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.     

Muscular Dystrophy in mdx Mice Despite Lack of Neuronal Nitric Oxide Synthase

Abstract: Neuronal nitric oxide synthase (nNOS) is a component of the dystrophin complex in skeletal muscle. The absence of dystrophin protein in Duchenne muscular dystrophy and in mdx mouse causes a redistribution of nNOS from the plasma membrane to the cytosol in muscle cells. Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers. To test the hypothesis that derangements in nNOS disposition mediate muscle damage in Duchenne dystrophy, we bred dystrophin-deficient mdx male mice and female mdx heterozygote mice that lack nNOS. We found that genetic deletion of nNOS does not itself cause detectable pathology and that removal of nNOS does not influence the extent of increased sarcolemmal permeability in dystrophin-deficient mice. Thus, histological analyses of nNOS-dystrophin double mutants show pathological changes similar to the dystrophin mutation alone. Taken together, nNOS defects alone do not produce muscular dystrophy in the mdx model.     

Crucial Role for Neuronal Nitric Oxide Synthase in Early Microcirculatory Derangement and Recipient Survival following Murine Pancreas Transplantation

Objective

Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation.

Background

Tetrahydrobiopterin donor treatment has been shown to specifically attenuate these derangements following pancreas transplantation, and tetrahydrobiopterin-mediated protective effects to rely on its NOS-cofactor activity, rather than on its antioxidant capacity. However, the NOS-isoform mainly involved in this process has still to be defined.

Methods

Using a murine pancreas transplantation model, grafts lacking one of the three NOS-isoforms were compared to grafts from wild-type controls. Donors were treated with either tetrahydrobiopterin or remained untreated. All grafts were subjected to 16 h cold ischemia time and transplanted into wild-type recipients. Following 4 h graft reperfusion, microcirculation was analysed by confocal intravital fluorescence microscopy. Recipient survival was monitored for 50 days.

Results

Transplantation of the pancreas from untreated wild-type donor mice resulted in microcirculatory damage of the transplanted graft and no recipient survived more than 72 h. Transplanting grafts from untreated donor mice lacking either endothelial or inducible NOS led to similar outcomes. In contrast, donor treatment with tetrahydrobiopterin prevented microcirculatory breakdown enabling long-term survival. Sole exception was transplantation of grafts from untreated donor mice lacking neuronal NOS. It resulted in intact microvascular structure and long-term recipient survival, either if donor mice were untreated or treated with tetrahydrobiopterin.

Conclusion

We demonstrate for the first time the crucial involvement of neuronal NOS in early microcirculatory derangements following solid organ transplantation. In this model, protective effects of tetrahydrobiopterin are mediated by targeting this isoform.     

Role of the Protective Antigen Octamer in the Molecular Mechanism of Anthrax Lethal Toxin Stabilization in Plasma

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-γ-D-glutamic acid capsule. Atx is comprised of three proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT can assemble on host cell surfaces or extracellularly in plasma. We show that, under physiological conditions in bovine plasma, LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel-forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration, which allows them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that could circulate freely in the blood.     

Inhibition of Neuronal Nitric Oxide Synthase by 7-Nitroindazole Protects Against MPTP-Induced Neurotoxicity in Mice

Abstract: Several studies suggest that nitric oxide (NO^•) contributes to cell death following activation of NMDA receptors in cultured cortical, hippocampal, and striatal neurons. In the present study we investigated whether 7-nitroindazole (7-NI), a specific neuronal nitric oxide synthase inhibitor, can block dopaminergic neurotoxicity seen in mice after systemic administration of MPTP. 7-NI dose-dependently protected against MPTP-induced dopamine depletions using two different dosing regimens of MPTP that produced varying degrees of dopamine depletion. At 50 mg/kg of 7-NI there was almost complete protection in both paradigms. Similar effects were seen with MPTP-induced depletions of both homovanillic acid and 3,4-dihydroxyphenylacetic acid. 7-NI had no significant effect on dopamine transport in vitro and on monoamine oxidase B activity both in vitro and in vivo. One mechanism by which NO^• is thought to mediate its toxicity is by interacting with superoxide radical to form peroxynitrite (ONOO⁻), which then may nitrate tyrosine residues. Consistent with this hypothesis, MPTP neurotoxicity in mice resulted in a significant increase in the concentration of 3-nitrotyrosine, which was attenuated by treatment with 7-NI. Our results suggest that NO^• plays a role in MPTP neurotoxicity, as well as novel therapeutic strategies for Parkinson's disease.     

