3-nitrotyrosine and linsidomine

Superoxide dismutases (SODs; EC 1.15.1.1) are a group of metalloenzymes which are essential to protect cells under aerobic conditions. In biological systems, it has been reported that SODs and other proteins are susceptible to be attacked by peroxynitrite (ONOO(-)) which can be originated from the reaction of nitric oxide with superoxide radical. ONOO(-) is a strong oxidant molecule capable of nitrating peptides and proteins at the phenyl side chain of the tyrosine residues. In the present work, bovine serum albumin (BSA) and recombinant iron-superoxide dismutase from the plant cowpea (Vu_FeSOD) are used as target molecules to estimate ONOO(-) production. The method employs the compound SIN-1, which simultaneously generates *NO and O(2)(-) in aerobic aqueous solutions. First, assay conditions were optimized incubating BSA with different concentrations of SIN-1, and at a later stage, the effect on the tyrosine nitration and catalytic activity of Vu_FeSOD was examined by in-gel activity and spectrophotometric assays. Both BSA and Vu_FeSOD are nitrated in a dose-dependent manner, and, at least in BSA nitration, the reaction seems to be metal catalyzed.

Topics: Antibodies; Biomarkers; Enzyme Activation; Immunohistochemistry; Molsidomine; Nitro Compounds; Nitrosation; Oxidative Stress; Recombinant Proteins; Serum Albumin, Bovine; Superoxide Dismutase; Tyrosine

In the present study, we hypothesized that postcon (postconditioning) confers cardioprotection in vivo by reducing the production of ONOO- (peroxynitrite) and nitro-oxidative stress subsequent to the inhibition of the iNOS (inducible NO synthase). Patients with AMI (acute myocardial infarct) were randomly assigned to undergo percutaneous coronary intervention without (control) or with ischaemic postcon by three episodes of 30-s inflation and 30-s deflation of the angioplasty balloon. Animal models of MI/R (myocardial ischaemia/reperfusion) injury were induced in rats by occluding the left coronary artery for 40 min followed by 4-h reperfusion. Rats were randomized to receive vehicle, postcon (three cycles of 10-s reperfusion and 10-s coronary re-occlusion preceding full reperfusion), the selective iNOS inhibitor 1400W or postcon plus 3-morpholinosydnonimine (an ONOO- donor). Postcon in patients reduced iNOS activity in white blood cells, decreased plasma nitrotyrosine, a fingerprint of ONOO- and an index of nitro-oxidative stress, and improved cardiac function (P<0.01 compared with control). In rats, postcon reduced post-ischaemic myocardial iNOS activity and nitrotyrosine formation, reduced myocardial infarct size (all P<0.05 compared with control) and improved cardiac function. Administration of 1400W resembled, whereas 3-morpholinosydnonimine abolished, the effects of postcon. In conclusion, reduction in ONOO--induced nitro-oxidative stress subsequent to the inhibition of iNOS represents a major mechanism whereby postcon confers cardioprotection in vivo.

Topics: Aged; Angioplasty, Balloon, Coronary; Animals; Apoptosis; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Humans; Ischemic Postconditioning; Leukocyte Count; Male; Malondialdehyde; Middle Aged; Molsidomine; Myocardial Infarction; Myocardial Reperfusion Injury; Nitric Oxide Synthase Type II; Oxidative Stress; Rats; Rats, Sprague-Dawley; Treatment Outcome; Tyrosine; Ventricular Function, Left

Hydrogen sulfide (H₂S) is an endogenous gaseous mediator affecting many physiological and pathophysiological conditions. Enhanced expression of H2S and reactive nitrogen/oxygen species (RNS/ROS) during inflammation alters cellular excitability via modulation of ion channel function. Sulfhydration of cysteine residues and tyrosine nitration are the posttranslational modifications induced by H₂S and RNS, respectively. The objective of this study was to define the interaction between tyrosine nitration and cysteine sulfhydration within the ATP-sensitive K(+) (KATP) channel complex, a significant target in experimental colitis. A modified biotin switch assay was performed to determine sulfhydration of the KATP channel subunits, Kir6.1, sulphonylurea 2B (SUR2B), and nitrotyrosine measured by immunoblot. NaHS (a donor of H₂S) significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. 3-Morpholinosydnonimine (SIN-1) (a donor of peroxynitrite) induced nitration of Kir6.1 subunit but not SUR2B. Pretreatment with NaHS reduced the nitration of Kir6.1 by SIN-1 in Chinese hamster ovary cells cotransfected with the two subunits, as well as in enteric glia. Two specific mutations within SUR2B, C24S, and C1455S prevented sulfhydration by NaHS, and these mutations prevented NaHS-induced reduction in tyrosine nitration of Kir6.1. NaHS also reversed peroxynitrite-induced inhibition of smooth muscle contraction. These studies suggest that posttranslational modifications of the two subunits of the KATP channel interact to alter channel function. The studies described herein demonstrate a unique mechanism by which sulfhydration of one subunit modifies tyrosine nitration of another subunit within the same channel complex. This interaction provides a mechanistic insight on the protective effects of H₂S in inflammation.

