂•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)₂ is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct¹⁴.     

Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA

Heat-induced formation of 8-oxoguanine was demonstrated in DNA solutions in 10^–3 M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chemical yield of 3.7 × 10^–2 µmol J^–1 for 8-oxoguanine production in DNA upon γ-irradiation was used as an adequate standard for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate constants for 8-oxoguanine formation were determined at elevated temperatures; the activation energy was found to be 27 ± 2 kcal/mol. Extrapolation to 37°C gave a value of k₃₇ = 4.7 × 10^–10 s^–1. Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced production of H₂O₂ in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H₂O₂. Gas saturation (O₂, N₂ and Ar), D₂O, scavengers of ¹O₂, O₂^–• and OH^• radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.     

Targeting Cancer Cells with Reactive Oxygen and Nitrogen Species Generated by Atmospheric-Pressure Air Plasma

The plasma jet has been proposed as a novel therapeutic method for cancer. Anticancer activity of plasma has been reported to involve mitochondrial dysfunction. However, what constituents generated by plasma is linked to this anticancer process and its mechanism of action remain unclear. Here, we report that the therapeutic effects of air plasma result from generation of reactive oxygen/nitrogen species (ROS/RNS) including H₂O₂, Ox, OH⁻, •O_2, NOx, leading to depolarization of mitochondrial membrane potential and mitochondrial ROS accumulation. Simultaneously, ROS/RNS activate c-Jun NH₂-terminal kinase (JNK) and p38 kinase. As a consequence, treatment with air plasma jets induces apoptotic death in human cervical cancer HeLa cells. Pretreatment of the cells with antioxidants, JNK and p38 inhibitors, or JNK and p38 siRNA abrogates the depolarization of mitochondrial membrane potential and impairs the air plasma-induced apoptotic cell death, suggesting that the ROS/RNS generated by plasma trigger signaling pathways involving JNK and p38 and promote mitochondrial perturbation, leading to apoptosis. Therefore, administration of air plasma may be a feasible strategy to eliminate cancer cells.     

Proliferation and wound healing of vascular cells trigger the generation of extracellular reactive oxygen species and LDL oxidation

Cell proliferation of vascular cells is a key feature in vascular biology, wound healing, and pathophysiological processes such as atherosclerosis and restenosis. In atherosclerotic intima, cell proliferation colocalizes with oxidized LDL that indicate a local oxidative stress. This study aims to investigate whether cell proliferation is causally related with extracellular ROS generation and subsequent LDL oxidation. Sparse proliferating endothelial and smooth muscle cells generate higher levels of extracellular ROS (O₂^•− and H₂O₂) and LDL oxidation than confluent contact-inhibited cells. During wound healing of confluent cell layer, cell proliferation associated with healing also induced enhanced extracellular ROS generation and LDL oxidation. Proliferation-associated extracellular ROS generation is mediated through mitogenic signaling pathways, involving ERK1/2 and PKC, but is independent of de novo DNA synthesis, gene expression and protein synthesis. Data obtained with inhibitors of oxidases suggest that proliferation-associated extracellular ROS are not generated by a single ROS-generating system and are not essential for cell proliferation. In conclusion, our data show that proliferating vascular cells (in sparse culture or during wound healing) generate high levels of extracellular ROS and LDL oxidation through regulation of ROS-generating systems by mitogenic signaling. This constitutes a link between proliferative events and oxidative stress/LDL oxidation in atherosclerotic lesions and restenosis.     

The effects of dopamine on antioxidant enzymes activities and reactive oxygen species levels in soybean roots

In the current work, we investigated the effects of dopamine, an neurotransmitter found in several plant species on antioxidant enzyme activities and ROS in soybean (Glycine max L. Merrill) roots. The effects of dopamine on SOD, CAT and POD activities, as well as H₂O₂, O₂^•−, melanin contents and lipid peroxidation were evaluated. Three-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), without or with 0.1 to 1.0 mM dopamine, in a growth chamber (25°C, 12 h photoperiod, irradiance of 280 μmol m⁻² s⁻¹) for 24 h. Significant increases in melanin content were observed. The levels of ROS and lipid peroxidation decreased at all concentrations of dopamine tested. The SOD activity increased significantly under the action of dopamine, while CT activity was inhibited and POD activity was unaffected. The results suggest a close relationship between a possible antioxidant activity of dopamine and melanin and activation of SOD, reducing the levels of ROS and damage on membranes of soybean roots.     