Protective Vascular and Cardiac Effects of Inducible Nitric Oxide Synthase in Mice with Hyperhomocysteinemia

Diet-induced hyperhomocysteinemia produces endothelial and cardiac dysfunction and promotes thrombosis through a mechanism proposed to involve oxidative stress. Inducible nitric oxide synthase (iNOS) is upregulated in hyperhomocysteinemia and can generate superoxide. We therefore tested the hypothesis that iNOS mediates the adverse oxidative, vascular, thrombotic, and cardiac effects of hyperhomocysteinemia. Mice deficient in iNOS (Nos2−/−) and their wild-type (Nos2+/+) littermates were fed a high methionine/low folate (HM/LF) diet to induce mild hyperhomocysteinemia, with a 2-fold increase in plasma total homocysteine (P<0.001 vs. control diet). Hyperhomocysteinemic Nos2+/+ mice exhibited endothelial dysfunction in cerebral arterioles, with impaired dilatation to acetylcholine but not nitroprusside, and enhanced susceptibility to carotid artery thrombosis, with shortened times to occlusion following photochemical injury (P<0.05 vs. control diet). Nos2−/− mice had decreased rather than increased dilatation responses to acetylcholine (P<0.05 vs. Nos2+/+ mice). Nos2−/− mice fed control diet also exhibited shortened times to thrombotic occlusion (P<0.05 vs. Nos2+/+ mice), and iNOS deficiency failed to protect from endothelial dysfunction or accelerated thrombosis in mice with hyperhomocysteinemia. Deficiency of iNOS did not alter myocardial infarct size in mice fed the control diet but significantly increased infarct size and cardiac superoxide production in mice fed the HM/LF diet (P<0.05 vs. Nos2+/+ mice). These findings suggest that endogenous iNOS protects from, rather than exacerbates, endothelial dysfunction, thrombosis, and hyperhomocysteinemia-associated myocardial ischemia-reperfusion injury. In the setting of mild hyperhomocysteinemia, iNOS functions to blunt cardiac oxidative stress rather than functioning as a source of superoxide.     

The Role of 5-HT1A Receptors and Neuronal Nitric Oxide Synthase in a Seizur Induced Kindling Model in Rats

Neurochemical Research - Dentate gyrus (DG) has a high density of 5-HT1A receptors. It has neural nitric oxide synthase (nNOS), which is involved in neural excitability. The purpose of this study...     

Hunting for Plant Nitric Oxide Synthase Provides New Evidence of a Central Role for Plastids in Nitric Oxide Metabolism