Topics: Animals; Calcium Chloride; CHO Cells; Cricetulus; Cysteine; Dose-Response Relationship, Drug; Ileum; KATP Channels; Mice; Molsidomine; Muscle Contraction; Muscle, Smooth; Mutation; Peroxynitrous Acid; Protein Processing, Post-Translational; Signal Transduction; Sulfides; Sulfonylurea Receptors; Transfection; Tyrosine

Glycation and nitrotyrosination are pathological posttranslational modifications that make proteins prone to losing their physiological properties. Since both modifications are increased in Alzheimer's disease (AD) due to amyloid-β peptide (Aβ) accumulation, we have studied their effect on albumin, the most abundant protein in cerebrospinal fluid and blood. Brain and plasmatic levels of glycated and nitrated albumin were significantly higher in AD patients than in controls. In vitro turbidometry and electron microscopy analyses demonstrated that glycation and nitrotyrosination promote changes in albumin structure and biochemical properties. Glycated albumin was more resistant to proteolysis and less uptake by hepatoma cells occurred. Glycated albumin also reduced the osmolarity expected for a solution containing native albumin. Both glycation and nitrotyrosination turned albumin cytotoxic in a cell type-dependent manner for cerebral and vascular cells. Finally, of particular relevance to AD, these modified albumins were significantly less effective in avoiding Aβ aggregation than native albumin. In summary, nitrotyrosination and especially glycation alter albumin structural and biochemical properties, and these modifications might contribute for the progression of AD.

Topics: Aged; Albumins; Alzheimer Disease; Amyloid beta-Peptides; Brain; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Cells; Female; Glycosylation; Humans; Male; Molsidomine; Neurons; Peptide Fragments; Protein Aggregates; Protein Processing, Post-Translational; tau Proteins; Trypsin; Tyrosine

Thin filament-associated proteins such as calponin, caldesmon, and smoothelin are believed to regulate acto-myosin interaction and thus, muscle contraction. Oxidative stress has been found to affect the normal contractile behavior of smooth muscle and is involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the expression of smooth muscle contractile proteins. The aim of the current study is to investigate the effect of oxidative stress on the expression of thin filament-associated proteins in rat gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague-Dawley rats were used. Muscle cells were treated with hydrogen peroxide (H2O2) (500 μM) for 30 min or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) (1 mM) for 90 min to induce oxidative stress. Calponin, caldesmon, and smoothelin expressions were measured via specifically designed enzyme-linked immunosorbent assay. We found that exposure to exogenous H2O2 or incubation of dispersed gastric muscle cells with SIN-1 significantly increased the expression of calponin, caldesmon, and smoothelin proteins. In conclusion: oxidative stress increases the expression of thin filament-associated proteins in gastric smooth muscle, suggesting an important role in gastrointestinal motility disorders associated with oxidative stress.

Topics: Animals; Gastrointestinal Motility; Gene Expression Regulation; Male; Microfilament Proteins; Molsidomine; Muscle, Smooth; Oxidative Stress; Rats; Rats, Sprague-Dawley; Stomach; Tyrosine

Nitrosylation of tyrosine (3-nitro tyrosine, 3-NT) has been implicated in the pathophysiology of various disorders particularly neurodegenerative conditions and aging. Cyperus rotundus rhizome is being used as a traditional folk medicine to alleviate a variety of disorders including neuronal stress. The herb has recently found applications in food and confectionary industries also. In current study, we have explored the protective effects of C. rotundus rhizome extract (CRE) through its oxido-nitrosative and anti apoptotic mechanism to attenuate peroxynitrite (ONOO(-)) induced neurotoxicity using human neuroblastoma SH-SY5Y cells. Our results elucidate that pre-treatment of neurons with CRE ameliorates the mitochondrial and plasma membrane damage induced by 500 μM SIN-1 to 80% and 24% as evidenced by MTT and LDH assays. CRE inhibited NO generation by downregulating i-NOS expression. SIN-1 induced depletion of antioxidant enzyme status was also replenished by CRE which was confirmed by immunoblot analysis of SOD and CAT. The CRE pre-treatment efficiently potentiated the SIN-1 induced apoptotic biomarkers such as bcl-2 and caspase-3 which orchestrate the proteolytic damage of the cell. The ONOO(-) induced damage to cellular, nuclear and mitochondrial integrity was also restored by CRE. Furthermore, CRE pre-treatment also regulated the 3-NT formation which shows the potential of plant extract against tyrosine nitration. Taken together, our findings suggest that CRE might be developed as a preventive agent against ONOO(-) induced apoptosis.

