Excessive Nitric Oxide Formation Research Articles

Serum ADMA levels were positively correlated with EDSS scores in patients with multiple sclerosis

Multiple sclerosis (MS) is an autoinflammatory, chronic central nervous system disease. In the pathogenesis of the disease increased nitric oxide (NO) levels play an important role. Nitric oxide (NO) has neuroprotective effects in physiological conditions, however, it is thought that excessive NO formation in MS may lead to demyelination and axonal damage. Derivatives of methylarginine including asymmetric dimethyl arginine (ADMA), L-N monomethyl arginine (L-NMMA), symmetric dimethyl arginine (SDMA) directly or indirectly reduce NO production. Our aim was to measure the levels of methylarginine derivatives and citrulline, ornithine, arginine, homoarginine levels, which are metabolites associated with NO metabolism, in MS subgroups.

Minocycline attenuates streptomycin-induced cochlear hair cell death by inhibiting protein nitration and poly (ADP-ribose) polymerase activation.

This study aimed to elucidate the protective effect of minocycline against streptomycin-induced damage of cochlear hair cells and its mechanism. Cochlear membranes were isolated from newborn Wistar rats and randomly divided into control, 500μmol/L streptomycin, 100μmol/L minocycline, and streptomycin and minocycline treatment groups. Hair cell survival was analyzed by detecting the expression of 3-nitrotyrosine (3-NT) in cochlear hair cells by immunofluorescence and an enzyme-linked immunosorbent assay. Expression of 3-NT and inducible nitric oxide synthase (iNOS), and poly (ADP-Ribose) polymerase (PARP) and caspase-3 activation were evaluated by western blotting. The results demonstrated hair cell loss at 24h after streptomycin treatment. No change was found in supporting cells of the cochleae. Minocycline pretreatment improved hair cell survival and significantly reduced the expression of iNOS and 3-NT in cochlear tissues compared with the streptomycin treatment group. PARP and caspase-3 activation was increased in the streptomycin treatment group compared with the control group, and pretreatment with minocycline decreased cleaved PARP and activated caspase-3 expression. Minocycline protected cochlear hair cells from injury caused by streptomycin in vitro. The mechanism underlying the protective effect may be associated with the inhibition of excessive formation of nitric oxide, reduction of the nitration stress reaction, and inhibition of PARP and caspase-3 activation in cochlear hair cells. Combined minocycline therapy can be applied to patients requiring streptomycin treatment.

PO-0052 Nitric Oxide, Immunoglobulins And Lipid Peroxidation In Type 1 Diabetic Children

<h3>Background and aims</h3> To assess the levels of lipid peroxidation and circulating levels of nitric oxide (NO), lipoproteins and immunoglobulins in type 1 diabetes children. <h3>Methods</h3> Thirty (30) type 1 diabetic patients newly diagnosed and 30 healthy control subjects, comparable for age (less than 15 years), sex and body mass index (BMI) were recruited in the Department of Paediatrics in the Mother and Child Hospital of Tlemcen. Lipid peroxidation was assessed by measuring the levels of malondialdehyde (MDA, CH2 (CHO) 2) using the thiobarbituric acid (TBA). The serum NOx (nitrate and nitrite, NOx [NO2-, NO3-]) was measured as an indirect marker of the formation of NO <i><i>in vivo</i></i> by the Griess method. Lipoproteins were measured by ultrasensitive gel electrophoresis (SEBIA, France). Immunoglobulins were determined by the radial immunodiffusion technique (IDR). <h3>Results</h3> Circulating levels of MDA and NO production were significantly higher in type 1 diabetic patients compared to controls (respectively, p = 0.001, p = 0.01). This was also the same for immunoglobulins A, G and M (for all comparisons, p &lt; 0.01). Circulating levels of alpha lipoprotein and Lp (a) were similar in both groups (p &gt; 0.05); however, those of the pre-beta and beta lipoproteins were significantly increased in patients compared to controls (respectively, p = 0.039, p = 0.018). <h3>Conclusion</h3> The onset of the DT1 is associated with nitrogen stress and oxidation of circulating lipids. Also, the excessive formation of NO and MDA may be the result of inflammatory conditions associated with the autoimmune disease process.

