Bcl-xL interrupts oxidative activation of neutral sphingomyelinase

Recent studies demonstrate a role for intracellular oxidation in the regulation of neutral sphingomyelinase (N-SMase). Glutathione (GSH) has been shown to regulate N-SMase in vitro and in cells. However, it has not been established whether the effects of GSH in cells are due to direct action on N-SMase. In this study, treatment of human mammary carcinoma MCF-7 cells with diamide, a thiol-depleting agent, caused a decrease in intracellular GSH and degradation of sphingomyelin (SM) to ceramide. The SM pool hydrolyzed in response to diamide belonged to the bacterial SMase-resistant pool of SM. Importantly, pretreatment of MCF-7 cells with GSH, N-acetylcysteine, an antioxidant, or GW69A, a specific N-SMase inhibitor, prevented diamide-induced degradation of SM to ceramide, suggesting that intracellular levels of GSH regulate the extent to which SM is degraded to ceramide and that this probably involves a GW69A-sensitive N-SMase. Unexpectedly, expression of Bcl-xL prevented tumor necrosis factor--induced SM hydrolysis and ceramide accumulation but not the decrease in intracellular GSH. Furthermore, Bcl-xL inhibited diamide-induced SM hydrolysis and ceramide accumulation but not the decrease in intracellular GSH. These results suggest that the site of action of Bcl-xL is downstream of GSH depletion and upstream of ceramide accumulation, and that GSH probably does not exert direct physiologic effects on N-SMase.     

Identification of multiple forms of membrane-associated neutral sphingomyelinase in bovine brain

Many different stimuli such as bioactive agents and environmental stresses are known to cause the activation of sphingomyelinase (SMase), which hydrolyzes sphingomyelin to generate ceramide as a second messenger playing a key role in differentiation and apoptosis in various cell types. Here we identified multiple forms of the membrane-associated neutral SMase (N-mSMase) activity in bovine brain. They could be classified into two groups according to extracting agents: group T-mSMase, extracted with 0.2% Triton X-100, and group S-mSMase, extracted with 0.5 M (NH(4))(2)SO(4). Group T-mSMase: alpha, beta, gamma, and delta, which were extensively purified from 40,000-g pellets of bovine brain homogenates by 3,150-, 5,275-, 1,665-, and 2,556-fold over the membrane extracts, respectively, by sequential use of several column chromatographies. On the other hand, S-mSMase was eluted as two active peaks of S-mSMase epsilon and zeta in a phenyl-5PW hydrophobic HPLC column and further purified by 1,119- and 976-fold over 40,000-g pellets of the homogenates, respectively. These highly purified N-mSMase enzyme preparations migrated as several bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed many different features in biochemical properties such as pH dependence, Mg(2+) requirements, and effects of detergents. Taken together, our data strongly suggest that mammalian brain N-mSMase may exist as multiple forms different in both its chromatographic profiles and biochemical properties.     

Apoptosis induced by capsaicin in prostate PC-3 cells involves ceramide accumulation,neutral sphingomyelinase,and JNK activation

Numerous studies have recently focused on the anticarcinogenic, antimutagenic, or chemopreventive activities of the main pungent component of red pepper, capsaicin (N-vanillyl-8-methyl-1-nonenamide). We have previously shown that, in the androgen-independent prostate cancer PC-3 cells, capsaicin inhibits cell growth and induces apoptosis through reactive oxygen species (ROS) generation [Apoptosis 11 (2006) 89–99]. In the present study, we investigated the signaling pathways involved in the antiproliferative effect of capsaicin. Here, we report that capsaicin apoptotic effect was mediated by ceramide generation which occurred by sphingomyelin hydrolysis. Using siRNA, we demonstrated that N-SMase expression is required for the effect of capsaicin on prostate cell viability. We then investigated the role of MAP kinase cascades, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in the antiproliferative effect of capsaicin, and we confirmed that capsaicin could activate ERK and JNK but not p38 MAPK. Pharmacological inhibition of JNK kinase, as well as inhibition of ROS by the reducing agent N-acetylcysteine, prevented ceramide accumulation and capsaicin-induced cell death. However, inhibition of ceramide accumulation by the SMase inhibitor D609 did not modify JNK activation. These data reveal JNK as an upstream regulator of ceramide production. Capsaicin-promoted activation of ERK was prevented with all the inhibitors tested. We conclude that capsaicin induces apoptosis in PC-3 cells via ROS generation, JNK activation, ceramide accumulation, and second, ERK activation.     