Topics: Apoptosis; Blotting, Western; Caspase 3; Catalase; Cell Death; Cell Line, Tumor; Cyperus; Cytoprotection; DNA Damage; Dose-Response Relationship, Drug; HSP70 Heat-Shock Proteins; Humans; Lipid Peroxidation; Molsidomine; Neurons; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Plant Extracts; Plants, Medicinal; Proto-Oncogene Proteins c-bcl-2; Rhizome; Superoxide Dismutase; Tyrosine

Superoxide has been shown to play a major role in ventricular remodeling and arrhythmias after myocardial infarction. However, the source of increased myocardial superoxide production and the role of superoxide in sympathetic innervation remain to be further characterized. Male Wistar rats, after coronary artery ligation, were randomized to vehicle, allopurinol, or apocynin for 4weeks. To determine the role of peroxynitrite in sympathetic reinnervation, we also used 3-morpholinosydnonimine (a peroxynitrite generator). The postinfarction period was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine, xanthine oxidase activity, NADPH oxidase activity, and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Sympathetic hyperinnervation was blunted after administration of allopurinol. Arrhythmic scores in the allopurinol-treated infarcted rats were significantly lower than those in vehicle. For similar levels of ventricular remodeling, apocynin had no beneficial effects on oxidative stress, sympathetic hyperinnervation, or arrhythmia vulnerability. Allopurinol-treated hearts had significantly decreased nerve growth factor expression, which was substantially increased after coadministration of 3-morpholinosydnonimine. These results indicate that xanthine oxidase but not NADPH oxidase largely mediates superoxide production after myocardial infarction. Xanthine oxidase inhibition ameliorates sympathetic innervation and arrhythmias possibly via inhibition of the peroxynitrite-mediated nerve growth factor pathway.

Topics: Acetophenones; Allopurinol; Animals; Arrhythmias, Cardiac; Coronary Vessels; Ethidium; Male; Molsidomine; Myocardial Infarction; Myocardium; NADPH Oxidases; Nerve Growth Factor; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Wistar; Superoxides; Sympathetic Nervous System; Tyrosine; Xanthine Oxidase

Drug-induced vascular injury (DIVI) is observed in rat mesenteric arterioles in response to treatment with phosphodiesterase-4 inhibitors (PDE4i). However, the mechanisms responsible for causing the characteristic vascular lesions are unclear. Nitrotyrosine (NT) adducts, markers of local nitric oxide (NO) production, have been observed in close proximity to the arterial lesions and in the inflammatory cells associated with DIVI. To determine if NO has a direct role in DIVI, rats were treated with the PDE4i CI-1044 at 10, 20, or 40 mg/kg alone or in combination with the nitric oxide synthase inhibitor L-NAME (60 mg/kg) or the nitric oxide donor SIN-1 (30 mg/kg). Mesenteries were collected and processed for microscopic evaluation. NT formation was evaluated in situ via immunohistochemical staining. Serum nitrite (SN), a marker of in vivo NO production, was measured. Compared with vehicle controls, treatment with CI-1044 alone resulted in dose-related increases in the frequency and severity of vascular injury, SN levels, and NT residues. SIN-1 coadministration caused vascular injury to occur at lower doses of CI-1044, compared with CI-1044 alone, with the overall incidence and severity of injury being greater across all CI-1044-dose groups. Following administration of 20 or 40 mg/kg CI-1044, there were also increases in NT immunoreactivity when SIN-1 was coadministered and significant increases in SN. Conversely, coadministration of L-NAME resulted in marked reduction of injury, NT, and SN when compared with CI-1044 alone. The present study suggests that NO production is closely linked to PDE4i-induced vascular injury.

Topics: Animals; Azepines; Biomarkers; Dose-Response Relationship, Drug; Enzyme Inhibitors; Immunohistochemistry; Male; Mesenteric Arteries; Molsidomine; NG-Nitroarginine Methyl Ester; Niacinamide; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Phosphodiesterase 4 Inhibitors; Rats; Rats, Sprague-Dawley; Tyrosine; Vascular System Injuries

The alteration and further damage of vascular smooth muscle function have been implicated in the development of vascular complications and diabetes. Little is known about protein tyrosine nitration in vascular smooth muscle cell injury induced by high glucose. In this article, vascular smooth muscle cell was exposed to 30 and 40 mM high glucose for 72 h, and then the cell injury in vascular smooth muscle cell induced by high glucose was studied. It was found that high glucose stimulated vascular smooth muscle cell injury in a dose-dependent manner, including decreasing intracellular and extracellular glutathione contents, increasing malondialdehyde and intracellular reactive oxygen species content, increasing the production of nitric oxide (increased nitrite content in cell and medium), as well as increasing protein tyrosine nitration. By comparing protein tyrosine nitration induced by high glucose conditions and extrinsic factors (hemin-nitrite-glucose oxidase system and 3-morpholinosydnonimine), it may be speculated that protein is nitrated selectively, and specific protein tyrosine nitration is involved in diabetic vascular complications.

