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     

Participation of Nitric Oxide in 24-Epibrassinolide-Induced Heat Resistance of Wheat Coleoptiles: Functional Interactions of Nitric Oxide with Reactive Oxygen Species and Ca Ions

Nitric oxide (NO) effects on heat resistance of wheat (Triticum aestivum L.) coleoptiles induced by 24-epibrassinolide (24-EB) have been investigated. Coleoptiles’ survival after damaging heating (43°С, 10 min) increased when they were treated preliminarily with 5–200 nM of 24-EB. After 24-EB treatment, transient amplification of nitric oxide (NO) and also ROS (superoxide anion-radical (O ₂ ^?? ) and hydrogen peroxide) generation by coleoptiles was noted. Coleoptiles pretreatment with inhibitors of nitrate reductase and an enzyme similar to animal NO-synthase partially removed the increase of NO content caused by the action of 24-EB. Amplification of superoxide anion-radical generation caused by 24-EB was depressed under the influence of imidazole (NADPH-oxidase inhibitor). Calcium antagonists (EGTA and neomycin) largely neutralized the 24-EB-induced increase in generation of both O ₂ ^?? and NO. The increase in NO content in coleoptile tissues caused by 24-EB was almost completely leveled by antioxidants and partly by imidazole. 24-EB-induced enhancement of the superoxide anion-radical generation was partially suppressed by the action of NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and the inhibitors of nitrate reductase and an enzyme similar to animal NO-synthase. Positive 24-EB effect on the heat resistance of wheat coleoptiles was leveled by PTIO, inhibitors of enzymes that generate NO, antioxidants, an inhibitor of NADPH-oxidase imidazole, and calcium antagonists. A conclusion was made on the role of NO in brassinosteroid signal transduction inducing heat resistance development of coleoptiles and on the functional interaction between NO, ROS, and calcium ions as the signal mediators.     

Role of Nitric Oxide in Methamphetamine Neurotoxicity: Protection by 7-Nitroindazole, an Inhibitor of Neuronal Nitric Oxide Synthase

Abstract: The role of nitric oxide (NO^•) in the neurotoxic effects of methamphetamine (METH) was evaluated using 7-nitroindazole (7-NI), a potent inhibitor of neuronal nitric oxide synthase. Treatment of mice with 7-NI (50 mg/kg) almost completely counteracted the loss of dopamine, 3,4-dihydroxyphenylacetic acid, and tyrosine hydroxylase immunoreactivity observed 5 days after four injections of 10 or 7.5 mg/kg METH. With the higher dose of METH, this protection at 5 days occurred despite the fact that combined administration of METH and 7-NI significantly increased lethality and exacerbated METH-induced dopamine release (as indicated by a greater dopamine depletion at 90 min and 1 day). Combined treatment with 4 × 10 mg/kg METH and 7-NI also slightly increased the body temperature of mice as compared with METH alone. Thus, the neuroprotective effects of 7-NI are independent from lethality, are not likely to be related to a reduction of METH-induced dopamine release, and are not due to a decrease in body temperature. These results indicate that NO^• formation is an important step leading to METH neurotoxicity, and suggest that the cytotoxic properties of NO^• may be directly involved in dopaminergic terminal damage.     

Reactive Oxygen Species-Dependent Nitric Oxide Production Contributes to Hydrogen-Promoted Stomatal Closure in Arabidopsis

