Nitrosation of uric acid induced by nitric oxide under aerobic conditions.

Uric acid is a well-established scavenger of reactive oxygen and nitrogen species such as hydroxyl radical and peroxynitrite. However, little attention has been paid to the relationship between uric acid and nitric oxide. This paper reports the identification and characterization of a reaction product of uric acid induced by nitric oxide. When uric acid was treated with nitric oxide gas in a neutral solution under aerobic conditions, uric acid was consumed, yielding an unknown product. The product was identified as nitrosated uric acid from mass spectrometric data, although the position of the nitroso group on the molecule was not determined. The nitrosated uric acid decomposed to several compounds including uric acid with a half-life of 2.2 min at pH 7.4 and 37 degrees C. The incubation of nitrosated uric acid with glutathione resulted in the formation of S-nitrosoglutathione. Nitrosated uric acid was also formed in the reaction with nitric oxide donors, but not with peroxynitrite. Nitrosated uric acid was detected in human serum and urine by in vitro treatment with a nitric oxide donor. In the reaction of glutathione with the nitric oxide donor, the addition of uric acid caused an increase in the yield of S-nitrosoglutathione. These results indicate that under aerobic conditions nitric oxide can convert uric acid into its nitroso derivative, which can give a nitroso group to glutathione. Uric acid may act as a vehicle of nitric oxide in humans.     

Inactivation of ribonucleotide reductase by nitric oxide.

Ribonucleotide reductase has been demonstrated to be inhibited by NO synthase product(s). The experiments reported here show that nitric oxide generated from sodium nitroprusside, S-nitrosoglutathione and the sydnonimine SIN-1 inhibits ribonucleotide reductase activity present in cytosolic extracts of TA3 mammary tumor cells. Stable derivatives of these nitric oxide donors were either inactive or much less inhibitory. EPR experiments show that the tyrosyl radical of the small subunit of E. Coli or mammalian ribonucleotide reductase is efficiently scavenged by these NO donors.     

Inactivation of macrophage nitric oxide synthase activity by NG-methyl-L-arginine

.N = O synthase catalyzes the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine to nitric oxide (.N = O). NG-Methyl-L-arginine has been previously characterized as a potent competitive inhibitor of both major types of .N = O synthases. Initial rate kinetics were performed with a spectrophotometric assay based on the oxidation of oxy- to methemoglobin by .N = O. NG-Methyl-L-arginine was a competitive inhibitor of .N = O synthase activity derived from activated murine macrophages with a Ki of 6.2 microM. When the enzyme was pre-incubated in the presence of the required cofactors NADPH and tetrahydrobiopterin, time- and concentration-dependent irreversible inactivation of the activity was observed. At 37 degrees C the kinact was 0.050 min-1. This inactivation process exhibited substrate protection, saturation kinetics and required the cofactors necessary for enzymatic turnover. These data indicate that NG-methyl-L-arginine acts as a mechanism-based enzyme inactivator of murine macrophage .N = O synthase.     

Inactivation of NADP(+)-dependent isocitrate dehydrogenase by nitric oxide

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. NO donors such as S-nitrosothiols, diethylamine NONOate, spermine NONOate, and 3-morpholinosydnomine N-ethylcarbamide (SIN-1)/superoxide dismutase inactivated ICDH in a dose- and time-dependent manner. The inhibition of ICDH by S-nitrosothiol was partially reversed by thiol, such as dithiothreitol or 2-mercaptoethanol. Loss of enzyme activity was associated with the depletion of the cysteine-reactive 5,5'-dithiobis-(2-nitrobenzoate) and the loss of fluorescent probe N,N'-dimethyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine accessible thiol groups. Using electrospray ionization mass spectrometry with tryptic digestion of protein, we found that nitric oxide forms S-nitrosothiol adducts on Cys305 and Cys387. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by NO. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and intrinsic tryptophan fluorescence. When U937 cells were incubated with 200 microM SNAP for 1 h, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Furthermore, stimulation with lipopolysaccharide significantly decreased intracellular ICDH activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N(omega)-methyl-L-arginine. This result indicates that ICDH was also inactivated by endogenous NO. The NO-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Inactivation of the cardiac ryanodine receptor calcium release channel by nitric oxide

