Nitrotyrosine Production Research Articles

Expression of nitrergic system and protein nitration in adult rat brains submitted to acute hypobaric hypoxia

Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, and NO synthase (NOS) activity assays in adult rats submitted for 30 min to hypoxia, in a hypobaric chamber at a simulated altitude of 38,000 ft (11000 m) (154.9 mm Hg). The cerebral cortex was studied after different survival times, 0 and 24 h, 5, 8, 15, and 30 days of reoxygenation. This situation led to morphological alterations in the large type I interneurons, as well as immunoreactive changes in the appearance and number of the small neurons (type II), both containing neuronal NOS (nNOS). Some of these small neurons showed immunoreactive cytoplasm and short processes; others, the more numerous during all reoxygenation periods, contained the immunoreactive product mainly related to a perinuclear ring. Ultrastructurally, these small neurons exhibited changes in nuclear structures as in the shape of the nuclear membrane, in the distribution of heterochromatin, and in the nucleolar morphology. The reaction product for nitrotyrosine, as a marker of protein nitration, showed modifications in distribution of the immunoreactive product. No expression was found for inducible NOS (iNOS). All these modifications were accompained by increased nNOS and nitrotyrosine production as demonstrated by Western blotting and calcium-dependent activity, returning to control conditions after 30 days of reoxygenation, suggesting a reversible NO mechanism of action.

Nitric oxide pathways in human bladder carcinoma. The distribution of nitric oxide synthases, soluble guanylyl cyclase, cyclic guanosine monophosphate, and nitrotyrosine.

Nitric oxide (NO) is produced by a group of synthase enzymes (NOS). By means of different pathways, NO exerts several functions in benign and malignant human bladder tissues. The current paper describes the NO/guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) and the NO/oxidative pathways in human bladder tissues. Bladder carcinoma tissues were collected from 18 patients by transurethral resection procedures. Normal benign vesical tissue specimens from a further eight patients with benign diseases served as controls. Immunohistochemistry was conducted for localization of sGC, cGMP, and nitrotyrosine in benign and malignant vesical tissues, evaluating two-three tissue sections per patient. Positive immunolabeling for sGC and cGMP was detected in vascular endothelial cells of normal and malignant vesical tissues. Those signals were most intense in bladder carcinoma tissues. Immunolabeling for sGC and cGMP was also detected in normal urothelial cells. In bladder carcinoma cells, a heterogeneous immunolabeling for sGC and cGMP was seen, with a wide spectrum of signal intensity. Positive immunostaining for sGC and cGMP was also observed in stromal round cells in benign and malignant bladder tissues. Immunolabeling for nitrotyrosine was mainly observed in endothelial cells, with a much stronger immunolabeling in bladder carcinoma tissues compared to normal benign controls. A weak immunolabeling for nitrotyrosine was also observed in bladder carcinoma cells. Normal urothelial cells did not show such nitrotyrosine expression. The current report provides evidences that NO play several roles through different pathways in benign and malignant vesical tissues. The influences generated by NO molecules can be divided into cGMP-mediated effects (those resulting from the NO/sGC/cGMP pathway) and non-cGMP-mediated effects (those resulting from the NO/oxidative pathway). Increased angiogenesis is a cGMP-mediated effect, while nitrotyrosine production is a non cGMP-mediated oxidative effect. Such an NO/oxidative pathway is observed more often in bladder carcinoma.

Contribution of peroxynitrite to fatal cardiovascular depression induced by overproduction of nitric oxide in rostral ventrolateral medulla of the rat

We evaluated the contribution of peroxynitrite to the fatal cardiovascular depression induced by overproduction of nitric oxide (NO) after activation of inducible NO synthase (iNOS) in the rostral ventrolateral medulla (RVLM), the origin of sympathetic vasomotor tone. In Sprague–Dawley rats maintained under propofol anesthesia, microinjection of E. coli lipopolysaccharide (LPS) bilaterally into the RVLM elicited significant hypotension, bradycardia, reduction in sympathetic vasomotor tone and mortality. There was also a discernible elevation of iNOS expression in the ventrolateral medulla, followed by a massive production of nitrotyrosine, an experimental index for peroxynitrite. Co-administration bilaterally into the RVLM of the selective iNOS inhibitor, S-methylisothiourea (50, 100 or 250 pmol), an active peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis- ( N-methyl-4′-pyridyl)-porphyrinato iron (III) (10 or 50 pmol), a peroxynitrite scavenger, l-cysteine (5, 50 or 100 pmol), or a superoxide dismutase mimetic, Mn(III)-tetrakis-(4-benzoic acid) porphyrin (1 or 10 pmol), significantly prevented mortality, reduced nitrotyrosine production and reversed the NO-induced cardiovascular suppression after application of LPS into the RVLM. We conclude that the formation of peroxynitrite by a reaction between superoxide anion and NO is primarily responsible for the fatal cardiovascular depression induced by overproduction of NO after activation of iNOS at the RVLM.