Influence of the dual COX-2/5-LOG inhibitor on the activity of L-arhinin/NO system in rat's blood with experimental ulcer of the stomach

A large spread of stomach ulcer causes the search for new effective gastroprotective drugs, which would possess antioxidant properties. It is established that the disease is accompanied by the imbalance in the system L-arhinin/NO. This is manifested by the activation of the NO-synthase pathway of L-arginine and is accompanied by excessive formation of nitric oxide. It is proved that the use of dual inhibitor COX-2/5-LOG normalizes deviation in the L-arhinin/NO system. This shows antioxidant and cytoprotective properties of the substance.

Sodium nitrite protects against kidney injury induced by brain death and improves post-transplant function

Renal injury induced by brain death is characterized by ischemia and inflammation and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 hours of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 hours of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain dead rats showed significant improvement in function over the first 2 to 4 days post transplantation compared to untreated brain dead animals. Gene microarray analysis after 2 hours of brain death without or with nitrite therapy showed the latter significantly altered the expression of about 400 genes. Ingenuity Pathway analysis indicated multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription and genes related to humoral immune responses. Thus, nitrite-therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.

Differential uptake and metabolism of nitrite in normoxic and hypoxic goldfish

Nitrite is a physiologically important nitric oxide donor at low concentrations but becomes toxic at high concentrations, as develops in freshwater fish exposed to environmental nitrite. We hypothesized that nitrite uptake across the gills differs between normoxic and hypoxic fish and that nitrite accumulation causes excess nitric oxide formation and nitrosative stress. Nitrite and its metabolites were measured via chemiluminescence in normoxic and hypoxic goldfish in control conditions and after 1 day of nitrite exposure. Exposure to nitrite produced much higher nitrite levels in plasma, red blood cells (RBCs) and muscle tissue of normoxic than hypoxic goldfish, suggesting that nitrite uptake was augmented by normoxia in spite of a predictable lower gill surface area. Elevation of nitrite was associated with increased concentrations of S-nitroso, N-nitroso and Fe-nitrosyl compounds in both extracellular and intracellular compartments, revealing nitrosative stress with extensive nitros(yl)ation of thiols, amines and heme groups. The degree of nitrosative stress correlated with nitrite load. Nitrate levels increased in all compartments, reflecting that a significant fraction of the nitrite taken up was converted to non-toxic nitrate. The generation of methemoglobin and nitrosylhemoglobin (assessed by spectral deconvolution) was more pronounced during normoxic nitrite exposure than during hypoxic nitrite exposure, in agreement with the higher nitrite load in normoxic fish. However, at any given nitrite load inside RBCs, the formation of S-nitroso compounds was augmented by hypoxia. We conclude that ambient oxygen conditions have profound influence on branchial nitrite uptake and that nitrosative stress is an integral part of nitrite toxicity at high nitrite concentrations.

Increased inducible nitric oxide synthase and arginase II expression in heart failure: no net nitrite/nitrate production and protein S-nitrosylation