We have recently reported [Mészáros L.G., Minarovic I., Zahradníková A. Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett 1996; 380: 49–52] that nitric oxide (NO) reduces the activity of the skeletal muscle ryanodine receptor Ca²⁺ release channel (RyRC), a principal component of the excitation-contraction coupling machinery in striated muscles. Since (i) as shown here, we have obtained evidence which indicates that the NO synthase (eNOS) of cardiac muscle origin co-purified with RyRC-containing sarcoplasmic reticulum (SR) fractions; and (ii) the effects of NO donors on the release channel, as well as on cardiac function, appear somewhat contradictory, we have made an attempt to investigate the response of the cardiac RyRC to NO that is generated in situ from L-arginine in the NOS reaction. We found that L-arginine-derived NO inactivates Ca²⁺ release from cardiac SR and reduces the steady-state activity (i.e. open probability) of single RyRCs fused into a planar lipid bilayer. This reduction was prevented by NOS inhibitors and the NO quencher hemoglobin and was reversed by 2-mercaptoethanol. We thus conclude that: (i) in isolated SR preparations, it is possible to assess the effects of NO that is generated from L-arginine in the NOS reaction; and (ii) cardiac RyRc responds to NO in a manner which is identical to that we have previously found with the skeletal channel. These findings suggest that the direct modulation of the RyRC by NO is a signaling mechanism which likely participates in earlier demonstrated NO-induced myocardial contractility changes.     

Inactivation of alpha 1-antiproteinase by hydroxyl radicals. The effect of uric acid

The elastase-inhibitory activity of alpha 1-antiproteinase is inactivated by hydroxyl radicals (.OH) generated by pulse radiolysis or by reaction of iron ions with H2O2 in the presence of superoxide or ascorbate. Uric acid did not protect alpha 1-antiproteinase against inactivation by .OH in pulse radiolysis experiments or in the superoxide/iron/H2O2 system, whereas it did in systems containing ascorbic acid. We propose that radicals formed by attack of .OH on uric acid are themselves able to inactivate alpha 1-antiproteinase, but that these uric acid radicals can be 'repaired' by ascorbic acid.     

Fatty acid control of nitric oxide production by macrophages

Modulation of macrophage functions by fatty acids (FA) has been studied by several groups, but the effect of FA on nitric oxide production by macrophages has been poorly examined. In the present study the effect of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on NF-kappaB activity and NO production in J774 cells (a murine macrophage cell line) was investigated. All FA tested stimulated NO production at low doses (1-10 microM) and inhibited it at high doses (50-200 microM). An increase of iNOS expression and activity in J774 cells treated with a low concentration of FA (5 microM) was observed. The activity of NF-kappaB was time-dependently enhanced by the FA treatment. The inhibitory effect of FA on NO production may be due to their cytotoxicity, as observed by loss of membrane integrity and/or increase of DNA fragmentation in cells treated for 48 h with high concentrations. The results indicate that, at low concentrations FA increase NO production by J774 cells, whereas at high concentrations they cause cell death.     

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O₂) conditions. The system measures the concentrations of O₂ and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O₂ concentration and electron turnover of the enzyme. At a high O₂ concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O₂. At low O₂ (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O₂ consumption. At both high and low O₂ concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.     

Altered circadian relationship between serum nitric oxide, carbon dioxide, and uric acid in multiple sclerosis

The free radical nitric oxide (NO*) is involved in a variety of diverse biological processes from acting as a vasodilator in the cardiovascular system to being the rate-limiting component in the production of peroxynitrite (ONOO-), a contributor to neurodegenerative disorders such as multiple sclerosis (MS). Uric acid (UA), the end product of purine metabolism in humans and a selective inhibitor of toxic reactions attributed to radicals formed by the interaction of ONOO- and CO2, is generally low in MS patients. We investigated the relationship between serum ONOO-, CO2, and UA in MS patients and normal controls by comparing the circadian characteristics of the NO* metabolites nitrite/ nitrate (NO), CO2, and UA. In this preliminary study, we found the functional relationship ascribed to the circadian timing of the peak and trough levels of NO, CO2, and UA in healthy subjects to be clearly altered in MS patients. These findings suggest that alterations in the temporal relationship between the 24h pattern in serum ONOO- formation and UA may either contribute to or reflect the disease processes in MS.     

Fatty acid transduction of nitric oxide signaling. Nitrolinoleic acid is a hydrophobically stabilized nitric oxide donor

The aqueous decay and concomitant release of nitric oxide (*NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO2) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of *NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO2 decay and *NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of *NO from LNO2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodide-based analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of *NO via pathways subject to regulation by the relative distribution of LNO2 to hydrophobic versus aqueous microenvironments.     

Electrochemical detection of uric acid via uricase-immobilized graphene oxide

Measurement of the uric acid level in the body can be improved by biosensing with respect to the accuracy, sensitivity and time consumption. This study has reported the immobilization of uricase onto graphene oxide (GO) and its function for electrochemical detection of uric acid. Through chemical modification of GO using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) as cross-linking reagents, the enzyme activity of the immobilized uricase was much comparable to the free enzyme with 88% of the activity retained. The modified GO-uricase (GOU) was then subjected to electrocatalytic detection of uric acid (UA) via cyclic voltammetry (CV). For that reason, a glassy carbon electrode (GCE) was modified by adhering the GO along with the immobilized uricase to facilitate the redox reaction between the enzyme and the substrate. The modified GOU/GCE outperformed a bare electrode through the electrocatalytic activity with an amplified electrical signal for the detection of UA. The electrocatalytic response showed a linear dependence on the UA concentration ranging from 0.02 to 0.49 mM with a detection limit of 3.45 μM at 3σ/m. The resulting biosensor also exhibited a high selectivity towards UA in the presence of other interference as well as good reproducibility.     