Nitric oxide (NO) has emerged as a central signaling molecule in plants and animals. However, the long search for a plant NO synthase (NOS) enzyme has only encountered false leads. The first works describing a pathogen-induced NOS-like plant protein were soon retracted. New hope came from the identification of NOS1, an Arabidopsis thaliana protein with an atypical NOS activity that was found to be targeted to mitochondria in roots. Although concerns about the NO-producing activity of this protein were raised (causing the renaming of the protein to NO-associated 1), compelling data on its biological role were missing until recently. Strong evidence is now available that this protein functions as a GTPase that is actually targeted to plastids, where it might be required for ribosome function. These and other results support the argument that the defective NO production in loss-of-function mutants is an indirect effect of interfering with normal plastid functions and that plastids play an important role in regulating NO levels in plant cells.A major revolution in biology took place by the early 1990s after the discovery that nitric oxide (NO), a free radical, was not a toxic by-product of oxidative metabolism but had a fundamental role as a signaling molecule regulating normal physiological processes in animal cells (Culotta and Koshland, 1992). A role of this volatile molecule in plant defense responses was subsequently reported, and it is now well established that NO is also a key player in the regulation of different plant developmental processes, including germination, root growth, vascular differentiation, stomatal closure, and flowering (Lamattina et al., 2003; Wendehenne et al., 2004; Crawford and Guo, 2005). Animal cells synthesize NO primarily by the activity of NO synthase (NOS) enzymes. There are several NOS isoforms, but all of them catalyze the same basic reaction: a NADPH-dependent oxidation of l-Arg to NO and l-citrulline. By contrast, the synthesis of NO in plant cells remains a matter of debate. The first reported mechanism to make NO in plants was the reduction of nitrite to NO catalyzed (with low efficiency) by nitrate reductase (NR), a cytosolic enzyme that normally reduces nitrate to nitrite (Yamasaki et al., 1999). But the contribution of NR to NO synthesis is still controversial.The analysis of the Arabidopsis thaliana nia1 nia2 double mutant, which shows substantially reduced NR activity levels, has shown that such activity is required for NO synthesis during flowering (Seligman et al., 2008), auxin-induced lateral root development (Kolbert et al., 2008), and abscisic acid (ABA)-induced stomatal closure (Desikan et al., 2002; Bright et al., 2006) but not during infection (Zhang et al., 2003), salicylic acid treatment (Zottini et al., 2007), or mechanical stress (Garces et al., 2001). Furthermore, foliar extracts of the mutant show the same capacity to produce NO as wild-type plants when nitrite is exogenously supplied (Modolo et al., 2005). These results indicate that additional mechanisms to reduce nitrite into NO exist in plant cells and that the decreased capability for NO synthesis of mutant plants with defective NR activity might result from their reduced nitrite levels (Modolo et al., 2005). Other enzymatic sources for nitrite-dependent NO synthesis exist in the plasma membrane (Stohr et al., 2001) and mitochondria (Planchet et al., 2005), whereas nonenzymatic production of NO from nitrite has been shown to occur in acidic and reducing environments, such as the apoplasm (Bethke et al., 2004) and plastids (Cooney et al., 1994). The highly reduced levels of l-Arg in the nia1 nia2 mutant (Modolo et al., 2006) might also compromise its ability to produce NO. This amino acid is a substrate for the production of polyamines, compounds that have been proposed to participate in NO synthesis (Tun et al., 2006). Additionally, plants have been found to synthesize NO by an Arg-dependent NOS activity similar to that present in animal cells, as detailed in the next section.     

胰岛素对PC12细胞神经型一氧化氮合酶表达及活性的影响

为探讨胰岛素对神经细胞中神经型一氧化氮合酶（nNOS）的表达及活性的影响,应用流式细胞术、原位杂交、电子自旋共振等技术方法研究胰岛素对PC12细胞中神经型一氧化氮合酶的影响.胰岛素作用PC12细胞9 h 后,神经型一氧化氮合酶的免疫荧光强度显著升高,且呈浓度依赖关系,其最大效应为对照的(155±13)%(P＜0.01, n=3, t-test).加入胰岛素(16 mU/L, 6 h)也能够显著上调nNOS mRNA的表达,为对照的(182±13)%(P＜0.01, n=3, t-test).另外加入胰岛素(16 mU/L)作用9 h后,神经型一氧化氮合酶的活性也显著升高,为对照的(167±15)%(P＜0.01, n=4, t-test).由上述结果可知,胰岛素对PC12细胞的神经型一氧化氮合酶的表达及活性有上调作用.     

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.     

早期经验对大鼠脑区一氧化氮合酶活性的影响

目的　探讨NO与早期饲养环境所引起脑效应的关系。方法　将断乳大鼠在丰富环境 (EC)和单调环境 (IC)中饲养 30d。环境暴露后通过NADPH -黄递酶组化方法对海马齿状回 (DEN)和大脑皮层NOS活性进行定量测定以及对大鼠进行Morris水迷宫作业训练。结果　EC大鼠与IC大鼠相比 ,海马齿状回 (DEN)和大脑皮层NOS活性明显下降 ,迷宫测试表明EC大鼠的空间认知显著优于IC大鼠。在环境暴露期间隔日注射一氧化氮合酶 (NOS)抑制物L -NAME(50mg/kg) ,未引起EC或IC大鼠认知行为的明显改变 ,但导致DEN和大脑皮层NOS活性的不同改变。结论　NO可能与早期经验脑效应有关。     

The Protective Effect of Dexanabinol (HU-211) on Nitric Oxide and Cysteine Protease-Mediated Neuronal Death in Focal Cerebral Ischemia

We hypothesized that dexanabinol can prevent neuronal death by protecting neuronal lysosomes from nitric oxide (NO)-mediated toxicity, and in turn, by suppressing the release of cathepsins during cerebral ischemia. Focal cerebral ischemia was induced in two sets of animals by permanent middle cerebral artery occlusion. The first set was used to monitor NO concentration and cathepsin activity, while the second was used for histological examination with hematoxylin and eosin, and TUNEL staining. In post-ischemic brain tissue, NO content and cathepsin B and L activity increased (p < 0.05). Dexanabinol treatment reduced NO concentration and cathepsin activity to the control level (p > 0.05). The number of eosinophilic and apoptotic neurons increased in the post-ischemic cerebral cortex (p < 0.05). However, dexanabinol treatment lowered both of these (p < 0.05). We conclude that dexanabinol might be a useful agent for the treatment of stroke patients.     

