Oxidation of DOPAC by nitric oxide: effect of superoxide dismutase

This study aimed to characterize the redox interaction between 3,4-dihydroxyphenylacetic acid (DOPAC) and nitric oxide (.NO), and to assess the reductive and oxidative decay pathways of the DOPAC semiquinone originating from this interaction. The reaction between DOPAC and.NO led to the formation of the DOPAC semiquinone radical, detected by electron paramagnetic resonance (EPR) and stabilized by Mg(2+), and the nitrosyl anion detected as nitrosylmyoglobin. The EPR signal corresponding to the DOPAC semiquinone was modulated as follows: (i) it was suppressed by glutathione and ascorbic acid with the formation of new EPR spectra corresponding to the glutathionyl and ascorbyl radical, respectively; (ii) it was enhanced by Cu,Zn-superoxide dismutase; the enzyme also accelerated the decay of the semiquinone species to DOPAC quinone. These results are interpreted as a one-electron oxidation of DOPAC by.NO; the reductive decay of the semiquinone back to DOPAC was facilitated by reducing agents, such as glutathione and ascorbate, whereas the oxidative decay to DOPAC quinone was facilitated by superoxide dismutase. The latter effect is understood in terms of a reversible conversion of nitrosyl anion to.NO by the enzyme. The biological relevance of these reactions is also discussed in terms of the reactivity of peroxynitrite towards DOPAC as a model with implications for aerobic conditions.     

In vitro effect of nanosilver on gene expression of superoxide dismutases and nitric oxide synthases in chicken sertoli cells

To evaluate effects of different concentrations of nanosilver colloid on the cell culture of Sertoli cells, the proportion of lipid peroxidation, antioxidant capacity, nitric oxide (NO) production and genes expression of superoxide dismutases (SOD1 and SOD2) and nitric oxide synthases (eNOS and iNOS) were measured. Sertoli cells were incubated at concentrations of 25, 75 and 125 ppm nanosilver for 48 h. There was progressive lipid peroxidation in treatments according to increasing of nanosilver. Lipid peroxidation, as indicated by malondialdehyde levels, was significantly elevated by the highest concentration of silver colloid (125 ppm), although antioxidant capacity, as measured by ferric ion reduction, was unaffected. Nitrite, as an index of NO production was reduced only in 125 ppm of nanosilver. Expression of SOD1 gene was reduced in nanosilver-treated cells at all concentrations, whereas expression of SOD2 gene was reduced only in cells treated with 125 ppm nanosilver. Expression of iNOS gene was progressively increased with higher concentrations of nanosilver. Expression of eNOS gene was also increased in 125 ppm of nanosilver. In conclusion, toxic effects of nanosilver could be due to high lipid peroxidation and suppression of antioxidant mechanisms via reduced expression of SOD genes and increased expression of NOS genes.     

Treatment with 7-nitroindazole enhances kainic acid induced cholinergic neurotoxicity in the rat striatum: a neuroprotective role for neuronal nitric oxide

1. In this study we investigated the effect of 7-nitroindazole (7-NI), a preferential inhibitor of neuronal nitric oxide synthase (nNOS), on kainic acid (KA) induced neurotoxicity in rats. Choline acetyltransferase activity (CAT), a cholinergic marker, and histological changes were employed to assess neurotoxicity.2. In control rats, the local intrastriatal injection of 0.5 g of KA reduced CAT from 22.9 ± 2.2 to 14.7 ± 2.0 nmol/h/mg tissue ((38 ± 6)% reduction) (P < 0.001). Greater reductions in CAT were observed with 1 and 2 g of KA ((70 ± 6)% and (80 ± 3)%, respectively). 7-NI aggravated KA-induced cholinergic and histological damage. KA reduced CAT by (68.2 ± 4)% in 7-NI-treated rats, by (38 ± 6)% in saline-treated controls, and by (41 ± 4)% in peanut-oil- (7-NI-vehicle-) treated rats (P = 0.0047).3. After KA, CAT activity averaged 14.3 ± 2.0 in peanut-oil-treated rats and 7.9 ± 1.0 nmol/h/mg tissue in 7-NI- (peanut-oil-) treated rats (P = 0.015). Similarly to changes in CAT, 7-NI treatment aggravated KA-induced histological changes indicative of neuronal damage (acute ischemic neuronal changes, disorganization of myelinated fibers bundle, and vacuolation changes of the neuropil). Treatment with 7-NI was not associated with increased mortality.4. Our findings suggest that neuronal NO plays a neuroprotective action on excitotoxicity.     