Our objective was to address the balance of inducible nitric oxide (NO) synthase (iNOS) and arginase and their contribution to contractile dysfunction in heart failure (HF). Excessive NO formation is thought to contribute to contractile dysfunction; in macrophages, increased iNOS expression is associated with increased arginase expression, which competes with iNOS for arginine. With substrate limitation, iNOS may become uncoupled and produce reactive oxygen species (ROS). In rabbits, HF was induced by left ventricular (LV) pacing (400 beats/min) for 3 wk. iNOS mRNA [quantitative real-time PCR (qRT-PCR)] and protein expression (confocal microscopy) were detected, and arginase II expression was quantified with Western blot; serum arginine and myocardial nitrite and nitrate concentrations were determined by chemiluminescence, and protein S-nitrosylation with Western blot. Superoxide anions were quantified with dihydroethidine staining. HF rabbits had increased LV end-diastolic diameter [20.0 + or - 0.5 (SE) vs. 17.2 + or - 0.3 mm in sham] and decreased systolic fractional shortening (11.1 + or - 1.4 vs. 30.6 + or - 0.7% in sham; both P < 0.05). Myocardial iNOS mRNA and protein expression were increased, however, not associated with increased myocardial nitrite or nitrate concentrations or protein S-nitrosylation. The serum arginine concentration was decreased (124.3 + or - 5.6 vs. 155.4 + or - 12.0 micromol/l in sham; P < 0.05) at a time when cardiac arginase II expression was increased (0.06 + or - 0.01 vs. 0.02 + or - 0.01 arbitrary units in sham; P < 0.05). Inhibition of iNOS with 1400W attenuated superoxide anion formation and contractile dysfunction in failing hearts. Concomitant increases in iNOS and arginase expression result in unchanged NO species and protein S-nitrosylation; with substrate limitation, uncoupled iNOS produces superoxide anions and contributes to contractile dysfunction.

GRAPE SEED PROANTHOCYANIDIN EXTRACT CHELATES IRON AND ATTENUATES THE TOXIC EFFECTS OF 6-HYDROXYDOPAMINE: IMPLICATIONS FOR PARKINSON'S DISEASE

ABSTRACT Proanthocyanidins are potent antioxidants associated with protection against diseases. We tested the reducing capacity, iron chelating activity, and anti-auto-oxidation ability of grape seed proanthocyanidin extract (GSPE). The mechanisms underlying GSPE attenuation of oxidative processes induced by 6-hydroxydopamine (6-OHDA), a neurotoxin used to induce Parkinson's disease, were investigated in cell-based systems. At high concentrations, GSPE (50 µg/µL) was a mild pro-oxidant in a Fenton-type reaction. GSPE (300 µg/mL) was as potent as 30 µM deferoxamine in its iron-chelating capacity, and as efficient as 5 mM ascorbic acid in delaying 6-OHDA auto-oxidation. In PC-12 cell cultures, 100 and 300 µg/mL GSPE significantly protected (P < 0.05) cells from 6-OHDA-induced (400 µM) toxicity. GSPE-induced cytoprotection is enhanced by a nitric oxide synthase inhibitor (NOSI), implying that the cytoprotective effect of GSPE does not require NOS activation. In conclusion, the iron-chelating activity of GSPE minimizes its pro-oxidant activity and delays 6-OHDA auto-oxidation to provide cytoprotection. PRACTICAL APPLICATIONS Parkinson's disease is a neurodegenerative disorder characterized by the degeneration of dopaminergic neurons. The recognized pharmacological strategies to prevent or treat Parkinson's disease include the minimization of oxidative stress, iron release and excitotoxicity resulting from excess nitric oxide formation. One of the best ways to delay or prevent the onset of the disease is to improve the biological antioxidant status by providing additional radical scavengers that are not pro-oxidants. The pro-oxidant activity, such as that of the antioxidant ascorbic acid, enhances radical cycling under certain conditions, and therefore may be detrimental. Grape seed proanthocyanidin extracts (GSPEs) are used as a dietary supplement in food products in several countries. Our current report provides evidence that GSPE has limited pro-oxidant activity, presumably because of its iron-chelating abilities, and protects cells from neurotoxic insults. GSPE may be effective as a dietary supplement for prophylactic use against the progressive neurodegeneration seen in Parkinson's disease.

Hypoxic conditioning suppresses nitric oxide production upon myocardial reperfusion.