The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore

Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited the function of the protective antigen (PA) pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA) domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.     

Characterization of Neuronal Amino Acid Transporters: Uptake of Nitric Oxide Synthase Inhibitors and Implication for Their Biological Effects

Abstract: In the present study we investigated uptake of the nitric oxide (NO) synthase inhibitors N ^G-methyl- l -arginine and N ^G-nitro- l -arginine by the mouse neuroblastoma × rat glioma hybrid cell line NG108-15. Uptake of N ^G-methyl- l -arginine was characterized by biphasic kinetics ( K _m1 = 8 µmol/L, V _max1 = 0.09 nmol × mg⁻¹× min⁻¹; K _m2 = 229 µmol/L, V _max2 = 2.9 nmol × mg⁻¹× min⁻¹) and was inhibited by basic but not by neutral amino acids. Uptake of N ^G-nitro- l -arginine followed Michaelis-Menten kinetics ( K _m = 265 µmol/L, V _max = 12.8 ± 0.86 nmol × mg⁻¹× min⁻¹) and was selectively inhibited by aromatic and branched chain amino acids. Further characterization of the transport systems revealed that uptake of N ^G-methyl- l -arginine is mediated by system y⁺, whereas systems L and T account for the transport of N ^G-nitro- l -arginine. In agreement with these data on uptake of the inhibitors, l -lysine and l -ornithine antagonized the inhibitory effects of N ^G-methyl- l -arginine on bradykinin-induced intracellular cyclic GMP accumulation, whereas l -tryptophan, l -phenylalanine, and l -leucine interfered with the effects of N ^G-nitro- l -arginine. These data suggest that rates of uptake are limiting for the biological effects of NO synthase inhibitors.     

Nitric Oxide Synthase and Breast Cancer: Role of TIMP-1 in NO-mediated Akt Activation

Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation.     

The Severity of Murray Valley Encephalitis in Mice Is Linked to Neutrophil Infiltration and Inducible Nitric Oxide Synthase Activity in the Central Nervous System

A study of immunopathology in the central nervous system (CNS) during infection with a virulent strain of Murray Valley encephalitis virus (MVE) in weanling Swiss mice following peripheral inoculation is presented. It has previously been shown that virus enters the murine CNS 4 days after peripheral inoculation, spreads to the anterior olfactory nucleus, the pyriform cortex, and the hippocampal formation at 5 days postinfection (p.i.), and then spreads throughout the cerebral cortex, caudate putamen, thalamus, and brain stem between 6 and 9 days p.i. (P. C. McMinn, L. Dalgarno, and R. C. Weir, Virology 220:414-423, 1996). Here we show that the encephalitis which develops in MVE-infected mice from 5 days p.i. is associated with the development of a neutrophil inflammatory response in perivascular regions and in the CNS parenchyma. Infiltration of neutrophils into the CNS was preceded by increased expression of tumor necrosis factor alpha and the neutrophil-attracting chemokine N51/KC within the CNS. Depletion of neutrophils with a cytotoxic monoclonal antibody (RB6-8C5) resulted in prolonged survival and decreased mortality in MVE-infected mice. In addition, neutrophil infiltration and disease onset correlated with expression of the enzyme-inducible nitric oxide synthase (iNOS) within the CNS. Inhibition of iNOS by aminoguanidine resulted in prolonged survival and decreased mortality in MVE-infected mice. This study provides strong support for the hypothesis that Murray Valley encephalitis is primarily an immunopathological disease.     

A Pivotal Role for Tryptophan 447 in Enzymatic Coupling of Human Endothelial Nitric Oxide Synthase (eNOS): EFFECTS ON TETRAHYDROBIOPTERIN-DEPENDENT CATALYSIS AND eNOS DIMERIZATION*

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.