Nitric Oxide Toxicity Research Articles

Arginases in parasitic diseases.

Abstract Parasites have elaborated a variety of strategies for invading hosts and escaping immune responses. This article proposes that a common mechanism whereby different parasites escape nitric oxide (NO) toxicity is the activation of arginase. This leads to a depletion of l-arginine (substrate of NO synthase, resulting in lower levels of cytotoxic NO) and increased production of polyamines (necessary for parasite growth and differentiation).

Protective effect of dopamine D2 agonists in cortical neurons via the phosphatidylinositol 3 kinase cascade.

Glutamate, one of the excitatory neurotransmitters, contributes to the neuronal death associated with neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, and with ischemia. In Alzheimer's disease brains, there is a decreased number of dopamine D2 receptors, which might cause neuronal dysfunction or death. In the present study, bromocriptine exerted a protective effect against glutamate-induced cytotoxicity in rat cortical neurons. This neuroprotective effect was mediated via D2 receptors, because it was attenuated by domperidone, a D2 dopaminergic receptor antagonist. Another dopamine D2 agonist, quinpirole, also protected cells against glutamate toxicity. D2 agonists protected cells from calcium influx, nitric oxide, and peroxynitrite toxicity, which are thought to be the mediators of glutamate toxicity. The phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) inhibited this neuroprotective effect of bromocriptine, in contrast to the mitogen-activated protein kinase kinase (MAPKK) inhibitor (PD98059), which did not counter the protective effect. Furthermore, Akt protein kinase, which is an effector of PI3K, was activated by bromocriptine, and the antiapoptotic protein Bcl-2 was up-regulated by bromocriptine treatment. These results suggest that D2 dopaminergic receptor activation plays an important role in neuroprotection against glutamate cytotoxicity and that the up-regulation of Bcl-2 expression via the PI3K cascade is, at least partially, involved in this effect.

Ethanol Consumption Increases Nitric Oxide Production in Rats, and Its Peroxynitrite-Mediated Toxicity Is Attenuated by Polyenylphosphatidylcholine

Background: Nitric oxide generally mediates beneficial responses but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent. Methods: To study the effects of ethanol and polyenylphosphatidylcholine on nitric oxide metabolism and toxicity, 36 rats were pair-fed liquid diets with 36% of energy either as ethanol or as additional carbohydrate for 24 days and were killed 90 min after intragastric feeding. Half received polyenylphosphatidylcholine in the diet (3 g/liter), and the other half equivalent amounts of essential fatty acids and choline. Nitric oxide was measured by chemiluminescence in arterial blood and liver cytosol and as a product of the inducible nitric oxide synthase activity. Peroxynitrite formation was assessed by the increase in nitrotyrosine protein residues, measured immunochemically. Results: In blood, administration of ethanol with or without polyenylphosphatidylcholine doubled nitric oxide levels. In the liver, ethanol increased nitric oxide by 52% (p < 0.01), and polyenylphosphatidylcholine attenuated this effect. Ethanol consumption increased the cytosolic activity of the inducible nitric oxide synthase and induced microsomal cytochromes P-450 capable of producing both nitric oxide and superoxide. This was associated with an 18% (p < 0.01) increase in nitrotyrosine protein residues, products of peroxynitrite toxicity, which occurred predominantly in steatotic hepatocytes. Polyenylphosphatidylcholine attenuated these changes by decreasing the ethanol effect on both the cytosolic and the microsomal activities, in addition to acting as a powerful antioxidant. Acute administration of the same ethanol dose increased nitric oxide levels, but did not affect nitrotyrosine protein residues. Conclusions: Chronic, but not acute, ethanol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide, both of which are prevented by polyenylphosphatidylcholine.

Ethanol Consumption Increases Nitric Oxide Production in Rats, and Its Peroxynitrite‐Mediated Toxicity Is Attenuated by Polyenylphosphatidylcholine