Inactivation of cytochrome c oxidase by mutant SOD1s in mouse motoneuronal NSC-34 cells is independent from copper availability but is because of nitric oxide

The copper-enzyme cytochrome c oxidase (Cytox) has been indicated as a primary molecular target of mutant copper, zinc superoxide dismutase (SOD1) in familial amyotrophic lateral sclerosis (fALS); however, the mechanism underlying its inactivation is still unclear. As the toxicity of mutant SOD1s could arise from their selective recruitment to mitochondria, it is conceivable that they might compete with Cytox for the mitochondrial copper pool causing Cytox inactivation. To investigate this issue, we used mouse motoneuronal neuroblastoma × spinal cord cell line-34, stably transfected for the inducible expression of low amounts of wild-type or mutant (G93A, H46R, and H80R) human SOD1s and compared the effects observed on Cytox with those obtained by copper depletion. We demonstrated that all mutants analyzed induced cell death and decreased the Cytox activity, but not the protein content of the Cytox subunit II, at difference with copper depletion that also affected subunit II protein. Copper supplementation did not counteract mutant hSOD1s toxicity. Otherwise, the treatment of neuroblastoma × spinal cord cell line-34 expressing G93A, H46R, or H80R hSOD1 mutants, and showing constitutive expression of iNOS and nNOS, with either a NO scavenger, or NOS inhibitors prevented the inhibition of Cytox activity and rescued cell viability. These results support the involvement of NO in mutant SOD1s-induced Cytox damage, and mitochondrial toxicity.     

Lipopolysaccharide-induced dopaminergic cell death in rat midbrain slice cultures: role of inducible nitric oxide synthase and protection by indomethacin

Glial cell activation associated with inflammatory reaction may contribute to pathogenic processes of neurodegenerative disorders, through production of several cytotoxic molecules. We investigated the consequences of glial activation by interferon-gamma (IFN-gamma)/lipopolysaccharide (LPS) in rat midbrain slice cultures. Application of IFN-gamma followed by LPS caused dopaminergic cell death and accompanying increases in nitrite production and lactate dehydrogenase release. Aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS), or SB203580, an inhibitor of p38 mitogen-activated protein kinase, prevented dopaminergic cell loss as well as nitrite production. SB203580 also suppressed expression of iNOS and cyclooxygenase-2 (COX-2) induced by IFN-gamma/LPS. A COX inhibitor indomethacin protected dopaminergic neurons from IFN-gamma/LPS-induced injury, whereas selective COX-2 inhibitors such as NS-398 and nimesulide did not. Notably, indomethacin was able to attenuate neurotoxicity of a nitric oxide (NO) donor. Neutralizing antibodies against tumour necrosis factor-alpha and interleukin-1beta did not inhibit dopaminergic cell death caused by IFN-gamma/LPS, although combined application of these antibodies blocked lactate dehydrogenase release and decrease in the number of non-dopaminergic neurons. These results indicate that iNOS-derived NO plays a crucial role in IFN-gamma/LPS-induced dopaminergic cell death, and that indomethacin exerts protective effect by mechanisms probably related to NO neurotoxicity rather than through COX inhibition.     

