Formation of NO and H2O2 in endometrial stromal cells under the effect of acetylcholine

While conducting the experimental work the possibility both the basal and activated by acetylcholine NO and H2O2 production by uterus endometrium stromal cells was estimated. The results obtained give a ground to suppose the existence of two types of cholinoreceptors in the tesyed cells via which two signal transduction ways are fulfilled. The concentrational and temporal dependancy of these metabolites synthesis have a cyclic character. The hypothesis is revealed about a principal possibility of NO and N2O2 to come forward in the role of secondary messengers of the endometrium stromal cells as well as prrovide for the paracrine regulation of myometrium functioning.     

ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-induced stomatal closure.     

植物MPK信号通路与信号分子H2O2和NO之间的相互关系

促分裂原活化蛋白激酶(MPK)级联途径是真核细胞中普遍存在且保守的信号传导通路,广泛参与植物生长发育和植物抵抗生物和非生物胁迫的防御反应。过氧化氢(H2O2)和一氧化氮(NO)作为重要的信使分子也广泛参与植物生长发育和防御反应的信号传导。近年来,研究也表明MPK信号通路与信号分子H2O2和NO之间存在着多种复杂的关系。一方面,在一些刺激的信号传递过程中,MPK信号通路参与了信号分子H2O2和NO的产生、清除或其信号的向下传递等过程;另一方面,在有些刺激的信号传递过程中,它们位于不同的信号传递途经中,行使不同的功能。本文就目前植物MPK信号通路与H2O2和NO之间相互关系的研究现状进行了综述和分析,并指出了该研究领域存在的问题。     

Hydrogen peroxide as a paracrine vascular mediator: regulation and signaling leading to dysfunction

Numerous studies have demonstrated the ability of a variety of vascular cells, including endothelial cells, smooth muscle cells, and fibroblasts, to produce reactive oxygen species (ROS). Until recently, major emphasis was placed on the production of superoxide anion (O(2)(-)) in the vasculature as a result of its ability to directly attenuate the biological activity of endothelium-derived nitric oxide (NO). The short half-life and radius of diffusion of O(2)(-) drastically limit the role of this ROS as an important paracrine hormone in vascular biology. On the contrary, in recent years, the O(2)(-) metabolite hydrogen peroxide (H(2)O(2)) has increasingly been viewed as an important cellular signaling agent in its own right, capable of modulating both contractile and growth-promoting pathways with more far-reaching effects. In this review, we will assess the vascular production of H(2)O(2), its regulation by endogenous scavenger systems, and its ability to activate a variety of vascular signaling pathways, thereby leading to vascular contraction and growth. This discussion will include the ability of H(2)O(2) to (i) initiate calcium flux as well as (ii) stimulate pathways leading to sensitization of contractile elements to calcium. The latter involves a variety of protein kinases that have also been strongly implicated in vascular hypertrophy. Previous intensive study has emphasized the ability of NADPH oxidase-derived O(2)(-) and H(2)O(2) to activate these pathways in cultured smooth muscle cells. However, growing evidence indicates a considerably more complex array of unique oxidase systems in the endothelium, media, and adventitia that appear to participate in these deleterious effects in a sequential and temporal manner. Taken together, these findings seem consistent with a paracrine effect of H(2)O(2) across the vascular wall.     

Sphingolipid signaling and redox regulation

Sphingolipids including ceramide and its derivatives such as ceramide-1-phosphate, glycosyl-ceramide, and sphinogosine (-1-phosphate) are now recognized as novel intracellular signal mediators for regulation of inflammation, apoptosis, proliferation, and differentiation. One of the important and regulated steps in these events is the generation of these sphingolipids via hydrolysis of sphingomyelin through the action of sphingomyelinases (SMase). Several lines of evidence suggest that reactive oxygen species (ROS; O2-, H2O2, and OH-,) and reactive nitrogen species (RNS; NO, and ONOO-) and cellular redox potential, which is mainly regulated by cellular glutathione (GSH), are tightly linked to the regulation of SMase activation. On the other hand, sphingolipids are also known to play an important role in maintaining cellular redox homeostasis through regulation of NADPH oxidase, mitochondrial integrity, and antioxidant enzymes. Therefore, this paper reviews the relationship between cellular redox and sphingolipid metabolism and its biological significance.     