Determination of nitrotyrosine and related compounds in biological specimens by competitive enzyme immunoassay

A gas mediator, nitric oxide is converted to peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of the tyrosine residues of proteins, and it enhances the late response of inflammation. The determination of nitrated tyrosine, nitrotyrosine, which is a stable final metabolite of peroxynitrite, provides an important indicator of tissue disorders caused by peroxynitrite. This paper reports a competitive solid-phase immunoassay for measuring nitrotyrosine in various biological specimens. In this study, peroxidase-conjugated nitrotyrosine was prepared by reaction of nitrotyrosine with 1,4-benzoquinone treatment, and then it was allowed to compete with nitrotyrosine on an anti-nitrotyrosine antibody-coated 96-well multiplate. No amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at the concentrations higher than 1000 times the concentration of nitrotyrosine. The limit of detection of free nitrotyrosine was approximately 500 pg/mL (2 nM) at a competition ratio ( B/ B o% of 80%. The newly developed enzyme immunoassay (EIA) method was used for assay of nitrotyrosine in biological specimens, with the following results: (i) Lipopolysaccharide (LPS) activation of RAW264.7 cells induced a significant increase in nitrotyrosine production compared to that with nonactivated cells. N ω-nitro- l-arginine methyl ester decreased nitrotyrosine production with either LPS-activated or nonactivated RAW cells. There is a relationship between nitrotyrosine production and nitrite ion. (ii) The nitrotyrosine level detected in the plasma specimens from healthy volunteers was 35.21±4.87 ng/ mL ( 135.4±18.7 nM ). (iii) The concentration of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients was 41.40±20.96 ng/ mL ( 159.02±80.6 nM ). Thus, the EIA method combines sensitivity and specificity with the ability to process a large number of specimens to quantify nitrotyrosine produced with in vivo and in vitro sources.

Role of hyperglycemia in nitrotyrosine postprandial generation.

Recently, much attention has been paid to the possibility that postprandial hyperglycemia may be a cardiovascular risk factor in diabetes. Oxidative stress has been involved in the pathogenesis of diabetic complications, and increased plasma levels of nitrotyrosine, a product of peroxynitrite action, have been found in the plasma of diabetic subjects. The aim of the present study was to evaluate whether postprandial hyperglycemia is accompanied by nitrotyrosine generation and, if so, to explore a possible direct role of hyperglycemia in such a phenomenon. A total of 23 type 2 diabetic patients and 15 matched normal healthy subjects were recruited for this study. Two different tests were performed in diabetic patients: a standard meal preceded by regular insulin (0.15 units/kg body wt) or insulin aspart (0.15 units/kg body wt) to achieve different levels of postprandial hyperglycemia. The meal test was also performed in healthy control subjects. At 0 min and 1, 2, 4, and 6 h after each meal, blood glucose, triglyceride, and nitrotyrosine levels were measured. Fasting nitrotyrosine was significantly increased in diabetic patients and was further increased during both meal tests in diabetic subjects but not normal subjects. As compared with regular insulin, aspart administration significantly reduced the area under the curve of both glycemia (P < 0.04) and nitrotyrosine (P < 0.03), whereas that of triglycerides was not significantly affected by the treatment. This study shows a direct correlation between postprandial hyperglycemia and the production of nitrotyrosine, a marker of oxidative stress, in patients with type 2 diabetes.

Intraplatelet tetrahydrobiopterin plays an important role in regulating canine coronary arterial thrombosis by modulating intraplatelet nitric oxide and superoxide generation.