The signaling role of hydrogen gas (H₂) has attracted increasing attention from animals to plants. However, the physiological significance and molecular mechanism of H₂ in drought tolerance are still largely unexplored. In this article, we report that abscisic acid (ABA) induced stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering intracellular signaling events involving H₂, reactive oxygen species (ROS), nitric oxide (NO), and the guard cell outward-rectifying K⁺ channel (GORK). ABA elicited a rapid and sustained H₂ release and production in Arabidopsis. Exogenous hydrogen-rich water (HRW) effectively led to an increase of intracellular H₂ production, a reduction in the stomatal aperture, and enhanced drought tolerance. Subsequent results revealed that HRW stimulated significant inductions of NO and ROS synthesis associated with stomatal closure in the wild type, which were individually abolished in the nitric reductase mutant nitrate reductase1/2 (nia1/2) or the NADPH oxidase-deficient mutant rbohF (for respiratory burst oxidase homolog). Furthermore, we demonstrate that the HRW-promoted NO generation is dependent on ROS production. The rbohF mutant had impaired NO synthesis and stomatal closure in response to HRW, while these changes were rescued by exogenous application of NO. In addition, both HRW and hydrogen peroxide failed to induce NO production or stomatal closure in the nia1/2 mutant, while HRW-promoted ROS accumulation was not impaired. In the GORK-null mutant, stomatal closure induced by ABA, HRW, NO, or hydrogen peroxide was partially suppressed. Together, these results define a main branch of H₂-regulated stomatal movement involved in the ABA signaling cascade in which RbohF-dependent ROS and nitric reductase-associated NO production, and subsequent GORK activation, were causally involved.Stomata are responsible for leaves of terrestrial plants taking in carbon dioxide for photosynthesis and likewise regulate how much water plants evaporate through the stomatal pores (Chaerle et al., 2005). When experiencing water-deficient conditions, surviving plants balance photosynthesis with controlling water loss through the stomatal pores, which relies on turgor changes by pairs of highly differentiated epidermal cells surrounding the stomatal pore, called the guard cells (Haworth et al., 2011; Loutfy et al., 2012).Besides the characterization of the significant roles of abscisic acid (ABA) in regulating stomatal movement, the key factors in guard cell signal transduction have been intensively investigated by performing forward and reverse genetics approaches. For example, both reactive oxygen species (ROS) and nitric oxide (NO) have been identified as vital intermediates in guard cell ABA signaling (Bright et al., 2006; Yan et al., 2007; Suzuki et al., 2011; Hao et al., 2012). The key ROS-producing enzymes in Arabidopsis (Arabidopsis thaliana) guard cells are the respiratory burst oxidase homologs (Rboh) D and F (Kwak et al., 2003; Bright et al., 2006; Mazars et al., 2010; Marino et al., 2012). Current available data suggest that there are at least two distinct pathways responsible for NO synthesis involved in ABA signaling in guard cells: the nitrite reductase (NR)- and l-Arg-dependent pathways (Desikan et al., 2002; Besson-Bard et al., 2008). Genetic evidence further demonstrated that removal of the major known sources of either ROS or NO significantly impairs ABA-induced stomatal closure. ABA fails to induce ROS production in the atrbohD/F double mutant (Kwak et al., 2003; Wang et al., 2012) and NO synthesis in the NR-deficient mutant nitrate reductase1/2 (nia1/2; Bright et al., 2006; Neill et al., 2008), both of which lead to impaired stomatal closure in Arabidopsis. Most importantly, ROS and NO, which function both synergistically and independently, have been established as ubiquitous signal transduction components to control a diverse range of physiological pathways in higher plants (Bright et al., 2006; Tossi et al., 2012).The guard cell outward-rectifying K⁺ channel (GORK) encodes the exclusive voltage-gated outwardly rectifying K⁺ channel protein, which was located in the guard cell membrane (Ache et al., 2000; Dreyer and Blatt, 2009). Expression profiles revealed that this gene is up-regulated upon the onset of drought, salinity, and cold stress and ABA exposure (Becker et al., 2003; Tran et al., 2013). Reverse genetic evidence further showed that GORK plays an important role in the control of stomatal movements and allows the plant to reduce transpirational water loss significantly (Hosy et al., 2003) and participates in the regulation of salinity tolerance by preventing salt-induced K⁺ loss (Jayakannan et al., 2013). Due to the high complexity of guard cell signaling cascades, whether and how ABA-triggered GORK up-regulation is attributed to the generation of cellular secondary messengers, such as ROS and NO, is less clear.Hydrogen gas (H₂) was recently revealed as a signaling modulator with multiple biological functions in clinical trails (Ohsawa et al., 2007; Itoh et al., 2009; Ito et al., 2012). It was previously found that a hydrogenase system could generate H₂ in bacteria and green algae (Meyer, 2007; Esquível et al., 2011). Although some earlier studies discovered the evolution of H₂ in several higher plant species (Renwick et al., 1964; Torres et al., 1984), it was also proposed that the eukaryotic hydrogenase-like protein does not metabolize H₂ (Cavazza et al., 2008; Mondy et al., 2014). Since the explosion limit of H₂ gas is about 4% to 72.4% (v/v, in the air), the direct application of H₂ gas in experiments is flammable and dangerous. Regardless of these problems to be resolved, the methodology, such as using exogenous hydrogen-rich water (HRW) or hydrogen-rich saline, which is safe, economical, and easily available, provides a valuable approach to investigate the physiological function of H₂ in animal research and clinical trials. For example, hydrogen dissolved in Dulbecco’s modified Eagle’s medium was found to react with cytotoxic ROS and thus protect against oxidative damage in PC12 cells and rats (Ohsawa et al., 2007). The neuroprotective effect of H₂-loaded eye drops on retinal ischemia-reperfusion injury was also reported (Oharazawa et al., 2010). In plants, corresponding results by using HRW combined with gas chromatography (GC) revealed that H₂ could act as a novel beneficial gaseous molecule in plant responses against salinity (Xie et al., 2012; Xu et al., 2013), cadmium stress (Cui et al., 2013), and paraquat toxicity (Jin et al., 2013). More recently, the observation that HRW could delay the postharvest ripening and senescence of kiwifruit (Actinidia deliciosa) was reported (Hu et al., 2014).Considering the fact that the signaling cascades for salt, osmotic, and drought stresses share a common cascade in an ABA-dependent pathway, it would be noteworthy to identify whether and how H₂ regulates the bioactivity of ABA-induced downstream components and, thereafter, biological responses, including stomatal closure and drought tolerance. To resolve these scientific questions, rbohD, rbohF, nia1/2, nitric oxide associated1 (noa1; Van Ree et al., 2011), nia1/2/noa1, and gork mutants were utilized to investigate the relationship among H₂, ROS, NO, and GORK in the guard cell signal transduction network. By the combination of pharmacological and biochemical analyses with this genetics-based approach, we provide comprehensive evidence to show that H₂ might be a newly identified bioeffective modulator involved in ABA signaling responsible for drought tolerance, that HRW-promoted stomatal closure was mainly attributed to the modulation of ROS-dependent NO generation, and that GORK might be the downstream target protein of H₂ signaling.     