Topics: Animals; Aorta; Cell Survival; Glucose; Glucose Oxidase; Glutathione; Hemin; Lipid Peroxidation; Male; Molsidomine; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitric Oxide; Primary Cell Culture; Rats; Rats, Wistar; Reactive Oxygen Species; Tyrosine

Reactive oxygen species have an important role in the control of platelet activity. Superoxide anion (O(2)(-)) is a free radical that can be converted into other reactive oxygen species such as peroxynitrite (ONOO(-)) that is formed from the reaction between O(2)(-) and nitric oxide (NO). There are conflicting data on ONOO(-) effects in platelets because it presents pro- or anti-aggregatory actions. 3-morpholinosydnonimine (SIN-1) co-generates NO and O(2)(-), yielding ONOO(-). Therefore, the present study aimed to investigate the mechanisms involved in the inhibition of human platelet adhesion by SIN-1. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8)platelets/ml) were added to adhere. Exposure of non-activated and thrombin-activated platelets to SIN-1 (0.001-100 microM) concentration-dependently inhibited adhesion, which was accompanied by marked increases in the cyclic GMP levels. In non-activated platelets, the soluble guanylate cyclase inhibitor ODQ prevented the SIN-1-induced cGMP elevations and adhesion inhibition. In thrombin-activated platelets, ODQ fully prevented the SIN-1-induced cGMP elevations, but only partly prevented the adhesion inhibition. The O(2)(-) and ONOO(-) scavengers superoxide dismutase (SOD) and -(-)epigallocatechin gallate, respectively, had minimal effects in non-activated platelets. The inhibition of activated platelets by SIN-1 was reversed by SOD and partly reduced by ECG. Western blot analysis of SIN-1-treated platelets showed a single 105 kDa-nitrated band. Nanospray LC-MS-MS identified the protein containing 3-nitrotyrosine residues as human alpha-actinin-1-cytoskeletal isoform. Our data show that platelet adhesion inhibition by SIN-1 in activated platelets involves cGMP-independent mechanism through O(2)(-) generation. Superoxide anion signaling pathway includes ONOO(-) formation and alpha-actinin nitration.

Topics: Blood Platelets; Blood Proteins; Cell Adhesion; Enzyme Inhibitors; Fibrinogen; Free Radical Scavengers; Guanylate Cyclase; Humans; Molsidomine; Nitrates; Peroxynitrous Acid; Solubility; Superoxides; Thrombin; Tyrosine

Many lines of evidence suggest that microgravity results in increased oxidative stress in the nervous system. In order to protect neuronal cells from oxidative damage induced by microgravity, we selected some flavonoids that might prevent oxidative stress because of their antioxidant activities. Among the 20 flavonoids we examined, we found that isorhamnetin and luteolin had the best protective effects against H(2)O(2) or SIN-1-induced cytotoxicity in SH-SY5Y cells. Using a clinostat to simulate microgravity, we found that isorhamnetin and luteolin treatment protected SH-SY5Y cells by preventing microgravity-induced increases in reactive oxygen species (ROS), nitric oxide (NO) and 3-nitrotyrosine (3-NT) levels, and a decrease in antioxidant power (AP). Moreover, isorhamnetin and luteolin treatment downregulated the expression of inducible nitric oxide synthase (iNOS), and oxidative stress was significantly inhibited by an iNOS inhibitor in SH-SY5Y cells exposed to simulated microgravity (SMG). These results indicate that isorhamnetin and luteolin could protect against microgravity-induced oxidative stress in neuroblastoma SH-SY5Y cells by inhibiting the ROS-NO pathway. These two flavonoids may have potential for preventing oxidative stress induced by space flight or microgravity.