The potential physiological implications of polygalacturonic acid-mediated production of superoxide

PGA/OGA/PF represent apoplastic signaling molecules implicated in the control of gene expression and the activity of enzymes involved in defense regulation. However, the underlying mechanisms behind such processes are lacking. Here we unequivocally show using EPR spectroscopy with DEPMPO spin-trap capable of differentiating between ^•OH and ^•O₂⁻ that PGA and PF can produce ^•O₂⁻ by transforming ^•OH. The potential physiological implications of this unique property are discussed. We propose that PGA/OGA/PF could represent the initiators of redox signaling cascades in stress response, with H₂O₂ being a downstream secondary messenger.Key words: polygalacturonic acid, pectin, superoxide, hydrogen peroxide, apoplast, redox signalingPGA/OGA/PF represent apoplastic signaling molecules involved in defense regulation.^1–5 For example, they induce de novo enzyme synthesis in the wound-inducible defense reaction⁶ and increase the resistance of plants to pathogens.⁷ However, the underlying mechanisms behind such processes are lacking. Aldington et al.⁸ have postulated that OGAs do not act through a receptor, but rather they owe their activity to some specific physical property. Pertinent to this is the fact that there is a broad range of active OGA structures.⁵ In addition, it has been reported that methylated OGAs are not able to trigger signaling pathways that are activated by OGAs possessing ‘free’ carboxyl groups.^9,10 In contrast to this concept, several research groups have showed that PGA/OGA/PF bind to wall-associated kinases (WAK1 and WAK2).^11–15 However, potential effects of PGA/OGA/PF on the activity of WAK1 or WAK2 have not been observed to date. We propose here that the specific property proposed by Aldington and co-workers,⁸ is in fact the ability of the polymers of galacturonic acid (PGA/OGA/PF) to produce ^•O₂⁻. By taking into account previously proposed mechanisms of reaction of PGA with ^•OH,^16,17 and thermodynamic properties of species potentially involved in the reaction,¹⁸ we hypothesized that PGA could transform the ^•OH radical into ^•O₂⁻. To test our hypothesis we investigated the effects of PGA and pectin on radical production in two different ^•OH-generating systems using EPR spectroscopy with the DEPMPO spin-trap capable of differentiating between ^•OH and ^•O₂⁻.¹⁹The results presented in Figure 1 document the ability of PGA to transform ^•OH to ^•O₂⁻. In addition, our experimental approach showed that pectin shares the ^•O₂⁻-producing ability of its constituent PGA. In the control Fenton system (Fe²⁺ + H₂O₂ → ^•OH + OH⁻ + Fe³⁺) only ^•OH radical was produced (Fig. 1A). However in the presence of PGA or pectin, a significant production of ^•O₂⁻ was detected. Haber-Weiss-like reaction (^•O₂⁻ + H₂O₂ → ^•OH + OH⁻ + O₂) generated ^•OH radical, accompanied by a low level of ^•O₂⁻ (Fig. 1B). The supplementation of PGA or pectin to this system led to sole or pronounced production of ^•O₂⁻, respectively. Under the same experimental settings, no ^•O₂⁻ production was observed for other two major extracellular carbohydrates—cellulose and mannan (Fig. 2).Open in a separate windowFigure 1The ability of PGA and pectin to transform ^•OH radical into ^•O₂⁻. Presented are characteristic EPR spectra of adducts of DEPMPO with the ^•OH radical (/OH) and the ^•O₂⁻ radical (/ooh) in two ^•OH-generating systems: (A) Fenton reaction; (B) Haber-Weiss-like reaction; in the absence (control) or presence of PGA or pectin (15 mgml⁻¹ final concentration). The downward triangle represents the characteristic line of the/OH adduct. The circular symbol represents the characteristic line of the/OOH adduct. The grey lines represent the spectral simulations based on signals of DEPMPO adducts contributing to each spectrum in specific percentages [mean values from four experiments (standard deviations were <5%)].Open in a separate windowFigure 2Characteristic EPR spectra of adducts of DEPMPO with the ^•OH radical (downward triangle) and the ^•O₂⁻ radical (circular symbol) in Haber-Weiss-like ^•OH generating system in the absence (control) or presence of cellulose or mannan (15 mgml⁻¹ final concentration). No ^•O₂⁻ production can be observed in the presence of cellulose or mannan.Presented results illustrate the ability of PGA and pectin to transform ^•OH radical into ^•O₂⁻. Other carbohydrates involved in plant metabolism, such as cellulose and mannan, but also glucose and fructose,²⁰ do not show such properties. In addition, methylated PGA do not produce ^•O₂⁻ in the reaction with ^•OH, but methane,²¹ probably with ^•CH₃ radical as an intermediate.