Influence of nitric oxide donors and peroxynitrite on the contractile effect and concentration of norepinephrine

Nitric oxide (NO) and peroxynitrite (ONOO) are said to destroy norepinephrine (NE). We studied the role of NE decomposition by NO donors and ONOO as they affect the contractile activity of NE in rat denuded thoracic aorta. First, we determined the relaxing effect of NO donors (SNAP, PROLI/NO, Sodium nitrite, SIN-1) and ONOO after precontraction by NE (1 microM). SNAP and SIN-1 (EC(50) 50-110 nM) were more active than PROLI/NO, Sodium nitrite or ONOO (EC(50) 19-30 microM). The relaxing effect of NO donors and ONOO were decreased by ODQ (10 microM), a guanylate cyclase inhibitor. Second, we compared the contractile activity of NE before and after preincubation with NO donors or ONOO in presence of ODQ. NE (1 microM) was incubated with NO donors or ONOO at the concentrations of 0.1 mM in both Krebs solution or phosphate buffer (pH 7.4; 0.1 M) for 10 minutes at 37 degrees C. NE evoked the aorta contraction in the same concentrations before and after preincubation with NO donors. In contrast, ONOO decreased effect of NE, EC(50) was measured at 4.3+/-0.3 nM and 13.4+/-1.6 nM, before and after preincubation of NE with ONOO respectively. Third, we measured the NE concentration using the HPLC method. We revealed that the concentration of NE after preincubation with NO donors was unaltered. However HPLC measurement revealed that NE concentration after preincubation with ONOO was reduced 2-3-fold. Therefore, under these experimental conditions ONOO, but not NO donors, was capable of destroying NE.     

Nitric oxide induces neutral ceramidase degradation by the ubiquitin/proteasome complex in renal mesangial cell cultures

The neutral ceramidase is a key enzyme in the regulation of cellular ceramide levels. Previously we have reported that stimulation of rat renal mesangial cells with nitric oxide (NO) donors leads to an inhibition of neutral ceramidase activity which is due to increased degradation of the enzyme. This and the concomitant activation of the sphingomyelinase results in an amplification of ceramide levels. Here, we show that the NO-triggered degradation of neutral ceramidase involves activation of the ubiquitin/proteasome complex. The specific proteasome inhibitor lactacystin completely reverses the NO-induced degradation of ceramidase protein and neutral ceramidase activity. As a consequence, the cellular amount of ceramide, which drastically increases by NO stimulation, is reduced in the presence of lactacystin. Furthermore, ubiquitinated neutral ceramidase accumulates after NO stimulation. In summary, our data clearly show that the ubiquitin/proteasome complex is an important determinant of neutral ceramidase activity and thereby regulates the availability of ceramide.     

Insulin renders diabetic rats resistant to acute ischemic stroke by arresting nitric oxide reaction with superoxide to form peroxynitrite

Background

The functions of free radicals on the effects of insulin that result in protection against cerebral ischemic insult in diabetes remain undefined. This present study aims to explain the contradiction among nitric oxide (NO)/superoxide/peroxynitrite of insulin in amelioration of focal cerebral ischemia–reperfusion (FC I/R) injury in streptozotocin (STZ)-diabetic rats and to delineate the underlying mechanisms. Long-Evans male rats were divided into three groups (age-matched controls, diabetic, and diabetic treated with insulin) with or without being subjected to FC I/R injury.

Results

Hyperglycemia exacerbated microvascular functions, increased cerebral NO production, and aggravated FC I/R-induced cerebral infarction and neurological deficits. Parallel with hypoglycemic effects, insulin improved microvascular functions and attenuated FC I/R injury in STZ-diabetic rats. Diabetes decreased the efficacy of NO and superoxide production, but NO and superoxide easily formed peroxynitrite in diabetic rats after FC I/R injury. Insulin treatment significantly rescued the phenomenon.

Conclusions

These results suggest that insulin renders diabetic rats resistant to acute ischemic stroke by arresting NO reaction with superoxide to form peroxynitrite.     