Topics: Animals; Antioxidants; Cell Line, Tumor; Cell Survival; Flavonols; Humans; Hydrogen Peroxide; Luteolin; Molecular Structure; Molsidomine; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Oxidants; Oxidative Stress; Quercetin; Reactive Oxygen Species; Tyrosine; Weightlessness

Surfactant protein D (SP-D) is an important effector of innate immunity. We have previously shown that SP-D accumulates at sites of acute bacterial infection and neutrophil infiltration, a setting associated with the release of reactive species such as peroxynitrite. Incubation of native SP-D or trimeric SP-D lectin domains (NCRDs) with peroxynitrite resulted in nitration and nondisulfide cross-linking. Modifications were blocked by peroxynitrite scavengers or pH inactivation of peroxynitrite, and mass spectroscopy confirmed nitration of conserved tyrosine residues within the C-terminal neck and lectin domains. Mutant NCRDs lacking one or more of the tyrosines allowed us to demonstrate preferential nitration of Tyr314 and the formation of Tyr228-dependent cross-links. Although there was no effect of peroxynitrite or tyrosine mutations on lectin activity, incubation of SP-D dodecamers or murine lavage with peroxynitrite decreased the SP-D-dependent aggregation of lipopolysaccharide-coated beads, supporting our hypothesis that defective aggregation results from abnormal cross-linking. We also observed nitration, cross-linking of SP-D, and a significant decrease in SP-D-dependent aggregating activity in the lavage of mice acutely exposed to nitrogen dioxide. Thus, modification of SP-D by reactive oxygen-nitrogen species could contribute to alterations in the structure and function of SP-D at sites of inflammation in vivo.

Topics: Amino Acid Sequence; Animals; Bronchoalveolar Lavage Fluid; Humans; Mice; Molsidomine; Nitrogen Dioxide; Peroxynitrous Acid; Protein Structure, Quaternary; Protein Structure, Tertiary; Pulmonary Surfactant-Associated Protein D; Rats; Recombinant Proteins; Tandem Mass Spectrometry; Tyrosine

In vitro stimulation of human intervertebral disc (IVD) cells.. To investigate the oxidative/nitrosative effects of peroxynitrite on human nucleus pulposus (NP) cells.. Peroxynitrite is an important tissue-damaging species generated at sites of inflammation and degeneration. The aim of this study was to examine the effects of oxidative/nitrosative stress caused by peroxynitrite and the peroxynitrite donor SIN-1 in human NP cells.. Degenerated human IVD tissue was analyzed for nitrosylation by immunofluorescence. In addition, human NP cells were isolated from IVDs, expanded and stimulated either with peroxynitrite itself or a stable peroxynitrite donor (SIN-1). Nitrosylation, accumulation of intracellular reactive oxygen species, NF-kappaB nuclear translocation, and cell viability were analyzed by fluorescence. Gene expression of TNF-alpha, IL-1beta, IL-6, IL-8, and IL-10 was quantified by real-time (RT)-PCR.. Degenerated IVD tissue showed strong nitrosylation, especially in the NP. Isolated human NP cells showed a strong signal for nitrosylation and intracellular reactive oxygen species on stimulation with peroxynitrite or SIN-1. NF-kappaB/p65 sustained nuclear translocation of NF-kappaB/p65 and stimulation of IL-1beta, IL-6, and IL-8 expression was noted on treatment of cells with SIN-1.. This study provides evidence that peroxynitrite may play a role in disc degeneration and discogenic back pain development by an increased synthesis of proinflammatory cytokines. Nuclear translocation of NF-kappaB was identified as the potential underlying pathway. Therefore, neutralizing peroxynitrite and its derivatives (e.g., via the use of antioxidants) may be a novel treatment option for discogenic back pain.

Topics: Active Transport, Cell Nucleus; Adolescent; Adult; Cell Nucleus; Cells, Cultured; Female; Gene Expression; Humans; Immunohistochemistry; Interleukin-10; Interleukin-1beta; Interleukin-6; Interleukin-8; Intervertebral Disc; Intervertebral Disc Displacement; Male; Middle Aged; Molsidomine; Nitric Oxide Donors; Peroxynitrous Acid; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Tyrosine

Elevated concentrations of nitric oxide (NO) and peroxynitrite (ONOO(-)) are present within the CNS following neurotrauma and are implicated in the pathogenesis of the accompanying neurologic deficits. We tested the hypothesis that elevated extracellular concentrations of ONOO(-), introduced by the donor 3-morpholinosydnonimine (SIN-1), induce reversible axonal conduction deficits in neurons of the guinea-pig spinal cord. The compound action potential (CAP) and compound membrane potential (CMP) of excised ventral cord white matter were recorded before, during, and after, bathing the tissue (30 min) in varying concentrations (0.125-2.0 mM) of SIN-1 (3.75-60 microM ONOO(-)). The principal results were rapid onset, concentration-dependent, reductions in amplitude of the CAP (P<0.05). At a concentration of 0.25 mM of SIN-1 the reduction in CAP amplitude was fully reversible and was not accompanied by any changes in CMP. At higher concentrations of SIN-1 (> or =0.5 mM) the reversibility was incomplete and there was concurrent depolarization of the CMP. These electrophysiological changes were not evident when the donor had been a priori depleted of ONOO(-) by uric acid or was co-administered with the ONOO(-) scavenger ebselen (3 mM). Immuno-fluorescence staining for nitrotyrosine (Ntyr) revealed extensive nitration of tyrosine residues in neurons exposed to higher concentrations of SIN-1. These results are the first to demonstrate that ONOO(-) induces reversible conduction deficits within axons of the spinal cord. The dissociation of CAP and CMP changes at low concentrations of SIN-1, when the CAP changes were reversible and there was no evidence of nitration of tyrosine residues, is consistent with ONOO(-)-induced alteration in Na+ channel conductance in the axolemma. The results support the view that ONOO(-) contributes to both reversible and non-reversible neurologic deficits following neurotrauma. The reversal of immune-mediated conduction deficits may contribute to spontaneous neurologic deficits following neurotrauma.