Serotonin binds to purified neuronal nitric oxide synthase: A possible explanation for ROS production induced by 5HT in the presence of nNOS

Serotonin (5HT) was shown to induce in vitro the production of ROS in the presence of neuronal nitric oxide synthase (nNOS) in addition to the basal NO^? formation. With the aim of understanding this mechanism, this study investigated the potential binding of 5HT to nNOS. By using [³H]5HT, it is reported here that 5HT binds to nNOS, but only when the enzyme is active and in a superoxide-dependent manner. This binding is prevented by DPI but not by L-NAME. The formation of 5HT-nNOS complex was shown to be very well correlated with the production of ROS by 5HT in the presence of nNOS. A mechanism involving nNOS only in its initial step is proposed to explain both the formation of 5HT-nNOS complex and the production of ROS observed in the presence of nNOS and 5HT.     

Endothelial nitric oxide synthase overexpression by neuronal cells in neurodegeneration: a link between inflammation and neuroprotection

The roles of neuronal and inducible nitric oxide synthases in neurones have been extensively investigated; by contrast, the biological significance of endothelial nitric oxide synthase (eNOS) overexpression that occurs in several pathological conditions has not yet been studied. We have started addressing this issue in a cell model of neurodegeneration, i.e. human SKNBE neuroblastoma cells transfected with a mutant form of alsin, a protein causing an early-onset type of amyotrophic lateral sclerosis, ALS2. We found that eNOS, which is endogenously expressed by these cells, was activated by tumour necrosis factor-α (TNF-α), a proinflammatory cytokine that plays important roles in ALS2 and several neurodegenerative diseases. The TNF-α-dependent eNOS activation occurred through generation, by sphingosine-kinase-1, of sphingosine-1-phosphate, stimulation of its membrane receptors and activation of Akt, as determined using small interference RNA and dominant negative constructs specific for the enzymes and receptors. eNOS activation by TNF-α conferred cytoprotection from excitotoxicity and neurotoxic cues such as reactive oxygen species, endoplasmic reticulum stress, DNA damage, and mutated alsin itself. Our results suggest that overexpression of eNOS by neurones is a broad-range protective mechanism activated during damage and establish a link of pathophysiological relevance between this enzyme and inflammation accompanying neurodegenerative diseases. These findings also question the concept that high NO output in the presence of oxidative stress leads always to peroxynitrite formation contributing to neurodegeneration.     

神经元型一氧化氮合酶在Ⅱ组代谢型谷氨酸受体介导的脑缺血耐受中的作用

目的：探讨神经元型一氧化氮合酶（nNOS）催化产生的一氧化氮（NO）在Ⅱ组代谢型谷氨酸受体（mGluR2／3）介导的脑缺血预处理（CIP）保护机制中的作用。方法：36只永久凝闭椎动脉的SD大鼠随机分为6组（n=6）：sham、CIP、损伤性缺血、CIP4-损伤性缺血、MqPG＋CIP和MTPG＋CIP＋损伤性缺血组。采用硫堇染色和免疫组化观察海马CA1区迟发性神经元死亡（DND）和nNOS表达的变化。结果：与Sham组相比,CIP组海马nNOS表达出现一定程度的上调,而损伤性脑缺血组则出现nNOS表达的明显上调,预先给与CIP可一定程度上防止损伤性脑缺血所致的nNOS表达的过度升高。在MTPG4-CIP组,预先侧脑室注射mGluR2／3阻断剂MTPG,可阻断CIP引起的nNOS表达增加,但对神经元的存活无影响。而在MTPG＋CIP＋损伤性缺血组中,出现大量锥体神经元DND,同时nNOS的表达较MTPG＋CIP组明显增加,该增加为损伤性脑缺血所致,而非MTPG的作用。结论：nNOS催化产生的NO作为mGluR2／3的下游分子参与脑缺血预处理过程中mGluR2／3介导的脑缺血耐受的形成。     

Retinoic acid decreases nitric oxide production in endothelial cells: a role of phosphorylation of endothelial nitric oxide synthase at Ser(1179)