Nitric oxide generally mediates beneficial responses but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent. To study the effects of ethanol and polyenylphosphatidylcholine on nitric oxide metabolism and toxicity, 36 rats were pair-fed liquid diets with 36% of energy either as ethanol or as additional carbohydrate for 24 days and were killed 90 min after intragastric feeding. Half received polyenylphosphatidylcholine in the diet (3 g/liter), and the other half equivalent amounts of essential fatty acids and choline. Nitric oxide was measured by chemiluminescence in arterial blood and liver cytosol and as a product of the inducible nitric oxide synthase activity. Peroxynitrite formation was assessed by the increase in nitrotyrosine protein residues, measured immunochemically. In blood, administration of ethanol with or without polyenylphosphatidylcholine doubled nitric oxide levels. In the liver, ethanol increased nitric oxide by 52% (p < 0.01), and polyenylphosphatidylcholine attenuated this effect. Ethanol consumption increased the cytosolic activity of the inducible nitric oxide synthase and induced microsomal cytochromes P-450 capable of producing both nitric oxide and superoxide. This was associated with an 18% (p < 0.01) increase in nitrotyrosine protein residues, products of peroxynitrite toxicity, which occurred predominantly in steatotic hepatocytes. Polyenylphosphatidylcholine attenuated these changes by decreasing the ethanol effect on both the cytosolic and the microsomal activities, in addition to acting as a powerful antioxidant. Acute administration of the same ethanol dose increased nitric oxide levels, but did not affect nitrotyrosine protein residues. Chronic, but not acute, ethanol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide, both of which are prevented by polyenylphosphatidylcholine.

Prostaglandin E 1 protects cultured spinal neurons against the effects of nitric oxide toxicity

The effects of prostaglandin (PG) E 1 on NO neurotoxicity were examined using rat cultured spinal neurons. Rat cultured spinal neurons exposed to the NO donor, 2,2’-(hydroxynitrosohydrazono) bis-ethanamine (NOC18), showed neurotoxic effects that were accompanied by apoptotic nuclear change, free radical generation, a reduction in glutathione, and mitochondrial dysfunction. PGE 1, at concentrations of 1–100 nM, protected cultured spinal neurons from NO toxicity by reversing the oxidative and pro-apoptotic properties elicited by NOC18 exposure. The administration of PGE 1 increased the intracellular cyclic AMP (cAMP) levels in cultured spinal neurons. In addition, reverse transcriptase–polymerase chain reaction (RT–PCR) analysis confirmed the existence of EP4, a cAMP-elevating PGE receptor, in cultured spinal neurons. The protective effects of PGE 1 against NO neurotoxicity was partially blocked by an inhibitor of MEK [the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase], suggesting that the MAPK/ERK pathway may play a significant role in the activity of PGE 1. PGE 1 up-regulated the expression of the anti-apoptotic protein, Bcl-2, as determined by Western blot analysis. PGE 1 also induced the expression of thioredoxin in cultured spinal neurons. Our data indicate that PGE 1 exerts a protective action against NO neurotoxicity in cultured spinal neurons, and suggests a therapeutic potential of PGE 1 against spinal cord disease, such as amyotrophic lateral sclerosis.

Soluble guanylyl cyclase activator YC-1 protects white matter axons from nitric oxide toxicity and metabolic stress, probably through Na(+) channel inhibition.

In the rat isolated optic nerve, nitric oxide (NO) activates soluble guanylyl cyclase (sGC), resulting in a selective accumulation of cGMP in the axons. The axons are also selectively vulnerable to NO toxicity. The experiments initially aimed to determine any causative link between these two effects. It was shown, using a NONOate donor, that NO-induced axonal damage occurred independently of cGMP. Unexpectedly, however, the compound YC-1, which is an allosteric activator of sGC, potently inhibited NO-induced axonopathy (IC(50) = 3 microM). This effect was not attributable to increased cGMP accumulation. YC-1 (30 microM) also protected the axons against damage by simulated ischemia, which (like NO toxicity) is sensitive to Na(+) channel inhibition. Although chemically unrelated to any known Na(+) channel inhibitor, YC-1 was effective in two biochemical assays for activity on Na(+) channels in synaptosomes. Electrophysiological recording from hippocampal neurons showed that YC-1 inhibited Na(+) currents in a voltage-dependent manner. At a concentration giving maximal protection of optic nerve axons from NO toxicity (30 microM), YC-1 did not affect normal axon conduction. It is concluded that the powerful axonoprotective action of YC-1 is unrelated to its activity on sGC but is explained by a novel action on voltage-dependent Na(+) channels. The unusual ability of YC-1 to protect axons so effectively without interfering with their normal function suggests that the molecule could serve as a prototype for the development of more selective Na(+) channel inhibitors with potential utility in neurological and neurodegenerative disorders.

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.

Dizocilpine inhibits amphetamine-induced formation of nitric oxide and amphetamine-induced release of amino acids and acetylcholine in the rat brain.