Nitric oxide inhibition of ERK1/2 activity in cells expressing neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (nNOS) is a constitutively expressed enzyme responsible for the production of nitric oxide (NO*) from l-arginine and O2. Nitric oxide is an intra- and intercellular messenger that mediates a diversity of signaling pathways in target cells. In the absence of l-arginine, nNOS has been shown to generate superoxide (O2*). Superoxide, either directly or through its self-dismutation to H2O2, is likewise believed to be a cell-signaling agent. Because nNOS can generate NO* and O2*, we examined the activation of cellular signal transduction pathways in nNOS-transfected cells grown in the presence or absence of l-arginine. Spin trapping/EPR spectroscopy confirmed that stimulated nNOS-transfected cells grown in an l-arginine environment secreted NO* into the surrounding milieu. Production of NO* blocked Ca2+ ionophore-induced activation of the ERK1/2 through a mechanism involving inhibition of the Ras G-protein and Raf-1 kinase. In contrast, ERK activation was largely unaffected in nNOS-transfected cells grown in l-arginine-free media. Inhibition of nNOS-generated NO* with the competitive NOS inhibitor, NG-nitro-l-arginine methyl ester, in cells grown in l-arginine restored ERK1/2 activation to levels similar to that found when nNOS was activated in l-arginine-free media. These findings indicate that nNOS can differentially regulate the ERK signal transduction pathway in a manner dependent on the presence of l-arginine and the production of NO*.     

Superoxide fluxes limit nitric oxide-induced signaling

Independently, superoxide (O2-) and nitric oxide (NO) are biologically important signaling molecules. When co-generated, these radicals react rapidly to form powerful oxidizing and nitrating intermediates. Although this reaction was once thought to be solely cytotoxic, herein we demonstrate using MCF7, macrophage, and endothelial cells that when nanomolar levels of NO and O2- were produced concomitantly, the effective NO concentration was established by the relative fluxes of these two radicals. Differential regulation of sGC, pERK, HIF-1alpha, and p53 were used as biological dosimeters for NO concentration. Introduction of intracellular- or extracellular-generated O2- during NO generation resulted in a concomitant increase in oxidative intermediates with a decrease in steady-state NO concentrations and a proportional reduction in the levels of sGC, ERK, HIF-1alpha, and p53 regulation. NO responses were restored by addition of SOD. The intermediates formed from the reactions of NO with O2- were non-toxic, did not form 3-nitrotyrosine, nor did they elicit any signal transduction responses. H2O2 in bolus or generated from the dismutation of O2- by SOD, was cytotoxic at high concentrations and activated p53 independent of NO. This effect was completely inhibited by catalase, suppressed by NO, and exacerbated by intracellular catalase inhibition. We conclude that the reaction of O2- with NO is an important regulatory mechanism, which modulates signaling pathways by limiting steady-state levels of NO and preventing H2O2 formation from O2-.     

NO可能作为H2O2的下游信号介导ABA诱导的蚕豆气孔关闭

ABA、H2O2和硝普钠(SNP)均能诱导蚕豆气孔关闭.NO的清除剂c-PTIO可以减轻由ABA或H2O2所诱导的蚕豆气孔关闭的程度,而过氧化氢酶(CAT)则不能减轻NO诱导的气孔关闭程度.激光共聚焦显微检测结果显示,10μmo1/L的ABA处理后,胞内H2O2的产生速率明显高于NO的产生速率;CAT几乎可完全抑制ABA所诱导的DAF的荧光增加;外源H2O2能显著诱导胞内DAF的荧光增加;c-PTIO对ABA诱导的DCF荧光略有促进作用,但外源SNP不能诱导胞内DCF荧光增加.这些结果表明,在ABA诱导气孔关闭过程中,H2O2可能在NO的上游起作用并受NO的负反馈调节.     