Topics: Action Potentials; Animals; Axons; Cell Membrane; Dose-Response Relationship, Drug; Extracellular Fluid; Female; Guinea Pigs; Molsidomine; Nerve Degeneration; Neural Conduction; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Peroxynitrous Acid; Sodium Channels; Spinal Cord; Spinal Cord Injuries; Tyrosine

Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL(-)) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL(-) assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC). The plasma LDL(-) fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL(-) revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the alpha-helical structures and beta(2) sheet as well as cysteine oxidation to cysteic acid in beta(1) sheet. Circular dichroism analyses showed that the alpha-helical content of LDL(-) was substantially lower ( approximately 25%) than that of native LDL ( approximately 90%); conversely, LDL(-) showed greater content of beta-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO(-)) or SIN-1: similar amino acid modifications as well as conformational changes (loss of alpha-helical structure and gain in beta-sheet structure) were observed. Both LDL(-) and ONOO(-)-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO(-)-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R. It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.

Topics: Animals; Aorta; Apolipoprotein B-100; Cattle; Cells, Cultured; Cysteine; Dose-Response Relationship, Drug; Endothelial Cells; Endothelium, Vascular; Humans; Lipid Peroxidation; Lipid Peroxides; Lipoproteins, LDL; Models, Chemical; Molsidomine; Oxidation-Reduction; Peroxynitrous Acid; Protein Conformation; Protein Denaturation; Protein Processing, Post-Translational; Protein Structure, Secondary; Proteins; Reproducibility of Results; Tyrosine

We synthesized a series of neoechinulin A derivatives and examined the structure-activity relationships in terms of their anti-nitration and anti-oxidant activities as well as their cytoprotective activity against peroxynitrite from SIN-1 (3-(4-morpholinyl)sydnonimine hydrochloride) using PC12 cells. Our results showed that the C-8/C-9 double bond, which constitutes a conjugate system with indole and diketopiperazine moieties of neoechinulin A is essential for anti-nitration and anti-oxidant activities as well as protection against SIN-1 cytotoxicity. The presence of an intact diketopiperazine moiety is an additional requirement for anti-nitration activity but not for the cytoprotective action. Our results suggest that the antioxidant activity or electrophilic nature of the C-8 carbon, both of which are afforded by the C-8/C-9 double bond, may play a role in the cytoprotective properties of this alkaloid.

Topics: Animals; Antioxidants; Brain Chemistry; Cell Survival; Indicators and Reagents; Indole Alkaloids; Lipid Peroxidation; Magnetic Resonance Spectroscopy; Molsidomine; Nitrates; PC12 Cells; Peroxynitrous Acid; Piperazines; Protective Agents; Rats; Structure-Activity Relationship; Tyrosine

In addition to serving as a biomarker of oxidative/nitrative stress, elevated levels of nitrotyrosine have been shown to cause DNA damage or trigger apoptosis. Whether the body is equipped with mechanisms for protecting against the potentially harmful nitrotyrosine remains unknown. The present study was designed to investigate the possibility that sulfation serves as a pathway for the metabolism/regulation of nitrotyrosine. Using metabolic labelling, nitrotyrosine O-[35S]sulfate was found to be produced and released into the medium of HepG2 human hepatoma cells labelled with [35S]sulfate in the presence of nitrotyrosine. To identify the enzyme(s) responsible for nitrotyrosine sulfation, a systematic study of all eleven known human cytosolic SULTs (sulfotransferases) was performed. Of the 11 enzymes tested, only SULT1A3 displayed sulfating activity toward nitrotyrosine. The pH-dependence and kinetic constants of SULT1A3 with nitrotyrosine or dopamine as substrate were determined. To examine whether the sulfation of nitrotyrosine occurs in the context of cellular physiology, HepG2 cells labelled with [35S]sulfate were treated with SIN-1 (morpholinosydnonimine), a peroxynitrite generator. Increments of nitrotyrosine O-[35S]sulfate were detected in the medium of HepG2 cells treated with higher concentrations of SIN-1. To gain insight into the physiological relevance of nitrotyrosine sulfation, a time-course study was performed using [3H]tyrosine-labelled HepG2 cells treated with SIN-1. The findings confirm that the bulk of free [3H]nitrotyrosine inside the cells was present in the unconjugated form. The proportion of sulfated [3H]nitrotyrosine increased dramatically in the medium over time, implying that sulfation may play a significant role in the metabolism of free nitrotyrosine.