The effects of retinoic acid (RA) on nitric oxide (NO) production are controversial. Furthermore, it has never been studied whether these effects are mediated by direct modulation of phosphorylation of endothelial nitric oxide synthase (eNOS). Using bovine aortic endothelial cells, we found that all-trans RA (atRA) dose- and time-dependently decreased NO production without alteration in eNOS expression. This decrease was accompanied by reduction in eNOS-Ser(1179) phosphorylation. However, atRA did not alter the phosphorylation of eNOS-Ser(116) or eNOS-Thr(497). Concurrently, atRA also decreased the expressions of vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1, and Akt phosphorylation. Co-treatment with troglitazone, an activator of VEGF expression, reversed the atRA-induced reductions in eNOS-Ser(1179) phosphorylation and NO production, with concomitant restoration in VEGF expression. Direct treatment with VEGF also reversed these inhibitory effects, suggesting an important role for VEGF. Nonetheless, the RARalpha antagonist Ro 41-5253 did not block all the inhibitory effects of atRA, indicating that these inhibitory effects are not mediated by the RA response element (RARE). Thus, atRA decreases eNOS-Ser(1179) phosphorylation through a mechanism that depends on VEGF-KDR/Flk-1-mediated Akt phosphorylation but is independent of RARE, leading to reduction in NO production.     

PIN-bodies: a new class of antibody-like proteins with CD4 specificity derived from the protein inhibitor of neuronal nitric oxide synthase

By inserting the CB1 paratope-derived peptide (PDP) from the anti-CD4 13B8.2 antibody binding pocket into each of the three exposed loops of the protein inhibitor of neuronal nitric oxide synthase (PIN), we have combined the anti-CD4 specificity of the selected PDP with the stability, ease of expression/purification, and the known molecular architecture of the phylogenetically well-conserved PIN scaffold protein. Such "PIN-bodies" were able to bind CD4 with a better affinity and specificity than the soluble PDP; additionally, in competitive ELISA experiments, CD4-specific PIN-bodies were more potent inhibitors of the binding of the parental recombinant antibody 13B8.2 to CD4 than the soluble PDP. The efficiency of CD4-specific CB1-inserted PIN-bodies was confirmed in biological assays where these constructs showed higher potencies to block antigen presentation by inhibition of IL-2 secretion and to inhibit the one-way and two-way mixed lymphocyte reactions, compared with soluble anti-CD4 PDP CB1. Insertion of the PDP into the first exposed loop (position 33/34) of PIN appeared to be the most promising scaffold. Taken together, our findings demonstrate that the PIN molecule is a suitable scaffold to expose new peptide loops and generate small artificial ligand-binding products with defined specificities.     

β‐amyloid decreases detectable endothelial nitric oxide synthase in human erythrocytes: a role for membrane acetylcholinesterase

Until few years ago, many studies of Alzheimer's disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Aβ) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Aβ is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Aβ on erythrocyte eNOS. We found that Aβ following to 24‐h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Aβ alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Aβ. These events could contribute to the vascular alterations associated with Alzheimer's disease disease. Copyright © 2012 John Wiley & Sons, Ltd.     

The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms

Nitric oxide (NO), which is produced from l-arginine by three isoforms of NO synthase (NOS), has been implicated in reproductive functions. However, the specific role of NOS isoforms in gamete function and fertilization is not clear. Three types of NOS knockout mice were super ovulated and fertilized in vitro and in vivo. The sperm count and motility, in vivo and in vitro fertilization rate as indicated by two-cell embryos and blastocyst rate were examined. The sperm count and motility from all three knockout mice were not significantly different from that of the wild type. Inducible NOS (iNOS) knockout mice were found to have the largest number of two-cell embryos/mouse collected after fertilization in vivo (P<0.01), but the rate of blastocyst formation from two-cell embryos in vitro was similar for all three knockouts. The rate of in vitro fertilization using either iNOS-deficient sperm or oocytes, but not those deficient in the other two NOS isoforms, was significantly elevated when compared to that in the wild type (P<0.001). While all three types of NOS do not seem to play a significant role in pre-ejaculated sperm function, iNOS may play an inhibitory role in sperm and oocyte functions affecting the process of fertilization and early embryo development.     