Effect of ascorbic acid on production of nitric oxide by leukocytes

The effect of ascorbic acid on suspensions of blood formic elements was studied by the ESR method. It was shown that incubation of a suspension of formic blood elements in the presence of ascorbic acid leads to the appearance of nitric oxide, which is produced by leukocytes and partially probably by thrombocytes. The formation of nitric oxide is evidenced by the appearance of nitrosyl complexes heme-NO. In this case, hemoglobin of erythrocytes serves as a natural trap for nitric oxide.     

Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin

In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.     

Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide

Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO(2)(*-)) and nitric oxide (J*NO). The inactivation was also observed with varying J*NO/JO(2)(*-) ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to J*NO and JO(2)(*-) in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant J*NO and JO(2)(*-). The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metal-catalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a *NO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions.     

Plasmodium-induced inflammation by uric acid

Infection of erythrocytes with the Plasmodium parasite causes the pathologies associated with malaria, which result in at least one million deaths annually. The rupture of infected erythrocytes triggers an inflammatory response, which is induced by parasite-derived factors that still are not fully characterized. Induced secretion of inflammatory cytokines by these factors is considered a major cause of malaria pathogenesis. In particular, the inflammatory cytokine tumor necrosis factor (TNF) is thought to mediate most of the life-threatening pathologies of the disease. Here we describe the molecular characterization of a novel pathway that results in the secretion of TNF by host cells. We found that erythrocytes infected by Plasmodium accumulate high concentrations of hypoxanthine and xanthine. Degradation of Plasmodium-derived hypoxanthine/xanthine results in the formation of uric acid, which triggers the secretion of TNF. Since uric acid is considered a "danger signal" released by dying cells to alert the immune system, Plasmodium appears to have co-evolved to exploit this warning system. Identifying the mechanisms used by the parasite to induce the host inflammatory response is essential to develop urgently needed therapies against this disease.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Modulation of nitric oxide formation by endothelial nitric oxide synthase gene haplotypes

Nitric oxide (NO) is a major regulator of the cardiovascular system. However, the effects of endothelial nitric oxide synthase (eNOS) gene polymorphisms or haplotypes on the circulating concentrations of nitrite (a sensitive marker of NO formation) and cGMP are unknown. Here we examined the effects of eNOS polymorphisms in the promoter region (T-786C), in exon 7 (Glu298Asp), and in intron 4 (4b/4a) and eNOS haplotypes on the plasma levels of nitrite and cGMP. We hypothesized that eNOS haplotypes could have a major impact on NO formation. We genotyped 142 healthy subjects by PCR-RFLP. To assess NO formation, the plasma concentrations of nitrite and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Haplotypes were inferred using the PHASE 2.1 program. No significant differences were found in age, body mass index, systolic and diastolic arterial blood pressure, heart rate, total cholesterol, triglycerides, cGMP, or nitrite among the genotype groups for the three polymorphisms studied here (all p>0.05). Interestingly, the C-4b-Glu haplotype was associated with lower plasma nitrite concentrations than those found in the other haplotype groups (p<0.05), but not with different cGMP levels (p>0.05). These findings suggest that eNOS gene variants combined within a specific haplotype modulate NO formation, although individual eNOS polymorphisms probably do not have major effects.     

Salicylic acid activates nitric oxide synthesis in Arabidopsis

The relationship between nitric oxide (NO) and salicylic acid (SA) was investigated in Arabidopsis thaliana. Here it is shown that SA is able to induce NO synthesis in a dose-dependent manner in Arabidopsis. NO production was detected by confocal microscopic analysis and spectrofluorometric assay in plant roots and cultured cells. To identify the metabolic pathways involved in SA-induced NO synthesis, genetic and pharmacological approaches were adopted. The analysis of the nia1,nia2 mutant showed that nitrate reductase activity was not required for SA-induced NO production. Experiments performed in the presence of a nitric oxide synthase (NOS) inhibitor suggested the involvement of NOS-like enzyme activity in this metabolic pathway. Moreover, the production of NO by SA treatment of Atnos1 mutant plants was strongly reduced compared with wild-type plants. Components of the SA signalling pathway giving rise to NO production were identified, and both calcium and casein kinase 2 (CK2) were demonstrated to be involved. Taken together, these results suggest that SA induces NO production at least in part through the activity of a NOS-like enzyme and that calcium and CK2 activity are essential components of the signalling cascade.