The role of neutral sphingomyelinase produced ceramide in lipopolysaccharide-mediated expression of inducible nitric oxide synthase

Lipopolysaccharide (LPS) and interferon-gamma (IFN) treatment of C6 rat glioma cells increased the intracellular ceramide level and the expression of the inducible nitric oxide synthase (iNOS) gene. To delineate the possible role of ceramide in the induction of iNOS, we examined the source of intracellular ceramide and associated signal transduction pathway(s) with the use of inhibitors of intracellular ceramide generation. The inhibitor of neutral sphingomyelinase (3-O-methylsphingomyelin, MSM) inhibited the induction of iNOS, whereas inhibitor of acidic sphingomyelinase (SR33557) or that of ceramide de novo synthesis (fumonisin B1) had no effect on the induction of iNOS. MSM-mediated inhibition of iNOS induction was reversed by the supplementation of exogenous C8-ceramide, suggesting that ceramide production by neutral sphingomyelinase (nSMase) is a key mediator in the induction of iNOS. The MSM-mediated inhibition of iNOS gene expression correlated with the decrease in the activity of ras. Inhibition of co-transfected iNOS promoter activity by dominant negative ras supported the role of ras in the nSMase-dependent regulation of iNOS gene. NF-kappaB DNA binding activity and its transactivity were also reduced by MSM pretreatment, and were completely reversed by the supplementation of C8-ceramide. As the dominant negative ras also reduced NF-kappaB transactivity, NF-kappaB activation may be downstream of ras. Our results suggest that ceramide generated by nSMase may be a critical mediator in the regulation of iNOS gene expression via ras-mediated NF-kappaB activation under inflammatory conditions.     

Antioxidant and pro-oxidant actions of flavonoids: effects on DNA damage induced by nitric oxide, peroxynitrite and nitroxyl anion

Antioxidant and pro-oxidant activities of flavonoids have been reported. We have studied the effects of 18 flavonoids and related phenolic compounds on DNA damage induced by nitric oxide (NO), peroxynitrite, and nitroxyl anion (NO⁻). Similarly to our previous findings with catecholamines and catechol-estrogens, DNA single-strand breakage was induced synergistically when pBR322 plasmid was incubated in the presence of an NO-releasing compound (diethylamine NONOate) and a flavonoid having an ortho-trihydroxyl group in either the B ring (e.g., epigallocatechin gallate) or the A ring (e.g., quercetagetin). Either NO or any of the above flavonoids alone did not induce strand breakage significantly. However, most of the tested flavonoids inhibited the peroxynitrite-mediated formation of 8-nitroguanine in calf-thymus DNA, measured by a new HPLC-electrochemical detection method, as well as the peroxynitrite-induced strand breakage. NO⁻ generated from Angeli’s salt caused DNA strand breakage, which was also inhibited by flavonoids but at only high concentrations. On the basis of these findings, we propose that NO⁻ and/or peroxynitrite could be responsible for DNA strand breakage induced by NO and a flavonoid having an ortho-trihydroxyl group. Our results indicate that flavonoids have antioxidant properties, but some act as pro-oxidants in the presence of NO.     

Inhibition of neutral Mg2+-dependent sphingomyelinase by ubiquinol-mediated plasma membrane electron transport

Summary.  Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, has emerged as an important lipid signaling molecule. Previous work carried out by our group has documented that plasma membrane Mg²⁺-dependent neutral sphingomyelinase can be effectively inhibited by exogenous ubiquinol. In this work, we have tested whether or not plasma-membrane-associated electron transport can also achieve this inhibition through endogenous ubiquinol. Our results have shown that Mg²⁺-dependent neutral sphingomyelinase in isolated plasma membranes was inhibited by NAD(P)H under conditions where ubiquinone is reduced to ubiquinol. This inhibition was potentiated in the presence of an extra amount of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2). Depletion of plasma membranes from lipophilic antioxidants by solvent extraction abolished the inhibition by reduced pyridine nucleotides without affecting the sensitivity of the neutral sphingomyelinase to exogenous ubiquinol. Reconstitution of plasma membranes with ubiquinone restored the ability of NAD(P)H to inhibit the enzyme. Our results support that the reduction of endogenous ubiquinone to ubiquinol by NAD(P)H-driven electron transport may regulate the activity of the plasma membrane neutral sphingomyelinase. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="**" ID="**" Present address: Department of Biomedical Engineering, School of Medicine, University of Baltimore, Maryland, U.S.A. RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio C-6, Campus Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Reduction of insulin-stimulated glucose uptake by peroxynitrite is concurrent with tyrosine nitration of insulin receptor substrate-1