活性氧中间体和NO在植物抗病中的作用

植物与病原菌互作时,活性氧中间体(reactiveoxygenintermediates,ROI)和一氧化氮(NO)参与了植物抗病性的建立。寄主与病原菌非亲合性互作产生二次氧爆发高峰,体内NO增加。许多氧化酶可以催化氧爆发产生ROI。ROI和NO通过氧化还原信号启动寄主细胞局部的过敏性坏死反应和全株系统获得性抗病性。     

Involvement of Oxygen Free Radicals in Ischaemia-Reperfusion Injury to Murine Turnours: Role of Nitric Oxide

Ischaemia-reperfusion (I/R) injury is a model system of oxidative stress and a potential anti-cancer therapy. Tumour cytotoxicity follows oxygen radical damage to the vasculature which is modulated by tumour production of the vasoactive agent, nitric oxide (NO*). in vivo hydroxylation of salicylate, to 2,3- and 2,5-dihydroxybenzoate (DHBs), was used to measure the generation of hydroxyl radicals (OH*) following temporary vascular occlusion in two murine tumours (with widely differing capacity to produce NO*) and normal skin. Significantly greater OH* generation followed I/R of murine adenocarcinoma CaNT tumours (low NO* production) compared to round cell sarcoma SaS tumours (high NO* production) and normal skin. These data suggest that tumour production of NO* confers resistance to I/R injury, in part by reducing production of oxygen radicals and oxidative stress to the vasculature. Inhibition of NO synthase (NOS), during vascular reperfusion, significantly increased OH* generation in both tumour types, but not skin. This increase in cytotoxicity suggests oxidative injury may be attenuation by tumour production of NO*. Hydroxyl radical generation following I/R injury correlated with vascular damage and response of tumours in vivo, but not skin, which indicates a potential therapeutic benefit from this approach.     