Glutamate receptor activation participates in mediation of neurotoxic effects in the striatum induced by the psychomotor stimulant amphetamine. The effects of the non-competitive NMDA receptor antagonist dizocilpine (MK-801) on amphetamine-induced toxicity and formation of nitric oxide (NO) in both striatum and cortex and on induced transmitter release in the nucleus accumbens were investigated. Repeated, systemic application of amphetamine elevated striatal and cortical lipid peroxidation and NO production. Moreover, amphetamine caused an immediate release of acetylcholine and aspartate and a delayed release of GABA in the nucleus accumbens. Surprisingly, glutamate release was not affected. Dizocilpine abolished the amphetamine-induced lipid peroxidation and NO production in striatum and cortex and diminished the elevation of neurotransmitter release. These findings suggest that amphetamine evokes neurotoxic effects in both striatal and cortical brain areas that are prevented by inhibiting NMDA receptor activation. The amphetamine-induced acetylcholine, aspartate and GABA release in the nucleus accumbens is also mediated through NMDA receptor-dependent mechanisms. Interestingly, the enhanced aspartate release might contribute to NMDA receptor activation in the nucleus accumbens, while glutamate does not seem to mediate amphetamine-evoked transmitter release in this striatal brain area.

Upregulation of phosphoinositide 3-kinase and protein kinase B in alveolar macrophages following ozone inhalation. Role of NF-kappaB and STAT-1 in ozone-induced nitric oxide production and toxicity.

Inhalation of toxic doses of ozone causes lung injury and inflammation in humans and experimental animals. Using a rodent model of ozone toxicity, we have previously demonstrated that macrophages recruited to the lung following exposure to this oxidant contribute to the pathogenesis of tissue injury. In the present studies we analyzed potential mechanisms regulating alveolar macrophage activity following ozone inhalation and the role of inflammatory mediators in toxicity. Treatment of mice with ozone (0.8 ppm, 3 h) resulted in increased expression of inducible nitric oxide synthase (iNOS) protein and production of nitric oxide (NO) and peroxynitrite by alveolar macrophages. In contrast, these effects were not observed in macrophages from transgenic mice with a targeted disruption of the gene for iNOS, or in mice overexpressing superoxide dismutase. Moreover, ozone toxicity, as measured by bronchoalveolar lavage protein levels and nitrotyrosine staining of the lung was prevented in both of these transgenic mouse strains. The promoter/enhancer region of the iNOS gene contains binding sites for the transcription factors NF-kappaB and STAT-1 which regulate the activity of the gene. Ozone inhalation resulted in a rapid and prolonged activation of NF-kappaB in alveolar macrophages. Phosphoinositide 3-kinase (PI 3-K) and its down stream target, protein kinase B (PKB), which are known to regulate NF-kappaB activity, also increased in alveolar macrophages following ozone inhalation. These data, together with our findings that inhibitors of PI 3-K block NO production, suggest that these proteins are important in controlling expression of iNOS. Furthermore, the fact that macrophages from NF-kappaB p50 knockout mice did not generate reactive nitrogen intermediates and that these mice were protected from ozone induced toxicity demonstrate the importance of the NF-kappaB signaling pathway in lung injury. We also found that STAT-1 nuclear binding activity and STAT-1 protein expression were upregulated in macrophages from ozone treated animals. Taken together, these data suggest that biochemical signaling pathways that control the expression of genes critical for the inflammatory process play a role in ozone toxicity.

Involvement of Ras in survival responsiveness to nitric oxide toxicity in pheochromocytoma cells.

Nitric oxide (NO) plays a key role in attenuation of tumor growth by activated macrophages that generate large amount of cytotoxic/cytostatic free radicals. However, some tumor cells may survive from NO cytotoxicity and continue to proliferate to malignant tumors. Since a protooncogene product Ras was shown to be activated by NO, this study investigated the involvement of Ras in the cell survival in response to NO cytotoxicity in pheochromocytoma (PC12) cells. Treatment with Ras inhibitor or constitutive expression of dominant negative Ras markedly increased NO-induced cell death. NO-resistant PC12 cells (PC12-NO-R) exhibited higher steady state Ras activity than the parental PC12 cells. Inducible expression using tetracycline-on (Tet-on) system of Ras mutants (dominant negative Ras or dominant active Ras) demonstrated that blockade of Ras activity increased NO-induced cell death whereas enhancement of Ras activity attenuated NO-induced cell death. Furthermore, inducible expression of NO-insensitive mutant Ras selectively increased cellular vulnerability to NO but not to ROS. NO, Ras inhibitor and extracellular signal-regulated kinase (Erk) blocker synergistically increased cell death. These observations suggest that Ras activity may be a critical factor for survival response of tumor cells to NO toxicity and pharmacological agents affecting Ras activity may enhance efficacy of NO-mediated tumor therapies.