Platelet-derived nitric oxide inhibits platelet aggregation via constitutive NO synthase (NOS). Tetrahydrobiopterin (BH(4)), a cofactor of NOS, augments NO formation, whereas its deficiency decreases NO bioactivity and increases superoxide generation by NOS. The roles of intraplatelet BH(4) in platelet aggregation and thrombus formation, however, are unknown. Accordingly, we investigated whether intraplatelet BH(4) is involved in regulating cyclic flow variations (CFVs) and platelet aggregation in a canine model with stenosed and endothelium-injured coronary arteries that mimics acute coronary syndromes in humans. After developing CFVs, dogs received saline or BH(4) (10 or 30 mg/kg) intravenously. Intraplatelet BH(4) and cGMP levels were decreased and intraplatelet nitrotyrosine production was increased during CFVs. ADP- and U46619-induced ex vivo platelet aggregation and platelet P-selectin expression were augmented during CFVs. BH(4) administration restored intraplatelet BH(4) and cGMP levels and decreased intraplatelet nitrotyrosine production, resulting in reduced CFVs and inhibited ex vivo platelet aggregation and platelet P-selectin expression. CFVs again developed after N(G)-monomethyl-L-arginine, an inhibitor of NOS, in BH(4)-treated dogs. Ex vivo platelet NOS activity at baseline, during CFVs, and after BH(4) administration did not differ. Intraplatelet BH(4) may play an important role in regulating thrombus formation by modulating platelet-derived nitric oxide and superoxide generation by platelet NOS.

Augmented oxidative stress of platelets in chronic smokers: Mechanisms of impaired platelet-derived nitric oxide bioactivity and augmented platelet aggregability

OBJECTIVESWe investigated whether impaired platelet-derived nitric oxide (PDNO) bioactivity and augmented platelet aggregability in chronic smokers are related to the imbalance of the intraplatelet redox state through increased oxidative stress.BACKGROUNDChronic smoking impairs PDNO release and augments platelet aggregability. However, their mechanisms are unknown.METHODSCollagen-induced PDNO release, platelet aggregation, plasma and intraplatelet vitamin C and reduced glutathione (GSH), intraplatelet cyclic guanosine 3′,5′-monophosphate (cGMP) and intraplatelet nitrotyrosine production, which is a marker of the peroxynitrite formation, were measured in 11 chronic smokers and 10 age-matched nonsmokers.RESULTSRelease of PDNO and levels of intraplatelet cGMP were lower, and platelet aggregation was greater, in smokers than in nonsmokers. Intraplatelet vitamin C and GSH levels were lower in smokers than in nonsmokers. Intraplatelet nitrotyrosine production was greater in smokers than in nonsmokers. Next, we investigated the effects of oral vitamin C administration (2 g). After vitamin C administration, intraplatelet vitamin C levels were increased and not different at 2 h between the two groups. Then, PDNO release, intraplatelet cGMP levels and platelet aggregation in smokers were restored to the levels of nonsmokers. In smokers, PDNO release and consumption of GSH during platelet aggregation were inversely correlated, and consumption was much less after vitamin C administration. Vitamin C administration decreased intraplatelet nitrotyrosine production in smokers.CONCLUSIONSImpaired PDNO bioactivity and augmented platelet aggregability may be caused by an imbalance of the intraplatelet redox state through increased oxidative stress in smokers.

Increased tyrosine nitration of the brain in chronic renal insufficiency: reversal by antioxidant therapy and angiotensin-converting enzyme inhibition.