Inducible nitric oxide synthetase plays an essential role in insulin resistance induced by a high-fat diet. The reaction of nitric oxide with superoxide leads to the formation of peroxynitrite (ONOO-), which can modify several proteins. In this study, we investigated whether peroxynitrite impairs insulin-signalling pathway. Our experiments showed that 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), a constitutive producer of peroxynitrite, dose-dependently inhibited insulin-stimulated glucose uptake. While SIN-1 did not affect the insulin receptor protein level and tyrosine phosphorylation, it reduced the insulin receptor substrate-1 (IRS-1) protein level, and IRS-1 associated phosphatidylinositol-3 kinase (PI-3 kinase) activity. Although SIN-1 did not induce Ser307 phosphorylation of IRS-1, tyrosine nitration of IRS-1 was detected in SIN-1-treated-Rat1 fibroblasts expressing human insulin receptors. Mass spectrometry showed that peroxynitrite induced at least four nitrated tyrosine residues in rat IRS-1, including Tyr939, which is critical for association of IRS-1 with the p85 subunit of PI-3 kinase. Our results suggest that peroxynitrite reduces the IRS-1 protein level and decreases phosphorylation of IRS-1 concurrent with nitration of its tyrosine residues.     

Nitric oxide donor-induced hyperpermeability of cultured intestinal epithelial monolayers: role of superoxide radical,hydroxyl radical,and peroxynitrite

Many of the cytopathic effects of nitric oxide (NO^·) are mediated by peroxynitrite (PN), a product of the reaction between NO^· and superoxide radical (O^·?₂). In the present study, we investigated the role of PN, O^·?₂ and hydroxyl radical (OH^·) as mediators of epithelial hyperpermeability induced by the NO^· donor, S-nitroso-N-acetylpenicillamine (SNAP), and the PN generator, 3-morpholinosydnonimine (SIN-1). Caco-2_BBe enterocytic monolayers were grown on permeable supports in bicameral chambers. Epithelial permeability, measured as the apical-to-basolateral flux of fluorescein disulfonic acid, increased after 24 h of incubation with 5.0 mM SNAP or SIN-1. Addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO^· scavenger, or Tiron, an O^·?₂ scavenger, reduced the increase in permeability induced by both donor compounds. The SNAP-induced increase in permeability was prevented by allopurinol, an inhibitor of xanthine oxidase (a source of endogenous O^·?₂). Diethyldithiocarbamate, a superoxide dismutase inhibitor, and pyrogallol, an O^·?₂ generator, potentiated the increase in permeability induced by SNAP. Addition of the PN scavengers deferoxamine, urate, or glutathione, or the OH^· scavenger mannitol, attenuated the increase in permeability induced by both SNAP and SIN-1. Both donor compounds decreased intracellular levels of glutathione and protein-bound sulfhydryl groups, suggesting the generation of a potent oxidant. These results support a role for PN, and possibly OH^·, in the pathogenesis of NO^· donor-induced intestinal epithelial hyperpermeability.     

Nitric oxide diffusion to red blood cells limits extracellular,but not intraphagosomal,peroxynitrite formation by macrophages

Macrophage-derived nitric oxide (^•NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by ^•NO itself or ^•NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O₂^•−). In vivo, the facile extracellular diffusion of ^•NO as well as different competing consumption routes limit its bioavailability for the reaction with O₂^•− and, hence, peroxynitrite formation. In this work, we evaluated the extent by which ^•NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O₂^•− and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of ^•NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of ^•NO and O₂^•−-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of ^•NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O₂^•− reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of ^•NO and O₂^•--forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, ^•NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.     