Differential role of superoxide and glutathione in S‐nitrosoglutathione-mediated apoptosis: a rationale for mild forms of familial amyotrophic lateral sclerosis associated with less active Cu,Zn superoxide dismutase mutants

SH-SY5Y cells transfected with the enzymatically inactive Cu,Zn superoxide dismutase mutant H46R were more resistant to S-nitrosoglutathione (GSNO)-induced apoptosis. Cytochrome c release from mitochondria, caspase 3 activation, p53 up-regulation, p21 cleavage and Bcl-2 modulation, all involved in the apoptotic process, were significantly less altered with respect to untransfected cells. The H46R resistance to NO was associated with a higher content of reduced glutathione (GSH) and was abolished by blockage of glutathione synthesis. On the other hand, H46R cells were as sensitive as SH-SY5Y cells to puromycin-induced apoptosis; furthermore, they were more susceptible to apoptosis elicited by the superoxide-generating drug paraquat and to cell necrosis provoked by t-butyl hydroperoxide. These results confirm that the level of superoxide dismutase activity is fundamental for protecting cells against oxygen free radical challenge. Its impairment is not detrimental to cells exposed to NO, as long as the overall reducing power represented by GSH is assured. These results are relevant to explain a milder progression of the familial amyotrophic lateral sclerosis disease when associated with the H46R mutation.     

Insulin enhances the expression of the endothelial nitric oxide synthase in native endothelial cells: a dual role for Akt and AP-1

Insulin-induced vasodilatation in vivo has been attributed to the activation of the endothelial nitric oxide (NO) synthase (eNOS). The present study addressed the effects of insulin on the activity and expression of eNOS in native and cultured endothelial cells. Insulin applied to native porcine aortic endothelial cells elicited the tyrosine phosphorylation of the insulin receptor and receptor substrate, the subsequent activation of phosphatidylinositol 3-kinase (PI 3-K), Akt (protein kinase B), and ERK1/2. Insulin did not activate eNOS in cultured endothelial cells nor relax endothelium-intact arterial segments. However, 4h after application of insulin to native endothelial cells eNOS mRNA was increased 2-fold. A comparable increase in eNOS protein was detected after 18-24h and associated with an increase in intracellular cyclic GMP. In native endothelial cells, insulin enhanced the DNA-binding activity of Sp1 and AP-1, but not that of NF-kappaB. The insulin-induced increase in eNOS expression was prevented by wortmannin as well as by AP-1 decoy oligonucleotides. The MEK1 inhibitor, PD 98059, also enhanced eNOS expression in native and cultured endothelial cells, an effect which was independent of ERK1/2 and associated with an increase in the DNA-binding activity of AP-1 and Sp1. These results demonstrate that insulin activates multiple signalling pathways in endothelial cells but does not acutely activate eNOS. Insulin however enhances eNOS mRNA and protein by a mechanism involving the combined activation of a PI 3-K- and AP-1-dependent pathway.     

Retinoic acid activates human inducible nitric oxide synthase gene through binding of RARalpha/RXRalpha heterodimer to a novel retinoic acid response element in the promoter

Human inducible nitric oxide synthase (hiNOS) catalyzes nitric oxide (NO) which has a significant effect on tumor suppression and cancer therapy. Here we revealed the detailed molecular mechanism involved in the regulation of hiNOS expression induced by retinoic acid (RA). We showed that RARalpha/RXRalpha heterodimer was important in hiNOS promoter activation, hiNOS protein expression, and NO production. Serial deletion and site-directed mutation analysis revealed two half-sites of retinoic acid response element (RARE) spaced by 5bp located at -172 to -156 in the hiNOS promoter. EMSA and ChIP assays demonstrated that RARalpha/RXRalpha directly bound to this RARE of hiNOS promoter. Our results suggested the identification of a novel RARE in the hiNOS promoter and the roles of the nuclear receptors (RARalpha/RXRalpha) in the induction of hiNOS by RA.     