大丽轮枝菌毒素与拟南芥互作反应中SA、NO与H2O2信号的相关性

本文研究了大丽轮枝菌毒素（VD-toxin)与拟南芥互作反应中外源SA、NO供体、NO合酶抑制剂等对拟南芥幼苗H2O2含量的影响,并对H2O2的积累部位进行了DAB组化染色检测。大丽轮枝菌毒素、外源SA、NO供体处理拟南芥幼苗均能诱导H2O2的积累,NO供体的诱导作用最强;NO合酶抑制剂处理则未表现出H2O2含量的增强;H2O2的积累部位主要在叶片的表皮毛和维管束组织。结果表明,在大丽轮枝菌毒素与拟南芥互作反应中,H2O2可能作为信号分子参与了SA和NO调控的拟南芥防卫反应,NO信号与H2O2信号间的关系可能更密切。     

Inhibitory and enhancing effects of NO on H(2)O(2) toxicity: dependence on the concentrations of NO and H(2)O(2)

Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H(2)O(2)) toxicity and protect cells against H(2)O(2) toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H(2)O(2) toxicity in cultured liver endothelial cells over a wide range of NO and H(2)O(2) concentrations. NO was generated by spermine NONOate (SpNO, 0.001-1 mM), H(2)O(2) was generated continuously by glucose/glucose oxidase (GOD, 20-300 U/l), or added as a bolus (200 microM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H(2)O(2)-induced cell death. SpNO concentrations >0.1 mM were injurious with low H(2)O(2) concentrations, but protective at high H(2)O(2) concentrations. Protection appeared to be mainly due to inhibition of lipid peroxidation, for which SpNO concentrations as low as 0.01 mM were sufficient. SpNO in high concentration (1 mM) consistently raised H(2)O(2) steady-state levels in line with inhibition of H(2)O(2) degradation. Thus, the overall effect of NO on H(2)O(2) toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H(2)O(2) levels and enhancement being predominant with high NO and low H(2)O(2) levels.     

Extracellular ATP-induced NO production and its dependence on membrane Ca2+ flux in Salvia miltiorrhiza hairy roots

Extracellular ATP (eATP) is a novel signalling agent, and nitric oxide (NO) is a well-established signal molecule with diverse functions in plant growth and development. This study characterizes NO production induced by exogenous ATP and examines its relationship with other important signalling agents, Ca(2+) and H(2)O(2) in Salvia miltiorrhiza hairy root culture. Exogenous ATP was applied at 10-500 microM to the hairy root cultures and stimulated NO production was detectable within 30 min. The NO level increased with ATP dose from 10-100 microM but decreased from 100-200 muM or higher. The ATP-induced NO production was mimicked by a non-hydrolysable ATP analogue ATPgammaS, but only weakly by ADP, AMP or adenosine. The ATP-induced NO production was blocked by Ca(2+) antagonists, but not affected by a protein kinase inhibitor. ATP also induced H(2)O(2) production, which was dependent on both Ca(2+) and protein kinases, and also on NO biosynthesis. On the other hand, ATP induced a rapid increase in the intracellular Ca(2+) level, which was dependent on NO but not H(2)O(2). The results suggest that NO is implicated in ATP-induced responses and signal transduction in plant cells, and ATP signalling is closely related to Ca(2+) and ROS signalling.     

Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium

The integrity of microvascular endothelium is an important regulator of myocardial contractility. Microvascular barrier integrity could be altered by increased reactive oxygen species (ROS) stress seen within minutes after cardiac arrest resuscitation. Akt and its downstream target nitric oxide (NO) synthase (NOS)3 can protect barrier integrity during ROS stress, but little work has studied these oxidant stress responses in human cardiac microvascular endothelial cells (HCMVEC). We, therefore, studied how ROS affects barrier function and NO generation via Akt and its downstream target NOS3 in HCMVEC. HCMVEC exposed to 500 microM H2O2 had increased Akt phosphorylation within 10 min at both Ser-473 and Thr-308 sites, an effect blocked by the phosphatidylinositol 3-kinase inhibitor LY-294002. H2O2 also induced NO generation that was associated with NOS3 Ser-1177 site phosphorylation and Thr-495 dephosphorylation, with Ser-1177 effects attenuated by LY-294002 and an Akt inhibitor, Akt/PKB signaling inhibitor-2 (API-2). H2O2 induced significant barrier disruption in HCMVEC within minutes, but recovery started within 30 min and normalized over hours. The NOS inhibitor Nomega-nitro-L-arginine methyl ester (200 microM) blocked NO generation but had no effect on H2O2-induced barrier permeability or the recovery of barrier integrity. By contrast, the Akt inhibitor API-2 abrogated HCMVEC barrier restoration. These results suggest that oxidant stress in HCMVEC activates NOS3 via Akt. NOS3/NO are not involved in the regulation of H2O2-affected barrier function in HCMVEC. Independent of NOS3 regulation, Akt proves to be critical for the restoration of barrier integrity in HCMVEC.     

Mechanisms of acetylcholine-dependent production of H2O2 and NO2- by stromal cells of endometrium

The metabolism of NO(NO2-) and H2O2(O2-) by stroma cells of pig endometrium is NAD(P)H and glutathione-dependent process. The efficiency of biosynthesis and utilization of these metabolites appreciably depends on the state of SH-groups of the conforming ferment systems. And the reversible oxidation of SH-groups (maybe by the reaction products) results in the drop of biosynthesis rate. The NO and H2O2 metabolism is also defined by the state of oxidative metabolism of arachidonic acid (depending on salicylate), and also intensity of a course of redox-processes on plasmalemma (is regulated by cytochrome c). The NO2- biosynthesis by stroma cells is strongly inhibited by the agents, which super produce H2O2(O2-) (salicylate and cytochrome c). The NO(NO2-) and H2O2(O2-) metabolism at stimulation by acetylcholine is of cyclic character, and the infringement of any link during biosynthesis or utilization of these compositions results in losses of cyclicity. In contrast to this the formation of nitrosoglutathione with time achieves the saturation, which reflects its buffer and depositing with respect to NO function and permits to consider formation of the latter as one of mechanisms of effective utilization of NO(NO2-)--by the stroma cells.     

Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging

The production of NO by heart mitochondria was 0.7-1.1 nmol NO/min.mg protein, an activity similar to the ones observed in mitochondrial membranes from other organs. Heart mtNOS seems to contribute with about 56% of the total cellular NO production. The immunological nature of the mtNOS isoform of cardiac tissue remains unclear; in our laboratory, heart mtNOS reacted with an anti-iNOS anti-body. Heart mtNOS expression and activity are regulated by physiological and pharmacological effectors. The state 4/state 3 transition regulates heart mtNOS activity and NO release in intact respiring mitochondria: NO production rates in state 3 were 40% lower than in state 4. Heart mtNOS expression was selectively regulated by O(2) availability in hypobaric conditions and the activity was 20-60% higher in hypoxic rats than in control animals, depending on age. In contrast, NADH-cytochrome c reductase and cytochrome oxidase activities were not affected by hypoxia. The activity of rat heart mtNOS decreased 20% on aging from 12 to 72 weeks of age. On the pharmacological side, mitochondrial NO production was increased after enalapril treatment (the inhibitor of the angiotensin converting enzyme) with modification of heart mtNOS functional activity in the regulation of mitochondrial O(2) uptake and H(2)O(2) production. Thus, heart mtNOS is a highly regulated mitochondrial enzyme, which in turn, plays a regulatory role through mitochondrial NO steady state levels that modulate O(2) uptake and O(2)(-) and H(2)O(2) production rates. Nitric oxide and H(2)O(2) constitute signals for metabolic control that are involved in the regulation of cellular processes, such as proliferation and apoptosis.     