Oxidative inactivation of the lymphoid tyrosine phosphatase mediated by both general and active site directed NO donors

Oxidative modification of protein tyrosine phosphatases (PTPs) has recently been recognized as an important regulatory mechanism in biological systems. Reported herein is the oxidative inactivation of the lymphoid tyrosine phosphatase (LYP) with both the general nitrosating reagent sodium nitroprusside (SNP) and also a novel peptide-based nitrosating reagent, Ac-ARLIEDNE(HcyNO)TAREG-NH₂, where HcyNO = S-nitrosohomocysteine. The SNP oxidatively inactivated LYP with a k_inact of 0.383 per min and a K_I of 27.4 μM and mixed-type inactivation kinetics. The peptide was a competitive LYP inactivator with a k_inact of 0.0472 per min and a K_I of 7.00 μM. LYP nitrosation by SNP was characterized by the addition of several NO moieties to the enzyme, while oxidation of LYP by the peptide did not result in the formation of a LYP-NO adduct. We propose that general NO donors promiscuously nitrosate any free cysteine residue while the active-site directed peptide selectively oxidizes the catalytic cysteine residue, resulting in the formation of a disulfide bond between the catalytic cysteine residue and a second cysteine in the active site.     

The role of reactive nitrogen species in secondary spinal cord injury: formation of nitric oxide, peroxynitrite, and nitrated protein

To determine whether reactive nitrogen species contribute to secondary damage in CNS injury, the time courses of nitric oxide, peroxynitrite, and nitrotyrosine production were measured following impact injury to the rat spinal cord. The concentration of nitric oxide measured by a nitric oxide-selective electrode dramatically increased immediately following injury and then quickly declined. Nitro-L-arginine reduced nitric oxide production. The extracellular concentration of peroxynitrite, measured by perfusing tyrosine through a microdialysis fiber into the cord and quantifying nitrotyrosine in the microdialysates, significantly increased after injury to 3.5 times the basal level, and superoxide dismutase and nitro-L-arginine completely blocked peroxynitrite production. Tyrosine nitration examined immunohistochemically significantly increased at 12 and 24 h postinjury, but not in sham-control sections. Mn(III) tetrakis(4-benzoic acid)-porphyrin (a novel cell-permeable superoxide dismutase mimetic) and nitro-L-arginine significantly reduced the numbers of nitrotyrosine-positive cells. Protein-bound nitrotyrosine was significantly higher in the injured tissue than in the sham-operated controls. These results demonstrate that traumatic injury increases nitric oxide and peroxynitrite production, thereby nitrating tyrosine, including protein-bound tyrosine. Together with our previous report that trauma increases superoxide, our results suggest that reactive nitrogen species cause secondary damage by nitrating protein through the pathway superoxide + nitric oxide peroxynitrite protein nitration.     

Inhibition of the catalytic activity of rhodanese by S-nitrosylation using nitric oxide donors

Rhodanese (EC 2.8.1.1.) from bovine liver contains four reduced cysteine groups. The –SH group of cysteine 247, located in a rhodanese active centre, transfers sulfane sulfur in a form of hydrosulfide (–S–SH) from appropriate donors to nucleophilic acceptors. We aimed to discover whether S-nitrosylation of critical cysteine groups in rhodanese can inhibit activity of the enzyme by covalent modification of –SH groups.

The inhibition of rhodanese activity was studied with the use of a number of nitric oxide (NO) donors. We have successfully confirmed using several methods that the inhibition of rhodanese activity is a result of the formation of stable S-nitrosorhodanese.

Low molecular weight NO donors, such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), inactivate rhodanese and are much more effective in this regard (100% inhibition at 2.5 mM) than such known inhibitors of this enzyme, as N-ethylmaleimide (NEM) (25 mM < 50%) or sulfates(IV) (90% inhibition at 5 mM). On the other hand, sodium nitroprusside (SNP) and nitrites inhibit rhodanese activity only in the presence of thiols, which suggests that S-nitrosothiols (RSNO) also have to participate in this reaction in this case.

A demonstration that rhodanese activity can be inhibited as a result of S-nitrosylation suggests the possible mechanism by which nitric oxide may regulate sulfane sulfur transport to different acceptors.     

Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death

Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO )-producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN-1 (200 microM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/ SIN-1-treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO-scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO-) was markedly enhanced in glucose-deprived/SIN-1 -treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose-deprived/SIN-1-treated astrocytes. The glutathione synthase inhibitor L-buthionine-(S,R)-sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1-treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose-deprived/SIN-1-treated astrocytes.     

Oxidation of nitric oxide by a new heterotrophic Pseudomonas sp.