Interaction of reactive oxygen species with nitric oxide promotes nitric oxide inactivation and generation of cytotoxic reactive nitrogen species that attack DNA, lipids, and proteins. Nitration of free tyrosine and tyrosine residues of proteins results in production of nitrotyrosine, which can lead to excitotoxicity and frequently is found in the brain of patients and animals with various degenerative, ischemic, toxic, and other neurologic disorders. According to earlier studies, reactive oxygen species activity is increased and neuronal NO synthase expression in the brain is elevated in animals with chronic renal failure (CRF). It was hypothesized, therefore, that tyrosine nitration must be increased in the uremic brain. This hypothesis was tested, through determination of nitrotyrosine abundance (by Western blot analysis), as well as distribution (by immunohistology), in the cerebrum of rats with CRF 6 wk after 5/6 nephrectomy. The results were compared with those of sham-operated controls and antioxidant (lazaroid)-treated and captopril-treated rats with CRF. Western blot analysis revealed a significant increase in nitrotyrosine abundance in the cerebral cortex of rats with CRF. This was accompanied by an intense nitrotyrosine staining of the neuronal processes, including proximal segments of dendrites, axons, and axon terminals of the cortical neurons. Both antioxidant therapy and captopril administration alleviated oxidative stress (as evidenced by normalization of plasma lipid peroxidation product malondialdehyde) and significantly reduced nitrotyrosine abundance in the cerebral cortex of the treated CRF group. In conclusion, CRF resulted in oxidative stress and increased tyrosine nitration in the cerebral cortex. Antioxidant therapy and angiotensin-converting enzyme inhibition alleviated the CRF-induced oxidative stress and mitigated tyrosine nitration in the rats with CRF.

Nitrotyrosination contributes minimally to toxicity of mutant SOD1 associated with ALS.

Enhanced production of nitrotyrosine and subsequent protein nitration has been proposed as the mechanism by which mutant SOD1 causes death of motor neurons in a familial form of amyotrophic lateral sclerosis (FALS-1). We have tested this hypothesis in a primary culture model in which mutant human SOD1 was expressed in motor neurons of dissociated spinal cord cultures. Preventing formation of nitrotyrosine by inhibiting nitric oxide synthase rescued cultured motor neurons from excitotoxic death induced by adding glutamate to the culture medium, but failed to significantly delay death of motor neurons expressing the G93A mutant SOD1. The results do not support generation of nitrotyrosine being the predominant lethal gain of function conferred by mutations in SOD1.

Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat.

Tamoxifen (TAM), a widely used non-steroidal anti-estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia-induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2 h rMCAo produced a time-dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7-nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC(50) values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [(3)H]arginine to [(3)H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100 microM TAM. These data suggest that TAM is an effective inhibitor of Ca(2+)/calmodulin-dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.

Intra and postischemic mild hypothermia : Effects on inducible nitric oxide synthase and peroxynitrite in experimental stroke

P86 Background: Mild hypothermia is protective even when delayed hours after stroke onset. Because metabolic depletion and glutamate release occur early after ischemia onset, other mechanisms may underlie the protection, particularly when cooling is delayed. Inflammation and free radicals mediate delayed ischemic injury. We examined whether mild hypothermia alters induction and activity of inducible nitric oxide synthase (iNOS, found primarily in inflammatory cells) and peroxynitrite generation. Methods: Rats were subjected to transient middle cerebral artery occlusion for 2 hours. Mild hypothermia began immediately (intraischemic, 33 O C-I, n=8) or 2 h (delayed, 33 O C-D, n=8) after ischemia onset during the reperfusion phase. Hypothermia was maintained for 2 h. Normothermic animals were maintained at 37 O C (n=8). Brains were examined 1 and 3 days later for infarction, NOS activity (NADPH diaphorase), iNOS induction and peroxynitrite (nitrotyrosine) production. Results: Mild hypothermia attenuated infarct size in both groups compared to normothermia (33 O C-I: 26.8±7.7%, 33 O C-D: 30.4±4.8%, 37 O C: 70.8±3.3%, P&lt;0.001). iNOS-positive cells appeared 1 d after ischemia in the infarct area and increased in number by 3 d, but NOS activity was only seen at 3 d. Nitrotyrosine-positive cells also increased by 3 d. Positive cells in all cases appeared microglia/monocyte/macrophage-like with ameboid morphology. Mild hypothermia significantly decreased NADPH diaphorase, iNOS and peroxynitrite densities at 3 d, but not 1 d. NADPH diaphorase-positive cells per hemisphere were: 37 O C: 1028±68, 33 O C-I: 152±12, 33 O C-D: 101±1 (P&lt;0.001). Densities of iNOS-positive cells were: 37 O C: 1265±83/mm 2 , 33 O C-I: 435±93/mm 2 , 33 O C-D: 688±59/mm2 (P&lt;0.001). Densities of nitrotyrosine-positive cells were: 37 O C: 585±14/mm 2 , 33 O C-I: 214±23/mm 2 , 33 O C-D: 106±12mm 2 (P&lt;0.001). No differences in any of the parameters were seen at 1 d. Conclusion: Mild hypothermic neuroprotection is related to attenuation of iNOS and peroxynitrite in inflammatory cells at 3 d but not earlier. This is evident even when cooling is delayed by 2 h.