Physiologically modulated concentrations of nitric oxide (NO) are generally beneficial, but excessive NO can injure myocardium by producing cytotoxic peroxynitrite. Recently we reported that intermittent, normobaric hypoxia conditioning (IHC) produced robust cardioprotection against infarction and lethal arrhythmias in a canine model of coronary occlusion-reperfusion. This study tested the hypothesis that IHC suppresses myocardial nitric oxide synthase (NOS) activity and thereby dampens explosive, excessive NO formation upon reperfusion of occluded coronary arteries. Mongrel dogs were conditioned by a 20 d program of IHC (FIO(2) 9.5-10%; 5-10 min hypoxia/cycle, 5-8 cycles/d with intervening 4 min normoxia). One day later, ventricular myocardium was sampled for NOS activity assays, and immunoblot detection of the endothelial NOS isoform (eNOS). In separate experiments, myocardial nitrite (NO(2)(-)) release, an index of NO formation, was measured at baseline and during reperfusion following 1 h occlusion of the left anterior descending coronary artery (LAD). Values in IHC dogs were compared with respective values in non-conditioned, control dogs. IHC lowered left and right ventricular NOS activities by 60%, from 100-115 to 40-45 mU/g protein (P < 0.01), and decreased eNOS content by 30% (P < 0.05). IHC dampened cumulative NO(2)(-) release during the first 5 min reperfusion from 32 +/- 7 to 14 +/- 2 mumol/g (P < 0.05), but did not alter hyperemic LAD flow (15 +/- 2 vs. 13 +/- 2 ml/g). Thus, IHC suppressed myocardial NOS activity, eNOS content, and excessive NO formation upon reperfusion without compromising reactive hyperemia. Attenuation of the NOS/NO system may contribute to IHC-induced protection of myocardium from ischemia-reperfusion injury.

CGMP-Dependent Protein Kinase I and Smooth Muscle Relaxation

See related article, pages 1096–1103 The maintenance of vascular tone is central to the regulation of blood pressure and tissue perfusion and plays a role in the pathogenesis of hypertension and atherosclerosis. Vascular tone is determined by the balance of vasodilator and vasoconstrictor stimuli. After several decades of research, the NO/cGMP/cGMP-dependent protein kinase (cGK) pathway is now recognized as an important mediator of vasodilation. However, the mechanisms by which cGK causes smooth muscle relaxation continue to be an important question. Smooth muscle contraction and relaxation are tightly coupled to the phosphorylation and dephosphorylation, respectively, of the regulatory myosin light chain.1 Myosin light chain phosphorylation state is determined by the relative activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). MLCK phosphorylates MLC leading to contraction,2 and MLCP dephosphorylates MLC, leading to relaxation3 (Figure). Both MLCK and MLCP activities are highly regulated. MLCK activity is activated by the binding of calcium/calmodulin and thus is the primary mechanism linking intracellular calcium concentration to smooth muscle contractility.4 MLCP activity is regulated by both vasodilator and vasoconstrictor stimuli, and is therefore responsible for much of the calcium-independent regulation of contractility (reviewed in5). Figure. MLC phosphorylation determines smooth muscle contractility. Contractile agonists lead to inositol 1,4,5 triphosphate (IP3) production or activation of RhoA (RhoA-GTP). IP3 binding to its receptor in the sarcoplasmic reticulum leads to release of Ca2+. Ca2+/calmodulin binds to and activates MLCK, which in turn phosphorylates MLC (calcium-dependent contraction). Activated RhoA binds to and activates ROCK, leading to phosphorylation and inhibition of MLCP, inhibiting MLC dephosphorylation (calcium-independent contraction). cGKI mediates relaxation by inhibiting both calcium-dependent and -independent contraction. cGKIα activates MLCP by a direct interaction and by inhibition of RhoA, and activates RGS2 to inhibit Gαq …

The effect of iNOS deletion on hepatic gluconeogenesis in hyperdynamic murine septic shock