Suppressor of cytokine signaling 3 (SOCS-3) protects beta -cells against interleukin-1beta - and interferon-gamma -mediated toxicity.

Suppressor of cytokine signaling 3 (SOCS-3) is a negative feedback regulator of IFN-gamma signaling, shown up-regulated in mouse bone marrow cells by the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and IFN-gamma. IL-1beta and IFN-gamma alone, or potentiated by TNF-alpha, are cytotoxic to the insulin producing pancreatic beta-cells and beta-cell lines in vitro and suggested to contribute to the specific beta-cell destruction in Type-1 diabetes mellitus (T1DM). Using a doxycycline-inducible SOCS-3 expression system in the rat beta-cell line INS-1, we demonstrate that the toxic effect of both IL-1beta or IFN-gamma at concentrations that reduced the viability by 50% over 3 days, was fully preventable when SOCS-3 expression was turned on in the cells. At cytokine concentrations or combinations more toxic to the cells, SOCS-3 overexpression yielded a partial protection. Whereas SOCS-3-mediated inhibition of IFN-gamma signaling is described in other cell systems, SOCS-3 mediated inhibition of IL-1beta signaling has not previously been described. In addition we show that SOCS-3 prevention of IL-1beta-induced toxicity is accompanied by inhibited transcription of the inducible nitric oxide synthase (iNOS) by 80%, resulting in 60% decreased formation of the toxic nitric oxide (NO). Analysis of isolated native rat islets exposed to IL-1beta revealed a naturally occurring but delayed up-regulated SOCS-3 transcription. Influencing SOCS-3 expression thus represents an approach for affecting cytokine-induced signal transduction at a proximal step in the signal cascade, potentially useful in future therapies aimed at reducing the destructive potential of beta-cell cytotoxic cytokines in T1DM, as well as other cytokine-dependent diseases.

Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia.

Brain subjected to acute ischemic attack caused by an arterial blockage needs immediate arterial recanalization. However, restoration of cerebral blood flow can cause tissue injury, which is termed reperfusion injury. It is important to inhibit reperfusion injury to achieve greater brain protection. Because oxidative stress has been shown to activate mitogen-activated protein kinases (MAPKs), and because oxidative stress contributes to reperfusion injury, MAPK may be a potential target to inhibit reperfusion injury after brain ischemia. Here, we demonstrate that reperfusion after forebrain ischemia dramatically increases phosphorylation level of extracellular signal-regulated kinase 2 (ERK2) in the gerbil hippocampus. In addition, i.v. administration of U0126 (100-200 mg/kg), a specific inhibitor of MEK (MAPK/ERK kinase), protects the hippocampus against forebrain ischemia. Moreover, treatment with U0126 at 3 h after ischemia significantly reduces infarct volume after transient (3 h) focal cerebral ischemia in mice. This protection is accompanied by reduced phosphorylation level of ERK2, substrates for MEK, in the damaged brain areas. Furthermore, U0126 protects mouse primary cultured cortical neurons against oxygen deprivation for 9 h as well as nitric oxide toxicity. These results provide further evidence for the role of MEK/ERK activation in brain injury resulting from ischemia/reperfusion, and indicate that MEK inhibition may increase the resistance of tissue to ischemic injury.

Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism.

Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co-culture system. Astrocyte-coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N-methyl-D-aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD(50) for glutamate by approximately four-fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S-nitroso-N-acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co-cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione-depleted astrocytes exhibited a two-fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione-dependent mechanism.

Akt Activation Protects Hippocampal Neurons from Apoptosis by Inhibiting Transcriptional Activity of p53

Survival factors suppress apoptosis by activating the serine/threonine kinase Akt. To investigate the molecular mechanism underlying activated Akt's ability to protect neurons from hypoxia or nitric oxide (NO) toxicity, we focused on the apoptosis-related functions of p53 and caspases. We eliminated p53 by employing p53-deficient neurons and increased p53 by infection with recombinant adenovirus capable of transducing p53 expression, and we now show that p53 is implicated in the apoptosis induced by hypoxia or NO treatments of primary cultured hippocampal neurons. Although hypoxia and NO induced p53, treatment with insulin-like growth factor-1 significantly inhibited caspase-3-like activation, neuronal death and transcriptional activity of p53. These insulin-like growth factor-1 effects are prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor. Adenovirus-mediated expression of activated-Akt kinase suppressed p53-dependent transcriptional activation of responsive genes such as Bax, suppressed caspase-3-like protease activity and suppressed neuronal cell death with no effect on the cellular accumulation and nuclear translocation of p53. In contrast, overexpression of kinase-defective Akt failed to suppress these same activities. These results suggest a mechanism where Akt kinase activation reduces p53's transcriptional activity that ultimately rescues neurons from hypoxia- or NO-mediated cell death.