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.

Ultrastructural Localization of Nitrotyrosine within the Caudate-Putamen Nucleus and the Globus Pallidus of Normal Rat Brain

Nitration of protein tyrosine residues by nitric oxide (NO)-derived reactive species results in the production of stable nitrotyrosine (NT) moieties that are immunochemically detectable in many regions of normal brain and enriched in those areas containing constitutive nitric oxide synthase (cNOS). These include the caudate-putamen nucleus (CPN) and the globus pallidus, which receives major inhibitory input from the CPN. To determine the functional sites for NT production in these critical motor nuclei, we examined the electron microscopic immunocytochemical localization of NT and cNOS in rat brain. In the CPN, NT was localized to the somata and dendrites of cNOS-containing interneurons and spiny neurons, some of which received input from cNOS-labeled terminals. The NT immunoreactivity was most prevalent on outer mitochondrial membranes and nearby segments of the plasma membranes in dendrites and within asymmetric synapses on dendritic spines. In the CPN and globus pallidus, there was also a prominent labeling of NT in astrocytic processes, small axons, and tubulovesicles and/or synaptic vesicles in axon terminals. These terminals formed mainly asymmetric synapses in the CPN and inhibitory-type synapses in the globus pallidus where they often apposed cNOS-containing terminals that also formed asymmetric, excitatory-type synapses. Our results suggest that NT is generated by mechanisms requiring the dual actions of excitatory transmitters and NO derived either from interneurons in the CPN or from excitatory afferents in the globus pallidus. The findings also implicate NT in the physiological actions of NO within the striatal circuitry and, particularly, in striatopallidal neurons severely affected in Huntington's disease.

Increased production of nitrotyrosine in lung tissue of rats with radiation-induced acute lung injury.

The purposes of this study were 1) to identify the nitric oxide (NO) synthase (NOS) isoform responsible for NO-mediated radiation-induced lung injury, 2) to examine the formation of nitrotyrosine, and 3) to see whether nitrotyrosine formation and lung injury are reduced by an inducible NOS (iNOS) inhibitor, aminoguanidine. The left hemithorax of rats was irradiated (20 Gy), and the degree of lung injury, the expression of NOS isoforms, and the formation of nitrotyrosine and superoxide were examined after 2 wk. iNOS mRNA was induced, and endothelial NOS mRNA was markedly increased in the irradiated lung. Nitrotyrosine was detected biochemically and immunohistochemically. Aminoguanidine prevented acute lung injury as indicated by decreased protein concentration and lactate dehydrogenase activity in bronchoalveolar lavage fluid and improved NMR parameters and histology. Furthermore, the formation of nitrotyrosine was significantly reduced in the aminoguanidine group. We conclude that iNOS induction is a major factor in radiation-induced lung injury and that nitrotyrosine formation may participate in the NO-induced pathogenesis.

Intra and postischemic mild hypothermia : Effects on inducible nitric oxide synthase and peroxynitrite in experimental stroke