To investigate the role of the inducible nitric oxide synthase activation-induced excess nitric oxide formation on the rate of hepatic glucose production during fully resuscitated murine septic shock. Prospective, controlled, randomized animal study. University animal research laboratory. Male C57Bl/6 and B6.129P2-Nos2(tm1Lau)/J (iNOS-/-) mice. Fifteen hours after cecal ligation and puncture, anesthetized, mechanically ventilated and instrumented mice (wild-type controls, n = 13; iNOS-/-, n = 12; wild-type mice receiving 5 mg.kg(-1) i.p. of the selective iNOS inhibitor GW274150 immediately after cecal ligation and puncture, n =8) received continuous i.v. hydroxyethylstarch and norepinephrine to achieve normotensive and hyperdynamic hemodynamics. Measurements were recorded 18, 21 and 24 h after cecal ligation and puncture. Liver microcirculatory perfusion and capillary hemoglobin O2 saturation (laser Doppler flowmetry and remission spectrophotometry) were well maintained in all groups. Despite significantly lower norepinephrine doses required to achieve the hemodynamic targets, the rate of hepatic glucose production (gas chromatography--mass spectrometry measurements of tissue isotope enrichment during continuous i.v. 1,2,3,4,5,6-13C6-glucose infusion) at 24 h after cecal ligation and puncture was significantly higher in both iNOS-/- and GW274150-treated mice, which was concomitant with a significantly higher hepatic phosphoenolpyruvate carboxykinase activity (spectrophotometry) in these animals. In normotensive, hyperdynamic septic shock, both pharmacologic and genetic deletion of the inducible nitric oxide synthase allowed maintenance of hepatic glucose production, most likely due to maintained activity of the key regulatory enzyme of gluconeogenesis, phosphoenolpyruvate carboxykinase.

Hypoxic Conditioning Suppresses Cytotoxic Nitric Oxide Production Upon Myocardial Reperfusion

Background Physiologocal concentrations of nitric oxide (NO) are generally beneficial, but excessive NO formation can injure ischemic myocardium by producing cytotoxic peroxynitrite upon reperfusion. Recently we reported that intermittent hypoxia conditioning (IHC) produced remarkable cardioprotection against infarction and lethal arrhythmias in a canine model of coronary occlusion-reperfusion. Hypothesis IHC suppresses myocardial formation of NO by the endothelial NO synthase isoform (eNOS) upon coronary artery reperfusion. Methods Mongrel dogs were conditioned by a 20 d program of intermittent normobaric hypoxia (FIO2 9.5- 10%; 5–10 min/cycle, 5–8 cycles/d with intervening 4 min normoxia), and compared with nonhypoxic control dogs. One day later, myocardium was sampled for measurement of left ventricular NOS activity (colorimetric assay) and eNOS content (Western blot). In other anesthetized dogs, myocardial nitrite release, an index of NO formation, was measured at baseline and during reperfusion following 1 h occlusion of the left anterior descending coronary artery (LAD). Results IHC lowered left ventricular NOS activity 45%, from 98 ± 10 to 55 ± 10 mU/g protein (P<0.01), and sharply decreased eNOS content (Figure). IHC reduced cumulative NO2− release during the first 5 min reperfusion from 26.4 ± 7.5 to 11.8 ± 1.8 μmol/g (P<0.05), but did not alter hyperemic LAD flow (15.2 ± 2.1 vs. 14.4 ± 2.4 ml/g). Conclusion IHC suppressed myocardial NOS activity and eNOS content. Reperfusion NO release was decreased in IHC myocardium without compromising reactive hyperemia. (supported by NIH grants HL-64785 and HL-71684)

Distribution and expression pattern of the nitrergic system in the cerebellum of the sheep