From no-confidence to nitric oxide acknowledgement: a story of bacterial nitric-oxide reductase.

The review briefly summarizes current knowledge of the bacterial nitric-oxide reductase (NOR). This membrane enzyme consists of two subunits, the smaller one contains haem C and the larger one two haems B and nonhaem iron. The protein sequence and structure of metal centres demonstrate the relationship of NOR to the family of terminal oxidases. The binuclear Fe-Fe reaction centre, consisting of antiferromagnetically coupled haem B and nonhaem iron, is analogous to Fe-Cu centre of terminal oxidases. The data on the structure and function of NOR and terminal oxidases suggest that all these enzymes are closely evolutionally related. The catalytic properties are determined most of all by the relatively high toxicity of nitric oxide as a substrate and the resulting strong need to maintain its concentration at nanomolar levels. A kinetic model of the action of the enzyme comprises substrate inhibition. NOR does not conserve the free energy of nitric oxide reduction because it does not work as a proton pump and, moreover, the protons coming into the reaction are taken from periplasm, i.e. they do not cross the membrane.

Kinetic characterization of the nitric oxide toxicity for PC12 cells: effect of half-life time of NO release

We investigated the effects of low concentrations of nitric oxide (NO) on cell viability using NO donors, (±)-( E)-4-methyl-2-[( E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR1), (±)-( E)-4-methyl-2-[( E)-hydroxyimino]-5-nitro-3-hexenamide (NOR2), (±)-( E)-4-ethyl-2-[( E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3) and (±)- N-[( E)-4-ethyl-2-[( Z)-hydroxyimino]-5-nitro-3-hexen-1-yl]-3-pyridine (NOR4). The half-life times of the NO release from these four NOR analogs, NOR1, NOR2, NOR3 and NOR4, were determined (6.5, 84, 105 and 340 min, respectively) by using 4,5-diaminofluorescein (DAF-2), a newly developed indicator of NO. Exposure of undifferentiated PC12 cells to low concentrations of NO donors, NOR2 or NOR3 (1–100 μM), but not NOR1 nor NOR4, resulted in cell death in a dose- and time-dependent manner, as determined from cell viability assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium (MTT) assay. After 24 h exposure to 50 μM NOR2 or NOR3, more than 90% of PC12 cells had died. Furthermore, while the toxic effect of NOR3 was attenuated by replacing the medium at 20 min, 1 or 2 h after drug addition, it was continued by replacing the medium at 3 h or later after drug addition. The cell death was characterized by DNA degradation, nuclear condensation and fragmentation, suggesting apoptosis-like cell death. Pretreatment with an antioxidant ascorbic acid (0.1–0.5 mM) completely prevented the cell death caused by NOR3, while glutathione (0.1–0.2 mM) and cysteine (0.2–0.4 mM) provided partial protection. These findings suggest that the cell toxicity induced by NO at low concentrations strongly depends upon the duration of expose to NO from NO donors, and these toxic effects are effectively prevented by the antioxidant, ascorbic acid.

Intrahepatic accumulation of nitrotyrosine in chronic viral hepatitis is associated with histological severity of liver disease

Background/Aims: The toxicity of nitric oxide is thought to be engendered, at least in part, by its reaction with superoxide yielding peroxynitrite, a potent oxidant that promotes the formation of nitrotyrosine within cells and tissue lesions. In this study we assessed the intrahepatic localization and distribution of the inducible nitric oxide synthase (iNOS) and nitrotyrosine (NTY) in patients with viral and non-viral liver disease. Methods: We carried out single and double immunostaining experiments on cryostat liver biopsy sections using monoclonal antibodies against iNOS and NTY. We also performed a comparative analysis between the intrahepatic immunostaining score of NTY and the histological activity index of chronic viral hepatitis. Results: We found a marked hepatocellular expression of iNOS with a diffuse lobular pattern in all liver samples from patients with viral liver disease, whereas NTY localization was mainly restricted to cellular foci consisting of hepatocytes and Kupffer cells. Interestingly, we demonstrated by means of double immunostaining experiments the existence of hepatocellular co-localization of iNOS and NTY in the majority of NTY-expressing liver cells. The amount of NTY was significantly higher in liver biopsies from viral liver disease than in non-viral liver disease. In addition, a statistically significant association between the intrahepatic amount of NTY and the severity of viral liver disease was found. Conclusions: Nitric oxide-mediated nitration of hepatocellular proteins is markedly induced in the inflamed liver tissue from patients with chronic viral hepatitis, and appears to be associated with the histological severity of viral chronic liver disease.