Topics: Arylsulfotransferase; Carcinoma, Hepatocellular; Cell Line, Tumor; Dopamine; Humans; Kinetics; Molsidomine; Sulfotransferases; Tyrosine

Peroxynitrite-mediated oxidative damage has been implicated in brain mitochondrial respiratory dysfunction after traumatic brain injury (TBI), which precedes the onset of neuronal loss. The aim of this study was to investigate the detrimental effects of the peroxynitrite donor SIN-1 (3-morpholinosydnonimine) on isolated brain mitochondria and to screen penicillamine, a stoichiometric (1:1) peroxynitrite-scavenging agent, and tempol, a catalytic scavenger of peroxynitrite-derived radicals, as antioxidant mitochondrial protectants. Exposure of the isolated mitochondria to SIN-1 caused a significant dose-dependent decrease in the respiratory control ratio and was accompanied by a significant increase in state II respiration, followed by significant decreases (P < 0.05) in states III and V. These functional alterations occurred together with significant increases in mitochondrial protein carbonyl (PC), lipid peroxidation-related 4-hydroxynonenal (4-HNE), and 3-nitrotyrosine (3-NT) content. Penicillamine hydrochloride (10 microM) partially but significantly (P < 0.05) protected against SIN-1-induced decreases in states III and V. However, a 2.5 microM concentration of tempol was able to significantly antagonize a 4-fold molar excess (10 microM) concentration of SIN-1 as effectively as were higher tempol concentrations, consistent with the likelihood that tempol works by a catalytic mechanism. The protection of mitochondrial respiration by penicillamine and tempol occurred in parallel with attenuation of PC, 4-HNE, and 3-NT. These results indicate that SIN-1 causes mitochondrial oxidative damage and complex I dysfunction and that antioxidant compounds that target either peroxynitrite or its radicals may be effective mitochondrial protectants in the treatment of neural injury.

Topics: Aldehydes; Animals; Cell Respiration; Cerebral Cortex; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Transport Complex I; Free Radical Scavengers; In Vitro Techniques; Male; Mice; Mice, Inbred Strains; Mitochondria; Molsidomine; Nitric Oxide Donors; Oxidative Stress; Oxygen Consumption; Penicillamine; Peroxynitrous Acid; Protein Carbonylation; Spin Labels; Tyrosine

Treatment of F-actin with the peroxynitrite-releasing agent 3-morpholinosydnonimine (SIN-1) produced a dose-dependent F-actin depolymerization. This is due to released peroxynitrite because it is not produced by 'decomposed SIN-1', and it is prevented by superoxide dismutase concentrations efficiently preventing peroxynitrite formation. F-actin depolymerization has been found to be very sensitive to peroxynitrite, as exposure to fluxes as low as 50-100nM peroxynitrite leads to nearly 50% depolymerization in about 1h. G-actin polymerization is also impaired by peroxynitrite although with nearly 2-fold lower sensitivity. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade of the ability of actin to stimulate myosin ATPase activity. Our results suggest that an imbalance of the F-actin/G-actin equilibrium can account for the observed structural and functional impairment of myofibrils under the peroxynitrite-mediated oxidative stress reported for some pathophysiological conditions.

Topics: Actins; Animals; Calorimetry, Differential Scanning; Enzyme Activation; Molsidomine; Myosins; Oxidation-Reduction; Peroxynitrous Acid; Protein Denaturation; Protein Folding; Rabbits; Temperature; Tyrosine

Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity, reaching 50% inhibition with 46.7 +/- 8.3 microM SIN-1 for 8.7 microM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca(2+)-dependent and K(+)/EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys(707) and Cys(697)), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5'-dithiobis(2-nitrobenzoic acid) and by the parallel decrease of Cys(707) labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADP.V(1) to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphatases; Animals; Catalysis; Cysteine; Dose-Response Relationship, Drug; Enzyme Stability; Magnesium; Molsidomine; Myosin Subfragments; Oxidation-Reduction; Peroxynitrous Acid; Protein Folding; Rabbits; Skeletal Muscle Myosins; Temperature; Time Factors; Tyrosine