The nitrergic system produces nitric oxide as an atypical neurotransmitter in the nervous system. Nitric oxide is produced from l-arginine through specific enzymes known as nitric oxide synthases. Of these, the more abundant form in neurons is the constitutive neuronal nitric oxide synthase, although the inducible isoform can be expressed as well, especially following stress or other injuries. The excessive formation of nitric oxide results in protein nitration, particularly at tyrosine residues, thus the presence of nitrotyrosine can be used as a marker of nitric oxide production. In previous studies we have shown the distribution of the components of the nitrergic system in the cerebellum of rodents, where neuronal nitric oxide synthase immunoreactivity was present in stellate and basket cells, and occasionally in granule cells. Here, we present evidence that in the sheep, as a model of larger mammals, most cerebellar neurons display an intense immunostaining for neuronal nitric oxide synthase, including unipolar brush cells, and Lugaro and Golgi neurons, which are not immunoreactive in rodents. In addition, weak immunoreactivity for inducible nitric oxide synthase and nitrotyrosine was found in particular cell types, indicating a basal expression for these markers. Our results suggest a larger dependence on the nitrergic system for the cerebella of larger mammals. Since this increase happens in both activating and inhibitory neurons of the cerebellar circuitry, we propose that in these animals there is a higher steady-state regulation of the cerebellum based on nitric oxide.

Release and hemodynamic influence of nitro-glycerine-derived nitric oxide in endotoxemic rats

Background and aim Nitric oxide released from nitro-glycerine (NG) has been considered to improve the microcirculation. Septic conditions are, however, associated with excessive formation of nitric oxide (NO), which is formed from l-arginine by the inducible NO synthase (iNOS) activity. Since the characteristics and influence of NG-derived NO in sepsis remains unclear, the major aims of the present study were to quantify the release and to determine the effects of NO formed from NG on systemic blood pressure under endotoxemic conditions. Material and methods Four hours following endotoxin challenge (10 mg/kg intraperitoneally), rats received an infusion of NG (0.5 or 5.0 μmol/kg/h) over 45 min. We determined the changes in blood pressure and the NO concentrations generated in brain, heart, intestine, kidney, liver, and lung by means of NO trapping and EPR technique. Results NG infusion in control rats and endotoxin challenge decreased systemic blood pressure to the same extent. However, in rats subjected to endotoxin challenge NG infusion did not affect the blood pressure. The endotoxin-induced increase in tissue NO concentrations were found to be 15-folds higher than tissue levels of NO following NG infusion. Conclusion Our results suggest that under endotoxic shock conditions in rats NG may not additionally affect the systemic blood pressure. This may relate to the excessive tissue NO levels induced by endotoxin that are not further increased by NG.

EFFECTS OF L-ARGININE AND NG-NITRO L-ARGININE METHYL ESTER ON LIPID PEROXIDE, SUPEROXIDE DISMUTASE AND NITRATE LEVELS AFTER EXPERIMENTAL SCIATIC NERVE ISCHEMIA-REPERFUSION IN RATS

Nitric oxide (NO) has been reported to function in both cytoprotective and cytotoxic tissue ischemia-reperfusion (I/R). In this study, we evaluated the effects of L-arginine, the substrate for NO, and NG-nitro L-arginine methyl ester (L-NAME), NO synthase (NOS) inhibitor on super--oxide dismutase (SOD) enzyme activity, malondialdehyde (MDA), a marker of lipid peroxidation, nitrate levels, and histopathological structure in rat sciatic nerve 2 h after ischemia, followed by 3 h of reperfusion. Reperfusion resulted in a significant increase in lipid peroxidation level and a decrease in nitrate level of the sciatic nerve. The increased level of lipid peroxidation was partly reduced by NOS inhibition. The decrease in sciatic nerve SOD level, observed in group subjected to I/R, was prevented by inhibition of NOS by L-NAME. These results were supported by histological findings that in the L-arginine-treated group, degenerations of both myelin sheath and axon were observed, while in the L--NAME-treated group, no pathological changes were detected. Our results suggested that excessive NO formation accelerates lipid peroxidation, as well as axonal degeneration on the early reperfusion period of the sciatic nerve

The role of inducible nitric oxide synthase inhibitor on the arteriolar hyporesponsiveness in hemorrhagic-shocked rats