A new bacterial strain isolated from soil consumed nitric oxide (NO) under oxic conditions by oxidation to nitrate. Phenotypic and phylogenetic characterization of the new strain PS88 showed that it represents a previously unknown species of the genus Pseudomonas, closely related to Pseudomonas fluorescens and Pseudomonas putida. The heterotrophic, obligately aerobic strain PS88 was not able to denitrify or nitrify; however, strain PS88 oxidized NO to nitrate. NO was not reduced to nitrous oxide (N₂O). Nitrogen dioxide (NO₂) and nitrite (NO₂ ^–) as possible intermediates of NO oxidation to nitrate (NO₃ ^–) could not be detected. NO oxidation was inhibited under anoxic conditions and by high osmolarity, but not by nitrite. NO oxidation activity was inhibited by addition of formaldehyde, HgCl₂, and antimycin, and by autoclaving or disintegrating the cells, indicating that the process was enzyme-mediated. However, the mechanism remains unclear. A stepwise oxidation at a metalloenzyme and a radical mechanism are discussed. NO oxidation in strain PS88 seems to be a detoxification or a co-oxidation mechanism, rather than an energy-yielding process. Received: 15 November 1995 / Accepted: 24 February 1996     

The discovery of novel, potent and highly selective inhibitors of inducible nitric oxide synthase (iNOS)

By careful analysis of experimental X-ray ligand crystallographic protein data across several inhibitor series we have discovered a novel, potent and selective series of iNOS inhibitors exemplified by compound 8.     

Taurine is a weak scavenger of peroxynitrite and does not attenuate sodium nitroprusside toxicity to cells in culture

Summary. Many studies have suggested an antioxidant role for taurine, but few studies have directly measured its free radical scavenging activity. The aim of the present study was to directly determine the action of taurine and taurine analogs to inhibit peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR) to rhodamine. Taurine was also tested to determine if it could attenuate the toxicity of sodium nitroprusside (SNP) to neuronal cultures. Taurine at concentrations above 30 mM had a modest ability to inhibit peroxynitrite formation derived from SIN-1. Hypotaurine could inhibit peroxynitrite formation from both SIN-1 (↓75%) and SNP (↓50%) at 10 mM. Other taurine analogs (homotaurine, β-alanine & isethionic acid) slightly potentiated DHR oxidation by SIN-1. Short-term (1-hour) treatment of PC12 cultures with either SNP (1–2 mM) or taurine (20–40 mM) appeared to induce cellular proliferation. In contrast, 24-hour treatment with SNP (1 mM) induced cell death. Combination treatments with taurine and SNP appeared to interact in an additive fashion for both cell proliferation and neurotoxic actions. It appears unlikely that taurine is a major endogenous scavenger of peroxynitrite. Received May 9, 2000 Accepted June 13, 2000     

Translesion synthesis past 2'-deoxyxanthosine, a nitric oxide-derived DNA adduct, by mammalian DNA polymerases

Cellular DNA is damaged by nitric oxide (NO), a multifunctional bioregulator and an environmental pollutant that has been implicated in diseases associated with cancer and chronic inflammation. 2'-Deoxyxanthosine (dX) is a major NO-derived DNA lesion. To explore the mutagenic potential of dX, a 38-mer oligodeoxynucleotide ((5')CATGCTGATGAATTCCTTCXCTTCTTTCCTCTCCCTTT) modified site-specifically with dX at the X position was prepared post-synthetically and used as a DNA template in primer extension reactions catalyzed by calf thymus DNA polymerase (pol) alpha and human DNA pol beta, eta, and kappa. Primer extension reactions catalyzed by pol alpha or beta in the presence of four dNTPs were retarded at the dX lesion while pol eta and kappa readily bypassed the lesion. The fully extended products were analyzed to quantify the miscoding specificity and frequency of dX using two-phase polyacrylamide gel electrophoresis (PAGE). With pol alpha, eta and kappa, incorrect dTMP was preferentially incorporated opposite the lesion, along with lesser amounts of dCMP, the correct base. When pol beta was used, direct incorporation of correct dCMP was primarily observed, accompanied by small amounts of misincorporation of dTMP, dAMP and dGMP. Steady-state kinetic analyses supported the results obtained from the two-phase PAGE assay. dX is a miscoding lesion capable of preferentially generating G-->A mutations. The miscoding frequency varied depending on DNA polymerase used.