²² This implies that carboxyl groups which are characteristic for PGA play a critical role in the production of ^•O₂⁻. Zegota¹⁶ has proposed that pectin and ^•OH react to produce pectin C(5) radical, which further reacts with molecular oxygen thus forming C(5) peroxyl radical. This radical is unstable, especially at physiological pH values,¹⁷ hence it is further decomposed to carbohydrate fragment(s) and superoxide, via ^•O₂⁻-elimination.^16,17Under in vivo settings, superoxide generated in the apoplast by PGA/OGA/PF can be further dismutated by SOD to H₂O₂, which represents a crucial signaling molecule in plants.^23,24 It is very interesting that signaling properties of H₂O₂ in the plant immune response remarkably overlap with the events initiated by OGA: (1) Similarly to the inverted H₂O₂ gradient across the plant plasma membrane,²⁴ OGA has been reported to activate calcium-dependent protein kinases,²⁵ to provoke membrane depolarization with H⁺ influx and K⁺ efflux,²⁶ and to induce activation of mitogen-activated protein kinases.²⁷ (2) Both, OGA^10,28 and H₂O₂^29–31 provoke an influx of Ca²⁺ from the apoplast into the intracellular compartment. (3) It has been documented that apoplastic generation of ^•O₂⁻ and H₂O₂ follows mechanical stress and the recognition of pathogens,^4,32–34 but also the supplementation of OGAs.^35–37 In addition, it has been reported that the supplementation of OGA to plant cells leads to the increase of apoplastic and total concentration of H₂O₂.^37,38 The enlisted results obtained by others and data presented here imply that PGA/OGA/PF could represent the initiators of redox signaling cascades in stress response, with H₂O₂ being a downstream secondary messenger.Hereby-proposed mechanism of apoplastic production of H₂O₂ by PGA/OGA/PF and SOD, depends on ^•OH radicals. Hence, the central question of our hypothesis is: “Where do apoplastic ^•OH radicals come from, under in vivo conditions?”. Hydrogen peroxide is continually generated in the apoplast by NAD(P) H oxidase/SOD, cell wall peroxidase and other sources during normal metabolism, as reviewed by Neill and co-workers.²⁴ The physiological concentrations of H₂O₂ in plants are not well established,⁴⁰ but it seems that apoplastic and total (fresh weight) concentrations are similar and maintained at around 1 µM.^38–40 In the extracellular compartment, continuous generation of H₂O₂ is balanced by its degradation in ^•OH-generating Fenton reaction which involves redox active metals, such as copper and iron.⁴¹ In principle, ^•OH radicals are further removed by apoplastic ascorbate or cell wall constituents (Fig. 3A).^41–43 However, mechanical wounding (e.g., provoked by cold⁴⁴), degradation of the cell wall by pathogenic enzymes (such as polygalacturonase or pectate lyase⁴⁵) or insect chewing could release PGA/OGA/PF from the cell wall into the apoplast. The presence of PGA/OGA/PF in the apoplast related with these events could drastically change apoplastic redox poise. Positively charged redox active metals readily bind to negatively charged polymers of galacturonic acid.⁴⁶ The close proximity of PGA/OGA/PF to the site of ^•OH production could change the fate of ^•OH. Instead of being scavenged, ^•OH could react with PGA/OGA/PF, which leads to ^•O₂⁻ production and subsequent H₂O₂ re-generation (Fig. 3B). Such ‘recycling’ of H₂O₂ could result in a higher steady-state H₂O₂ concentration in the apoplast and consequent H₂O₂ influx, as H₂O₂ is capable of passing the membrane via passive diffusion and specific aquaporins.⁴⁷ In the stress response, H₂O₂ can traverse the membrane, induce Ca²⁺ influx or diffuse into surrounding healthy tissue to modulate enzyme activity⁴⁸ and initiate gene expression,^23,24,49 crucial for subsequent phases of defense and adaptation. To conclude, PGA/OGA/PF could provide the cell with information about the status of the cell wall affected by stressors, via H₂O₂ signaling.Open in a separate windowFigure 3Schematic presentation of potential effects of PGA/OGA/PF released from stressed wall on apoplastic redox poise and H₂O₂ and Ca²⁺ signaling cascades. (A) Redox processes in apoplast under physiological settings. (B) Redox processes in the apoplast of plant cell exposed to stress. PGA/OGA/PF are released from the cell wall into the apoplast changing the redox poise by transforming ^•OH to ^•O₂⁻ and H₂O₂ (‘H₂O₂ recycling’). This could lead to H₂O₂ accumulation, H₂O₂ influx (via diffusion or peroxiporins) or the activation of Ca²⁺ influx, which leads to the activation of different intracellular responses.     