Hemorrhagic shock (HS) has been implicated in the induction of inducible nitric oxide synthase (iNOS) that leads to increase production of nitric oxide (NO). Recently, NO has been implicated to cause hyporesponsiveness of blood vessel in vitro towards vasoconstrictors in refractory (decompensated) HS. In our in vivo model, we examined the effects of aminoguanidine (AG), a known iNOS inhibitor, with angiotensin II (ANG II), a vasoconstrictor, following hemorrhagic shock decompensatory phase (HSDP) on percentage survival, vascular responsiveness, mean arterial blood pressure (MABP), heart rate and mean nitrate/nitrite levels in anaesthetized rats. HSDP (3 h) was achieved via constant pressure method (40–45 mmHg). MABP and heart rate was measured via the left carotid artery. Plasma collected from HSDP rats was used to measure nitrate/nitrite levels. Vascular hyporeactivity to ANG II was carried out using HSDP aortic strips, precontracted with KCl and noradrenaline. Sham-operated rats served as controls. HSDP rats decreased percentage survival, vascular contractility to ANG II and noradrenaline, MABP, heart rate while showing increased levels of nitrate/nitrite. Infusion of AG with ANG II, increased percentage survival and had reversed these cardiovascular effects of HSDP rats. This study indicates that excessive NO formation from iNOS activity induces vascular hyporeactivity and decompensation in HSDP. This might suggest that selective NOS inhibitor, AG, when coupled with ANG II, show reduction in NO's effect in HSDP.

Does nitric oxide play a pathogenic role in hepatitis C virus infection?

In spite of important progress in understanding the mechanisms of hepatitis C virus (HCV) pathogenesis in the last decade, we still lack firm experimental evidence to explain the two major features of the natural history of this viral infection: viral persistence and hepatic damage. Nevertheless, recent information from both in vitro HCV replication systems and animal models of HCV infection have led to a plethora of critical observations showing that host immune responses play a critical role influencing the outcome of HCV-induced liver disease, such as favoring virus persistence and by mediating liver cell injury.1 In an attempt to reconcile these apparently opposite pathogenic pathways, the increasing evidence that nitric oxide (NO) is one of the most versatile mediators in the control of viral infections, being the earliest host's antiviral response2, as well as in the pathogenesis of many human infectious and inflammatory diseases,3,4 acting as a pro-apoptotic inducer in some cell types or as an anti-apoptotic modulator in other cell types including hepatocytes,5 makes it reasonable to consider this multifunctional molecule as a potential player in HCV pathogenesis. This notion has been reinforced since the observation in the liver of HCV-infect ed patients of an enhanced iNOS expression, implying an excessive NO formation, positively correlated with viral load and hepatic inflammation.6,7,8 The ultimate effect of iNOS-derived NO on HCV pathogenesis, however, still remains far from completely understood but, in this commentary, we will try to shed light on the NO paradox in the pathogenesis of HCV infection.

PPA250 [3-(2,4-Difluorophenyl)-6-{2-[4-(1H-imidazol-1-ylmethyl) Phenoxy]ethoxy}-2-phenylpyridine], a Novel Orally Effective Inhibitor of the Dimerization of Inducible Nitric-Oxide Synthase, Exhibits an Anti-Inflammatory Effect in Animal Models of Chronic Arthritis