Toxicity of nitric oxide and peroxynitrite to bacterial pathogens of fish.

The inhibitory effect of the nitric oxide (NO) donor S-nitroso-acetyl-penicillamine (SNAP) and the NO and O2- donor 3-morpholino-sydnonimine hydrochloride (SIN-1) was tested in a cell-free assay. Strains of the bacterial fish pathogens Aeromonas salmonicida, Renibacterium salmoninarum and Yersinia ruckeri were exposed to different concentrations of the NO donors for 24 h. The results showed that NO possesses inhibitory properties, while peroxynitrite had no effect. However, when SIN-1 was used in combination with superoxide dismutase (SOD) alone or with catalase, an inhibitory effect comparable to that caused by SNAP was seen. The implications of these results are discussed.

Genetic responses against nitric oxide toxicity.

The threat of free radical damage is opposed by coordinated responses that modulate expression of sets of gene products. In mammalian cells, 12 proteins are induced by exposure to nitric oxide (NO) levels that are sub-toxic but exceed the level needed to activate guanylate cyclase. Heme oxygenase 1 (HO-1) synthesis increases substantially, due to a 30- to 70-fold increase in the level of HO-1 mRNA. HO-1 induction is cGMP-independent and occurs mainly through increased mRNA stability, which therefore indicates a new NO-signaling pathway. HO-1 induction contributes to dramatically increased NO resistance and, together with the other inducible functions, constitutes an adaptive resistance pathway that also defends against oxidants such as H2O2. In E. coli, an oxidative stress response, the soxRS regulon, is activated by direct exposure of E. coli to NO, or by NO generated in murine macrophages after phagocytosis of the bacteria. This response is governed by the SoxR protein, a homodimeric transcription factor (17-kDa subunits) containing [2Fe-2S] clusters essential for its activity. SoxR responds to superoxide stress through one-electron oxidation of the iron-sulfur centers, but such oxidation is not observed in reactions of NO with SoxR. Instead, NO nitrosylates the iron-sulfur centers of SoxR both in vitro and in intact cells, which yields a form of the protein with maximal transcriptional activity. Although nitrosylated SoxR is very stable in purified form, the spectroscopic signals for the nitrosylated iron-sulfur centers disappear rapidly in vivo, indicating an active process to reverse or eliminate them.

Activation of Akt Kinase Inhibits Apoptosis and Changes in Bcl‐2 and Bax Expression Induced by Nitric Oxide in Primary Hippocampal Neurons

Emerging data indicate that growth factors such as insulin-like growth factor-1 (IGF-1) prevent neuronal death due to nitric oxide (NO) toxicity. On the other hand, growth factors can promote cell survival by acting on phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, serine-threonine kinase Akt, in various types of cells. Here, we examined the mechanism by which IGF-1 inhibits neuronal apoptosis induced by NO in primary hippocampal neurons. IGF-1 was capable of preventing apoptosis and caspase-3-like activation induced by a NO donor, sodium nitroprusside or 3-morpholin-osydnonimine. Incubation of neurons with a P13-kinase inhibitor, wortmannin or LY294002, blocked the effects of IGF-1 on NO-induced neurotoxicity and caspase-3-like activation. In addition, the P13-kinase inhibitors blocked the effect of IGF-1 on down-regulation in Bcl-2 and upregulation in Bax expression induced by NO. Adenovirus-mediated overexpression of the activated form of Akt significantly inhibited NO-induced cell death, caspase-3-like activation, and changes in Bcl-2 and Bax expression. Moreover, expression of the kinase-defective form of Akt almost completely blocked the effects of IGF-1. These findings suggest that activation of Akt is necessary and sufficient for the effect of IGF-1 and is capable of preventing NO-induced apoptosis by modulating the NO-induced changes in Bcl-2 and Bax expression.