Regulation of peroxiredoxins by nitric oxide in immunostimulated macrophages

Reactive oxygen species and nitric oxide (NO) are capable of both mediating redox-sensitive signal transduction and eliciting cell injury. The interplay between these messengers is quite complex, and intersection of their signaling pathways as well as regulation of their fluxes requires tight control. In this regard, peroxiredoxins (Prxs), a recently identified family of six thiol peroxidases, are central because they reduce H2O2, organic peroxides, and peroxynitrite. Here we provide evidence that endogenously produced NO participates in protection of murine primary macrophages against oxidative and nitrosative stress by inducing Prx I and VI expression at mRNA and protein levels. We also show that NO prevented the sulfinylation-dependent inactivation of 2-Cys Prxs, a reversible overoxidation that controls H2O2 signaling. In addition, studies using macrophages from sulfiredoxin (Srx)-deficient mice indicated that regeneration of 2-Cys Prxs to the active form was dependent on Srx. Last, we show that NO increased Srx expression and hastened Srx-dependent recovery of 2-Cys Prxs. We therefore propose that modulation by NO of Prx expression and redox state, as well as up-regulation of Srx expression, constitutes a novel pathway that contributes to antioxidant response and control of H2O2-mediated signal transduction in mammals.     

Nitric oxide and reactive oxygen species do not elicit hypersensitive cell death but induce apoptosis in the adjacent cells during the defense response of oat

Nitric oxide (NO) acts as a signaling molecule in many cellular responses in plants and animals. Oat plants (Avena sativa L.) evoke the hypersensitive response (HR), which shares morphological and biochemical features with mammalian apoptosis, such as DNA laddering and heterochromatin condensation, in response to the avirulent crown rust fungus (Puccinia coronata f. sp. avenae). We examined the role of NO and reactive oxygen species (ROS) in the initiation of hypersensitive cell death, which is induced by direct contact with the pathogen, and apoptotic cell death in the adjacent cells. Cytofluorimetric analysis using the fluorescent NO probe DAF and the H2O2 probe DCF demonstrated that NO and H2O2 were generated simultaneously in primary leaves at an early stage of the defense response. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) markedly enhanced H2O2 accumulation detected by 3,3-diaminobenzidine staining and DCF, whereas treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) strongly suppressed it. Superoxide dismutase (SOD) increased NO accumulation, suggesting that endogenous NO may modulate the level of H2O2 by interacting with O2- in the HR lesion. Cytological observation showed that administration of cPTIO, SNAP, or SOD had no effect on elicitation of hypersensitive cell death, but clearly reduced heterochromatin condensation in the nearby cells and DNA laddering. These findings indicate that NO and ROS are not essential mediators for the initiation of hypersensitive cell death. However, NO and O2- but not H2O2 are required for the onset of apoptotic cell death in the adjacent cells, where excess NO may exert its anti-apoptotic function by regulating cellular redox state.     

Redox modulation of protein kinase/phosphatase balance in melanoma cells: the role of endogenous and gamma-glutamyltransferase-dependent H2O2 production

Alterations of protein kinase and protein phosphatase activities have been described in a number of tumors. Redox changes, such as in conditions of oxidant stress, have been reported to affect the cellular protein kinase/phosphatase balance. A basal production of reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)), exists in tumor cells, and the membrane-bound ecto-enzyme gamma-glutamyltransferase (GGT)-overexpressed in a variety of malignant tumors-is one of the mechanisms capable of promoting such a production. The present study was aimed to verify the interactions of GGT activity with protein phosphatase and kinase activities in Me665/2/60 melanoma cells, expressing high levels of this enzyme and exhibiting both basal and GGT-dependent production of hydrogen peroxide. An increase of total phosphatase as well as tyrosine phosphatase activities was observed after treatment of cells with both micromolar H(2)O(2) and GGT stimulation. Accordingly, stimulation of GGT resulted in decreased levels of phosphotyrosine. On the other hand, when serine/threonine phosphatase activities were selectively analyzed, both H(2)O(2) treatment and GGT stimulation caused their down-regulation.The data reported suggest that basal conditions of oxidant stress in melanoma may represent a factor contributing to the redox regulation of protein phosphorylation, and that GGT-mediated prooxidant reactions may participate in the process. As basal oxidant stress and expression of GGT activity are present in a variety of malignant tumors besides melanoma, these phenomena likely represent general mechanisms participating in the alteration of intracellular transduction during carcinogenesis.