Integrated Analysis of COX-2 and iNOS Derived Inflammatory Mediators in LPS-Stimulated RAW Macrophages Pre-Exposed to Echium plantagineum L. Bee Pollen Extract

Oxidative stress and inflammation play important roles in disease development. This study intended to evaluate the anti-inflammatory and antioxidant potential of Echium plantagineum L. bee pollen to support its claimed health beneficial effects. The hydromethanol extract efficiently scavenged nitric oxide (^•NO) although against superoxide (O₂ ^•−) it behaved as antioxidant at lower concentrations and as pro-oxidant at higher concentrations. The anti-inflammatory potential was evaluated in LPS-stimulated macrophages. The levels of ^•NO and L-citrulline decreased for all extract concentrations tested, while the levels of prostaglandins, their metabolites and isoprostanes, evaluated by UPLC-MS, decreased with low extract concentrations. So, E. plantagineum bee pollen extract can exert anti-inflammatory activity by reducing ^•NO and prostaglandins. The extract is able to scavenge the reactive species ^•NO and O₂ ^•− and reduce markers of oxidative stress in cells at low concentrations.     

Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction

The role of the oxidative burst, transient production of activated oxygen species such as H₂O₂ and superoxide (O₂⁻) in elicitation of phytoalexins and the hypersensitive reaction (HR) was investigated in white clover (Trifolium repens L.) and tobacco (Nicotiana tabacum L.). H₂O₂ and O₂⁻ production was measured as chemiluminescence (CL) mediated by luminol, which was added to suspension-cultured white clover just before measurement in an out-of-coincidence mode scintillation counter. Maximum CL occurred between 10 and 20 min after addition of 0.4 × 10⁸ colony-forming units/mL of incompatible Pseudomonas corrugata or 158 μm HgCl₂. Autoclaved P. corrugata produced a slightly higher response. Elicitation of cells with 25 μm HgCl₂ did not produce CL. Preincubation of plant cells in superoxide dismutase, which converts O₂⁻ to H₂O₂, for 2 min before addition of bacteria did not significantly increase maximum CL levels (P ≥ 0.05). Preincubation of plant cells with catalase for 2 min before addition of bacteria prevented the increase in CL, confirming that H₂O₂ is the substrate for the luminol reaction. Addition of live bacteria or HgCl₂ (25 and 158 μm) to white clover increased levels of the phytoalexin medicarpin during a 24-h period, but addition of autoclaved bacteria did not elicit formation of medicarpin. Preincubation of plant cells with catalase, which quenched the bacteria-induced oxidative burst, did not decrease phytoalexin accumulation. Live bacteria infiltrated into Havana 44 tobacco leaf panels induced development of the HR, but autoclaved bacteria did not. Incubation of live bacteria with superoxide dismutase and catalase before infiltration into tobacco leaves did not interfere with development of the HR. Tobacco leaf panels infiltrated with up to 158 μm HgCl₂ did not develop an HR. These results suggest that an oxidative burst consisting of H₂O₂ and O₂⁻ does occur during these two plant defense responses, but it may not be a necessary element of the signaling system for HR and phytoalexin formation.     

Ferricytochrome c Directly Oxidizes Aminoacetone to Methylglyoxal,a Catabolite Accumulated in Carbonyl Stress

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH₄ ⁺ ion, and H₂O₂ coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. The participation of O₂ ^•− and HO^• radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5′-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E₀ values = −0.51 and −1.0 V) to ferricytochrome c (E₀ = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH₄ ⁺ ion. In the presence of oxygen, aminoacetone enoyl and O₂ ^•− radicals propagate aminoacetone oxidation to methylglyoxal and H₂O₂. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.     

Kinin B1 Receptor Enhances the Oxidative Stress in a Rat Model of Insulin Resistance: Outcome in Hypertension,Allodynia and Metabolic Complications

Background

Kinin B₁ receptor (B₁R) is induced by the oxidative stress in models of diabetes mellitus. This study aims at determining whether B₁R activation could perpetuate the oxidative stress which leads to diabetic complications.