Association of polymorphisms of endothelial nitric oxide synthase (eNOS) gene with the risk of primary open angle glaucoma in a Brazilian population

The present study aimed to investigate the association of endothelial nitric oxide synthase (eNOS) gene polymorphisms with primary open angle glaucoma (POAG). We conducted a case-control study that included 90 patients with POAG and 127 healthy controls whose blood samples were genotyped for the functional polymorphisms T-786C and Glu298Asp of the eNOS gene by Taqman fluorescent allelic discrimination assay. The T-786C polymorphism was significantly associated as a risk factor for POAG among women (OR: 2.28; 95% CI: 1.11 to 4.70, p=0.024) and marginally associated to the risk of POAG in the patients ≥52 years of age at diagnosis (OR: 2.11; 95% CI: 0.98 to 4.55, p=0,055). However, these results was not confirmed after adjustments for gender, age, self-declared skin color, tobacco smoking and eNOS genotypes by multivariate logistic regression model (OR: 2.08; 95% CI: 0.87 to 5.01, p=0.101 and OR: 2.20; 95% CI: 0.95 to 5.12, p=0.067, respectively). The haplotype CG of T-786C and Glu298Asp showed a borderline association with risk of POAG in the overall analysis (OR: 1.76; 95% CI: 0.98 to 3.14, p=0.055) and among women (OR: 2.02; 95% CI: 0.98 to 4.16, p=0.052). Furthermore, the CG haplotype was significantly associated with the development of POAG for the age at diagnosis group ≥52 years (OR: 3.48; 95% CI: 1.54 to 7.84, p=0.002).We suggested that haplotypes of the polymorphisms T-786C and Glu298Asp of eNOS may interact with gender and age in modulating the risk of POAG.     

Functions and dysfunctions of nitric oxide in brain

Nitric oxide (NO) works as a retrograde neurotransmitter in synapses, allows the brain blood flow and also has important roles in intracellular signaling in neurons from the regulation of the neuronal metabolic status to the dendritic spine growth. Moreover NO is able to perform post-translational modifications in proteins by the S-nitrosylation of the thiol amino acids, which is a physiological mechanism to regulate protein function. On the other hand, during aging and pathological processes the behavior of NO can turn harmful when reacts with superoxide anion to form peroxynitrite. This gaseous compound can diffuse easily throughout the neuronal membranes damaging lipid, proteins and nucleic acids. In the case of proteins, peroxynitrite reacts mostly with the phenolic ring of the tyrosines forming nitro-tyrosines that affects dramatically to the physiological functions of the proteins. Protein nitrotyrosination is an irreversible process that also yields to the accumulation of the modified proteins contributing to the onset and progression of neurodegenerative processes such as Alzheimer's disease or Parkinson's disease.     

Nitrite as the major source of nitric oxide production by Arabidopsis thaliana in response to Pseudomonas syringae

The origin of nitric oxide (*NO) in plants is unclear and an *NO synthase (NOS)-like enzyme and nitrate reductase (NR) are claimed as potential sources. Here we used wild-type and NR-defective double mutant plants to investigate *NO production in Arabidopsis thaliana in response to Pseudomonas syringae pv maculicola. NOS activity increased substantially in leaves inoculated with P. syringae. However, electron paramagnetic resonance experiments showed a much higher *NO formation that was dependent on nitrite and mitochondrial electron transport rather than on arginine or nitrate. Overall, these results indicate that NOS, NR and a mitochondrial-dependent nitrite-reducing activity cooperate to produce *NO during A. thaliana-P. syringae interaction.