P86 Background: Mild hypothermia is protective even when delayed hours after stroke onset. Because metabolic depletion and glutamate release occur early after ischemia onset, other mechanisms may underlie the protection, particularly when cooling is delayed. Inflammation and free radicals mediate delayed ischemic injury. We examined whether mild hypothermia alters induction and activity of inducible nitric oxide synthase (iNOS, found primarily in inflammatory cells) and peroxynitrite generation. Methods: Rats were subjected to transient middle cerebral artery occlusion for 2 hours. Mild hypothermia began immediately (intraischemic, 33OC-I, n=8) or 2 h (delayed, 33OC-D, n=8) after ischemia onset during the reperfusion phase. Hypothermia was maintained for 2 h. Normothermic animals were maintained at 37OC (n=8). Brains were examined 1 and 3 days later for infarction, NOS activity (NADPH diaphorase), iNOS induction and peroxynitrite (nitrotyrosine) production. Results: Mild hypothermia attenuated infarct size in both groups compared to normothermia (33OC-I: 26.8±7.7%, 33OC-D: 30.4±4.8%, 37OC: 70.8±3.3%, P<0.001). iNOS-positive cells appeared 1 d after ischemia in the infarct area and increased in number by 3 d, but NOS activity was only seen at 3 d. Nitrotyrosine-positive cells also increased by 3 d. Positive cells in all cases appeared microglia/monocyte/macrophage-like with ameboid morphology. Mild hypothermia significantly decreased NADPH diaphorase, iNOS and peroxynitrite densities at 3 d, but not 1 d. NADPH diaphorase-positive cells per hemisphere were: 37OC: 1028±68, 33OC-I: 152±12, 33OC-D: 101±1 (P<0.001). Densities of iNOS-positive cells were: 37OC: 1265±83/mm2, 33OC-I: 435±93/mm2, 33OC-D: 688±59/mm2 (P<0.001). Densities of nitrotyrosine-positive cells were: 37OC: 585±14/mm2, 33OC-I: 214±23/mm2, 33OC-D: 106±12mm2 (P<0.001). No differences in any of the parameters were seen at 1 d. Conclusion: Mild hypothermic neuroprotection is related to attenuation of iNOS and peroxynitrite in inflammatory cells at 3 d but not earlier. This is evident even when cooling is delayed by 2 h.

Pterin-6-aldehyde, Xanthine Oxidase Inhibitor and Superoxide Scavenger, Directly React with Peroxynitrite

The effect of pterin-6-aldehyde (P6A), xanthine oxidase inhibitor and superoxide scavenger, on the production of nitrotyrosine as a footprint of tyrosine nitration by peroxynitrite, was compared with that of uric acid, a peroxynitrite scavenger. The amounts of tyrosine, P6A and nitrotyrosine were quantified using reversed-phase high-performance liquid chromato-graphy (RP-HPLC). P6A suppressed nitrotyrosine formation less effectively than uric acid, that is, 0.25 mM P6A reduced nitrotyrosine formation to 67.9± 10.8%, while 0.025 mM uric acid reduced it to 34.2± 1.6%. In living systems, peroxynitrite is generated by the reaction of super-oxide with nitric oxide and has a variety of toxic effects. Our results show that P6A is not necessarily a strong scavenger of peroxynitrite. However, since P6A is a potent scavenger of superoxide, P6A is thought to totally suppress peroxynitrite generation. Compounds that scavenge both superoxide and peroxynitrite may be useful in tissue damage in which reactive oxygen species are involved.

Expression of inducible nitric oxide synthase in human coronary atherosclerotic plaque.

Macrophages in atherosclerotic plaque may express the inducible isoform of NO synthase (iNOS), which produces large amounts of NO. On one hand, the production of NO can be protective by its vasodilatory, antiaggregant and antiproliferative effects. On the other hand, the formation of peroxynitrite from NO may favour vasospasm and thrombogenesis. In this study, we investigated whether iNOS is present in human coronary atherosclerotic plaque, and we correlated these data with the clinical instability of the patients. Fragments were retrieved by coronary atherectomy from 24 patients with unstable angina and 12 patients with stable angina. The presence of macrophages, and the production of TNF alpha, iNOS and nitrotyrosine were detected by immunocytochemistry. Macrophage clusters were found in 67% of stable patients and 87% of patients with unstable angina (NS). TNF alpha was expressed in about 50% of cases in both groups. iNOS was not expressed in fragments from stable patients but was found in macrophages from 58% of unstable patients (P < 0.001). The expression of iNOS was associated with the presence of nitrotyrosine residues, a marker of peroxynitrite formation. Expression of iNOS was correlated both with complaints of angina at rest (P < 0.05) and with the presence of thrombus at morphological examination (P < 0.001). The expression of iNOS may be induced in human coronary atherosclerotic plaque and is associated with different factors of instability.