Methods and Findings

Young Sprague-Dawley rats were fed with 10% D-Glucose or tap water (controls) for 8–12 weeks. A selective B₁R antagonist (SSR240612) was administered acutely (3–30 mg/kg) or daily for a period of 7 days (10 mg/kg) and the impact was measured on systolic blood pressure, allodynia, protein and/or mRNA B₁R expression, aortic superoxide anion (O₂ ^•−) production and expression of superoxide dismutase (MnSOD) and catalase. SSR240612 reduced dose-dependently (3–30 mg/kg) high blood pressure in 12-week glucose-fed rats, but had no effect in controls. Eight-week glucose-fed rats exhibited insulin resistance (HOMA index), hypertension, tactile and cold allodynia and significant increases of plasma levels of glucose and insulin. This was associated with higher aortic levels of O₂ ^•−, NADPH oxidase activity, MnSOD and catalase expression. All these abnormalities including B₁R overexpression (spinal cord, aorta, liver and gastrocnemius muscle) were normalized by the prolonged treatment with SSR240612. The production of O₂ ^•− in the aorta of glucose-fed rats was also measured in the presence and absence of inhibitors (10–100 µM) of NADPH oxidase (apocynin), xanthine oxidase (allopurinol) or nitric oxide synthase (L-NAME) with and without Sar[D-Phe⁸]des-Arg⁹-BK (20 µM; B₁R agonist). Data show that the greater aortic O₂ ^•− production induced by the B₁R agonist was blocked only by apocynin.

Conclusions

Activation of kinin B₁R increased O₂ ^•− through the activation of NADPH oxidase in the vasculature. Prolonged blockade of B₁R restored cardiovascular, sensory and metabolic abnormalities by reducing oxidative stress and B₁R gene expression in this model.     

Redox Signaling via Oxidative Inactivation of PTEN Modulates Pressure-Dependent Myogenic Tone in Rat Middle Cerebral Arteries

The present study examined the level of generation of reactive oxygen species (ROS) and roles of inactivation of the phosphatase PTEN and the PI3K/Akt signaling pathway in response to an increase in intramural pressure-induced myogenic cerebral arterial constriction. Step increases in intraluminal pressure of cannulated cerebral arteries induced myogenic constriction and concomitant formation of superoxide (O₂ ^.−) and its dismutation product hydrogen peroxide (H₂O₂) as determined by fluorescent HPLC analysis, microscopic analysis of intensity of dihydroethidium fluorescence and attenuation of pressure-induced myogenic constriction by pretreatment with the ROS scavenger 4,hydroxyl-2,2,6,6-tetramethylpiperidine1-oxyl (tempol) or Mito-tempol or MitoQ in the presence or absence of PEG-catalase. An increase in intraluminal pressure induced oxidation of PTEN and activation of Akt. Pharmacological inhibition of endogenous PTEN activity potentiated pressure-dependent myogenic constriction and caused a reduction in NPo of a 238 pS arterial K_Ca channel current and an increase in [Ca²⁺]_i level in freshly isolated cerebral arterial muscle cells (CAMCs), responses that were attenuated by Inhibition of the PI3K/Akt pathway. These findings demonstrate an increase in intraluminal pressure induced increase in ROS production triggered redox-sensitive signaling mechanism emanating from the cross-talk between oxidative inactivation of PTEN and activation of the PI3K/Akt signaling pathway that involves in the regulation of pressure-dependent myogenic cerebral arterial constriction.     

Sublethal concentrations of salicylic acid decrease the formation of reactive oxygen species but maintain an increased nitric oxide production in the root apex of the ethylene-insensitive Never ripe tomato mutants