Nitric oxide (NO) plays an important role in various physiological processes. Excessive NO production is closely related to inflammatory and autoimmune diseases such as septic shock and rheumatoid arthritis. Suppression of excess NO formation in participating cells may be helpful in improving disease status. In this study, we examined the effects of a newly synthesized imidazole derivative, 3-(2,4-difluorophenyl)-6-[2-[4-(1H-imidazol-1-ylmethyl) phenoxy]ethoxy]-2-phenylpyridine (PPA250), on NO production in vitro and in vivo, as well as on the dimerization of inducible nitric-oxide synthase (iNOS). PPA250 at concentrations of 25 nM and higher inhibited NO production in activated mouse macrophage-like RAW264.7 cells. The IC(50) was approximately 82 nM. Western blot analysis revealed that PPA250 prevents dimerization of iNOS but has no effect on transcription and translation. In addition, oral administration of PPA250 (10 mg/kg and higher) reduced the NO concentration in serum from mice in which sepsis was induced by bacterial lipopolysaccharide. Since the inhibitory activity was observed not only in vitro but also in vivo, we examined the therapeutic potential of PPA250 in two animal models of arthritis, collagen-induced arthritis in mice and adjuvant arthritis in rats. PPA250 suppressed the development of a destructive polyarthritis in both models after the appearance of clinical signs. These results indicate that inhibitors of iNOS homodimerization, including PPA250, could be useful therapeutic agents for inflammatory and autoimmune diseases, such as rheumatoid arthritis, in which NO is involved.

Nitric oxide toxicity in CNS white matter: an in vitro study using rat optic nerve

Excessive nitric oxide formation may contribute to the pathology occurring in diseases affecting central white matter, such as multiple sclerosis. The rat isolated optic nerve preparation was used to investigate the potential toxicity of the molecule towards such tissue. The nerves were exposed to a range of concentrations of different classes of nitric oxide donor for up to 23 h, with or without a subsequent period of recovery, and the damage assessed by quantitative histological methods. Degeneration of axons and macroglia occurred in a time- and concentration-dependent manner, the order of susceptibility being: axons>oligodendrocytes>astrocytes. Use of NONOate donors differing in half-life indicated that nitric oxide delivered in an enduring manner at relatively low concentration was more toxic than the same amount supplied rapidly at high concentration. The mechanism by which nitric oxide affects axons was studied using a donor [3-( n-propylamino)propylamine/NO adduct, PAPA/NO] with an intermediate half-life that produced selective axonopathy after a 2-h exposure (plus 2 h recovery). Axon damage was abolished if, during the exposure, Na + or Ca 2+ was removed from the bathing medium or the sodium channel inhibitors tetrodotoxin or BW619C89 (sipatrigine) were added. In electrophysiological experiments, the donor elicited a biphasic depolarisation. The second, larger component (occurring after 7–10 min) was associated with a block of nerve conduction and could be inhibited by tetrodotoxin. Coincident with the secondary depolarisation was a reduction in ATP levels by about 50%, an effect that was also inhibited by tetrodotoxin. It is concluded that nitric oxide, in submicromolar concentrations, can kill axons and macroglia in white matter. The findings lend support to the hypothesis that nitric oxide may be of importance to white matter pathologies, particularly those in which inducible nitric oxide synthase is expressed. The axonopathy, at least when elicited over relatively short time intervals, is likely to be caused by metabolic inhibition. As in anoxia and anoxia/aglycaemia, nitric oxide-induced destruction of axons is likely to be caused by the Ca 2+ overload that follows a reduction in ATP levels in the face of continued influx of Na + through voltage-dependent channels.

Impairment of brain mitochondrial function by reactive nitrogen species: the role of glutathione in dictating susceptibility

Mitochondrial enzymes involved in energy metabolism display varying degrees of sensitivity towards reactive nitrogen species such as peroxynitrite (ONOO −). With regards to the electron transport chain, cytochrome oxidase appears particularly sensitive. Inhibition of this component may lead to an increase in mitochondrial superoxide formation, exacerbation of cellular oxidative stress and further mitochondrial damage. Impairment of the electron transport chain may lead to a loss of membrane potential, ATP deficiency, opening of the permeability transition pore and the release of factors capable of initiating apoptosis. Reduced glutathione will react, via a number of diverse reactions, with reactive nitrogen species and hence is capable of limiting mitochondiral damage. Loss of brain glutathione may therefore be an important factor in those neurological conditions in which there is evidence of excessive nitric oxide formation and mitochondrial damage.