Nitric Oxide Transport in Normal Human Thoracic Aorta: Effects of Hemodynamics and Nitric Oxide Scavengers

Despite the crucial role of nitric oxide (NO) in the homeostasis of the vasculature, little quantitative information exists concerning NO transport and distribution in medium and large-sized arteries where atherosclerosis and aneurysm occur and hemodynamics is complex. We hypothesized that local hemodynamics in arteries may govern NO transport and affect the distribution of NO in the arteries, hence playing an important role in the localization of vascular diseases. To substantiate this hypothesis, we presented a lumen/wall model of the human aorta based on its MRI images to simulate the production, transport and consumption of NO in the arterial lumen and within the aortic wall. The results demonstrated that the distribution of NO in the aorta was quite uneven with remarkably reduced NO bioavailability in regions of disturbed flow, and local hemodynamics could affect NO distribution mainly via flow dependent NO production rate of endothelium. In addition, erythrocytes in the blood could moderately modulate NO concentration in the aorta, especially at the endothelial surface. However, the reaction of NO within the wall could only slightly affect NO concentration on the luminal surface, but strongly reduce NO concentration within the aortic wall. A strong positive correlation was revealed between wall shear stress and NO concentration, which was affected by local hemodynamics and NO reaction rate. In conclusion, the distribution of NO in the aorta may be determined by local hemodynamics and modulated differently by NO scavengers in the lumen and within the wall.     

Nitric Oxide: A Unique Endogenous Signaling Molecule in Vascular Biology

The properties of nitric oxide as an endogenous cell signaling molecule in vascular biology are described.     

NMDA Receptor Activation Produces Concurrent Generation of Nitric Oxide and Reactive Oxygen Species: Implications for Cell Death

Abstract: The ability of glutamate to stimulate generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescent dye 2,7-dichlorofluorescin (DCF). Exposure of cells to glutamate (10 µM) produced a rapid generation of oxidants that was blocked ～70% by MK-801 (a noncompetitive NMDA-receptor antagonist). To determine if nitric oxide (NO) or reactive oxygen species (ROS) contributed to the oxidation of DCF, cells were treated with compounds that altered their generation. NO production was inhibited with N^G-nitro-l -arginine methyl ester (l -NAME) (nitric oxide synthase inhibitor) and reduced hemoglobin (NO scavenger). Alternatively, cells were incubated with superoxide dismutase (SOD) and catalase, which selectively metabolize O₂^?· andH₂O₂. Concurrent inhibition of O₂^?· and NO production nearly abolished intracellular oxidant generation. Pretreatment of cells with either chelerythrine (1 µM, protein kinase C inhibitor) or quinacrine (5 µM, phospholipase A₂ inhibitor) before addition of glutamate also blocked oxidation of DCF. Generation of oxidants by glutamate was significantly reduced by incubating the cells in Ca²⁺-free buffer. In cytotoxicity studies, a positive correlation was observed between glutamate-induced death and oxidant generation. Glutamate-induced cytotoxicity was blocked by MK-801 and attenuated by treatment with l -NAME, chelerythrine, SOD, or quinacrine. It is concluded that glutamate induces concurrent generation of NO and ROS by activation of both NMDA receptors and non-NMDA receptors through a Ca²⁺-mediated process. Activation of NO synthase and phospholipaseA₂ contribute significantly to this response. It is proposed that simultaneous generation of NO and ROS results in formation of peroxynitrite, which initiates the cellular damage.     

A Search for the Intermediate Radical, ONOO', in the Reaction Between Oxygen and Nitric Oxide in Solution

The most probable initial reaction between NO and O₂ is direct addition to give the peroxyl radical ONOO. In view of the potential importance of this radical in biology, we have searched extensively for its formation, using EPR spectroscopy and rapid freezing techniques. At best, only extremely low concentrations were detected, and in most systems, no signals were detectable. We conclude that this radical is unlikely to be of major importance per se in biological systems, in contrast with its one electron adduct, the peroxynitrite anion.     