The pattern of salicylic acid (SA)-induced production of reactive oxygen species (ROS) and nitric oxide (NO) were different in the apex of adventitious roots in wild-type and in the ethylene-insensitive Never ripe (Nr) mutants of tomato (Solanum lycopersicum L. cv Ailsa Craig). ROS were upregulated, while NO remained at the control level in apical root tissues of wildtype plants exposed to sublethal concentrations of SA. In contrast, Nr plants expressing a defective ethylene receptor displayed a reduced level of ROS and a higher NO content in the apical root cells. In wild-type plants NO production seems to be ROS(H₂O₂)-dependent at cell death-inducing concentrations of SA, indicating that ROS and NO may interact to trigger oxidative cell death. In the absence of significant ROS accumulation, the increased NO production caused moderate reduction in cell viability in root apex of Nr plants exposed to 10⁻³ M SA. This suggests that a functional ethylene signaling pathway is necessary for the control of ROS and NO production induced by SA.Key words: ethylene receptor mutant, never ripe, nitric oxide, reactive oxygen species, root apex, salicylic acid, tomatoSeveral signal molecules, including salicylic acid (SA) have been implicated in the response of plants to biotic^1–3 and abiotic stressors.^4–6 SA was identified as a central regulator of local defense against (hemi)biotophic pathogens inducing a hypersensitive response (HR), which is characterized by the development of lesions that restrict pathogen spread. It has also emerged as a possible signaling component involved in the activation of certain plant defense responses in non-infected part of the plants establishing the systemic acquired resistance (SAR).⁷The SA-induced biotic and abiotic stress adaptation most likely involves reactive oxygen species (ROS) and nitric oxide (NO) in primary signaling events that activate multiple signal transduction pathways. SA-induced ROS is required for the activation of antioxidant defense mechanisms⁴ and if the generation of ROS exceeds the capacity of antioxidant systems, the cells die.⁸ NO is another important player that is required for the induction of defense mechanisms⁹ or for ROS-induced cell death.¹⁰Accumulation of SA, and two other plant hormones, ethylene (ET) and jasmonic acid (JA) are intimately associated with the initiation or spread of cell death. In HR SA and ROS have been proposed to be on a positive feedback loop that amplifies signals and leads to programmed cell death (PCD). Ethylene caused increased spreading of cell death, while lesion containment can be achieved by JA through decreasing the sensitivity of the cells to ethylene and through the suppression of SA biosynthesis and signaling.⁸Ethylene evolution is associated with diverse physiological processes such as leaf and flower senescence, abscission of organs and fruit ripening.¹¹ The biosynthesis of ethylene is stimulated by a variety of abiotic and biotic stress factors. Ethylene overproducing mutants (eto1 and eto3) of Arabidopsis were found to be more sensitive to O₃, an abiotic stressor which induces ROS-dependent cell death.¹² Cadmium-induced cell death was also accompanied by increased production of ethylene and simultaneously by H₂O₂ accumulation in tomato cell suspension, and based on the effect of specific inhibitors of ethylene biosynthesis and action the authors concluded that the cell death process required H₂O₂ production and a functional ethylene signaling pathway.¹³ Ethylene signaling is also required for the susceptible disease response of tomato plants infected with Xanthomonas campestris pv vesicatoria.¹⁴ It was found that the accumulation of SA and increased production of ethylene were important components of the disease symptoms of this pathogen in wild-type plants, while in Never ripe (Nr) mutants, which have a non-functional ethylene receptor, the infected plants failed to accumulate SA, produced less ethylene, and the leaves exhibited reduced necrotic lesions.It has been also shown that SA enhances NO synthesis in a dose-dependent manner.¹⁵ ROS, such as ^·O₂⁻ and H₂O₂ as well as NO can act together in the cell death regulation and propagation.^8,16 The compartment-specific (down)regulation of ROS can be controlled by NO, accordingly, ROS and NO homeostasis may be essential for the induction or for the avoidance of cell death.     

Lack of K-Dependent Oxidative Stress in Cotton Roots Following Coronatine-Induced ROS Accumulation

Coronatine [COR] is a novel type of plant growth regulator with similarities in structure and property to jasmonate. The objective of this study was to examine the relationship between increased root vitality induced by 10nM COR and reactive oxygen species scavenging under potassium (K)-replete (2.5mM) and K-deficient (0.05mM) conditions in hydroponic cultured cotton seedlings. K-replete and K-deficient conditions increased root vitality by 2.7- and 3.5-fold, respectively. COR treatment significantly decreased lipid peroxidation in cotton seedlings determined by reduction in MDA levels. These results suggest that COR improves the functioning of both enzymatic and non-enzymatic antioxidant systems. Under K-replete and K-deficient conditions, COR significantly increased the activities of antioxidant enzymes SOD (only for K-repletion), CAT, GPX, and APX comparing; COR also significantly increased DPPH-radical scavenging activity. However, COR led to 1.6- and 1.7-fold increases in superoxide anion (O₂^•-) concentrations, and 5.7- and 2.1-fold increases in hydrogen peroxide (H₂O₂) levels, respectively. Additionally, COR intensified the DAB staining of H₂O₂ and the NBT staining of O₂^•-. Therefore, our results reveal that COR-induced ROS accumulation stimulates the activities of most antioxidant enzymes but does not induce oxidative stress in cotton roots.