Role of Peroxynitrite in the Vasoactive and Cytotoxic Effects of Alzheimer's β-Amyloid1–40Peptide

Increasing evidence implicates oxidative stress as partially responsible for the neurodegenerative process of Alzheimer's disease (AD). Recent reports show an increased production of nitrotyrosine in AD brains, suggesting that peroxynitrite is produced in excess in this disease. Furthermore, incidence of cerebral amyloid angiopathy in AD cases is very frequent (83%), strongly suggesting a vascular component of AD pathogenesis. We have evaluated the hypothesis that peroxynitrite could be responsible for mediating the cytotoxicity and vasoactivity induced by the amyloid-β1–40(Aβ) peptide. Rat brain endothelial cells (RBE-4) appear to be sensitive to Aβ-induced toxicity but not to the cytotoxicity induced by peroxynitrite. Addition of Cu/Zn superoxide dismutase to cell culture media, which is only able to clear extracellular superoxide, was not effective in blocking Aβ-induced toxicity. However, we were able to partially block Aβ-induced cytotoxicity by using Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) which dismutes superoxide intracellularily. Yet, MnTBAP was not able to prevent the vasoactivity triggered by Aβ. Moreover, addition of peroxynitrite to rat aortae did not modulate the vasotension induced by Aβ. We conclude that intracellular superoxide radicals may contribute to Aβ-induced cytotoxicity. Our results also indicate that peroxynitrite does not significantly contribute to Aβ-induced cytotoxicity in rat brain endothelial cells (RBE-4) or vasoactivity in rat aortae. These results suggest that therapeutic efforts aimed at removal of reactive oxygen species with SOD is unlikely to be beneficial for treatment of Aβ-induced endothelial dysfunction. However, compounds that clear free radicals intracellularly may well be beneficial.

Inhibition of Inducible Nitric Oxide Synthase Prevents LPS-Induced Acute Lung Injury in Dogs

Nitric oxide (NO) is produced by inducible NO synthase (iNOS) after LPS stimulation, and reacts with superoxide to form peroxynitrite. We hypothesize that in LPS-induced lung injury, NO generated by iNOS plays a key role through the formation of peroxynitrite. We developed an acute lung injury dog model by injecting LPS, and examined the effects of selective iNOS inhibitors, aminoguanidine (AG) and S-methylisothiourea sulfate (SMT), on the LPS-induced lung injury. At 24 h after LPS injection, arterial oxygen tension and mean arterial pressure decreased, and shunt ratio and lung wet-to-dry weight ratio increased. On histology, the LPS group had marked neutrophil infiltration and widening of the alveolar septa. On immunohistochemistry, iNOS and nitrotyrosine, a major product of nitration of protein by peroxynitrite, were observed in the interstitium, capillary wall, and neutrophils in the airspaces of the LPS group. Treatments with AG and SMT prevented worsening of gas exchange, hemodynamics, and wet-to-dry weight ratio. On histology, AG and SMT treatments markedly suppressed lung injury, iNOS protein, and nitrotyrosine production. We conclude that NO released by iNOS may play a critical role in the pathogenesis of LPS-induced acute lung injury. This study suggests that iNOS inhibitors may have potential in the treatment of LPS-induced acute respiratory distress syndrome.

Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by alveolar inflammation, progressive proliferation of septal cells, increased production of septal matrix, and loss of lung architecture. The process of cellular injury in lung fibrosis is thought to be mediated by oxygen radicals produced by infiltrating inflammatory cells. Peroxynitrite is a potent oxidant produced by the rapid reaction of nitric oxide (NO) and superoxide. We investigated the production of nitrotyrosine, a byproduct of protein nitration by peroxynitrite, and the expression of the enzymes responsible for generating NO, in lungs of patients with IPF and compared them with lungs of normal control subjects. We used immunohistochemistry, histochemistry, and in situ hybridization to study the production of nitrotyrosine and the expression of inducible (iNOS) and constitutive endothelial (eNOS) nitric oxide synthases in 48 lungs of patients with different stages of IPF and 21 normal lungs. In lungs of control subjects, there was little expression of iNOS and nitrotyrosine in the airway epithelium and alveolar macrophages, and abundant expression of eNOS in the airway epithelium and vascular endothelium. By contrast, in lungs of patients with IPF, strong expression of nitrotyrosine and NOS was seen in macrophages, neutrophils, and alveolar epithelium. A significant increase in the expression of these molecules was only seen in lungs of patients with the early to intermediate stage of the disease. The active stage of IPF is associated with increased inflammatory and alveolar expression of nitrotyrosine and NOS. Increased production of NO and peroxynitrite may be responsible for the oxidative damage seen in this disease.