抗黄体酮联合Aromatase抑制剂或iNOS终止恒河猴妊娠

[目的]评价抗黄体酮(mifepristone)联合Aromatase抑制剂(letrozole或aminoglutethimide)或iNOS抑制剂(aminoguandine)是否能有效终止恒河猴早期妊娠。[方法]将30只猴子随机分为5组(治疗组每组6只,对照组6只),并在妊娠30,31和32天进行如下处理:对照组,每只动物1ml安慰剂;A组,Mifepristone(1mg/kg,sc.);B组,Mifepristone(sc.)+Letrozole(2.5mg/只sc.);C组,Mifepristone(1mg/kg,sc.)+aminoglute-chimide(50mg/kgsc.,bid);D组,Mifepristone(1mg/kg,sc.)+aminoguanidine(150mg/kg,sc.,bid)。所有妊娠猴在妊娠29天通过超声波确认。[结果]在B、C、D组,所有的动物的妊娠都在妊娠早期被终止(6/6)。A组和对照组的妊娠终止率分别为3/6和2/6。同时,联合用药能够有效排空子宫腔和减少出血。[结论]该处理能有效地终止恒河猴早期妊娠。联合用药比用于女人的妊娠治疗更有效,并减少了流血时间,或许可以代替目前的终止妊娠的医疗方法。     

Plasma Total Nitric Oxide and Endothelial Constitutive Nitric Oxide Synthase (ecNOS) Gene Polymorphism: A Study in a South Indian Population

In an analysis of the possible association of endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphism and plasma nitric oxide levels in patients with acute coronary syndrome, we investigated 106 patients with the syndrome and 100 healthy controls. Genotype was determined using the polymerase chain reaction; plasma nitric oxide levels were found using ELISA. The genotype frequencies for the a/b polymorphism in the control group were 77% for bb, 19% for ab, and 4% for aa. In the patients, genotype frequencies were 55% bb, 34% ab, and 11% aa. The allele frequencies were 28% a and 72% b among the patients and 13% a and 87% b among control subjects. Our findings showed a significant association of the ecNOS gene polymorphism with acute coronary syndrome in the South Indian population.     

A Novel A3 Group Aconitase Tolerates Oxidation and Nitric Oxide

Achromobacter denitrificans YD35 is an NO₂⁻-tolerant bacterium that expresses the aconitase genes acnA3, acnA4, and acnB, of which acnA3 is essential for growth tolerance against 100 mm NO₂⁻. Atmospheric oxygen inactivated AcnA3 at a rate of 1.6 × 10⁻³ min⁻¹, which was 2.7- and 37-fold lower compared with AcnA4 and AcnB, respectively. Stoichiometric titration showed that the [4Fe-4S]²⁺ cluster of AcnA3 was more stable against oxidative inactivation by ferricyanide than that of AcnA4. Aconitase activity of AcnA3 persisted against high NO₂⁻ levels that generate reactive nitrogen species with an inactivation rate constant of k = 7.8 × 10⁻³ min⁻¹, which was 1.6- and 7.8-fold lower than those for AcnA4 and AcnB, respectively. When exposed to NO₂⁻, the acnA3 mutant (AcnA3Tn) accumulated higher levels of cellular citrate compared with the other aconitase mutants, indicating that AcnA3 is a major producer of cellular aconitase activity. The extreme resistance of AcnA3 against oxidation and reactive nitrogen species apparently contributes to bacterial NO₂⁻ tolerance. AcnA3Tn accumulated less cellular NADH and ATP compared with YD35 under our culture conditions. The accumulation of more NO by AcnA3Tn suggested that NADH-dependent enzymes detoxify NO for survival in a high NO₂⁻ milieu. This novel aconitase is distributed in Alcaligenaceae bacteria, including pathogens and denitrifiers, and it appears to contribute to a novel NO₂⁻ tolerance mechanism in this strain.