Peroxynitrite (ONOO(-)) is a compound formed by reaction of superoxide (O(2) (-)) with nitric oxide (NO) and is expected to possess characteristics of both O(2) (-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOO(-) in defense responses in plants, it has been very difficult to detect ONOO(-) in bioimaging due to its short half-life or paucity of methods for ONOO(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOO(-) in bioimaging, was used for intracellular ONOO(-) detection. ONOO(-) generation in tobacco BY-2 cells treated with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, occurred within 1 h and reached a maximum level at 6-12 h after INF1 treatment. Urate, a ONOO(-) scavenger, abolished INF1-induced ONOO(-) generation. It is well known that ONOO(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOO(-)-specific reaction. Western blot analysis using anti-nitrotyrosine antibodies recognized nitrotyrosine-containing proteins in 20 and 50 kDa bands in BY-2 protein extract containing SIN-1 [3-(4-morpholinyl) sydnonimine hydrochloride; an ONOO(-) donor]. These bands were also recognized in INF1-treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOO(-) detection and tyrosine nitration in defense responses in plants.

Topics: Algal Proteins; Aniline Compounds; Antibodies, Anti-Idiotypic; Cells, Cultured; Cyclic GMP; Fluoresceins; Fungal Proteins; Molsidomine; Nicotiana; Nitric Oxide; Peroxynitrous Acid; Phytophthora; Proteins; Reactive Oxygen Species; Superoxides; Tyrosine; Uric Acid

Peroxynitrite formed by the reaction of superoxide and nitric oxide is a highly reactive species with a role in various pathological processes such as cancer, chronic inflammation, and cardiovascular and neurological diseases. In the present study, the effect of the carotenoids, lycopene and beta-carotene, on peroxynitrite-mediated modifications in plasmid DNA as well as cellular DNA and proteins were investigated. In pUC18 plasmid DNA, these carotenoids strongly inhibited DNA strand breaks caused by peroxynitrite generated from 3-morpholinosydnonimine (SIN-1). SIN-1 was also used to determine effects on DNA damage and protein tyrosine nitration in Chinese hamster lung fibroblasts. SIN-1 dose-dependently increased nitration of proteins in cells above basal levels as determined by Western blotting. This nitration was inhibited in the presence of the uric acid as well as lycopene. Physiological concentrations (0.31-10 microM) of lycopene and beta-carotene also had protective effects on DNA damage, as measured by the comet assay. Lycopene significantly reduced DNA damage particularly, in the median range of concentrations (2.5 microM). The protective effects of lycopene and beta-carotene could be due to their scavenging of reactive oxygen (ROS) and/or nitrogen species (RNS) as they reduce the amount of intracellular ROS/RNS produced following treatment with SIN-1 by as much as 47.5% and 42.4%, respectively. The results obtained in this study suggest that carotenoids may alleviate some of the deleterious effects of peroxynitrite and possibly other reactive nitrogen species as well in vivo.

Topics: Animals; Antioxidants; beta Carotene; Carotenoids; Cell Line; Cell Survival; Comet Assay; Cricetinae; Cricetulus; DNA Damage; Lycopene; Molsidomine; Nitric Oxide Donors; Peroxynitrous Acid; Tyrosine

Peroxynitrite is thought to contribute to the progression of many diseases including cardiovascular disease, cancer, and neurodegenerative disorders. We report that pre-treatment of fibroblasts with the citrus flavanone, hesperetin, prior to peroxynitrite exposure protects against peroxynitrite-mediated cytotoxicity. This protection was partially mediated by the intracellular scavenging of peroxynitrite by hesperetin as exposure of fibroblasts to peroxynitrite following hesperetin loading led to the formation of two intracellular nitro-hesperetin derivatives. In addition, protection appeared to be mediated by hesperetin-induced changes in MAP kinase signalling. Exposure of fibroblasts to hesperetin led to concentration-dependent increases in the phosphorylation of ERK1/2 and was observed to restore peroxynitrite-mediated decreases in ERK1/2 phosphorylation. We propose that the protective potential of hesperetin in fibroblasts may be mediated both by intracellular scavenging of peroxynitrite and by modulation of fibroblast signalling.

Topics: Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Hesperidin; Humans; Molsidomine; Nitric Oxide Donors; Peroxynitrous Acid; Signal Transduction; Tyrosine

Thioredoxin (Trx) is an oxidoreductase that prevents free radical-induced cell death in cultured cells. Here we assessed the mechanism(s) underlying the cardioprotective effects of Trx in vivo.. The effects of myocardial ischemia (30 min) and reperfusion were measured in mice, with assays of myocardial apoptosis, superoxide production, NOx and nitrotyrosine content, and myocardial infarct size. Recombinant human Trx (rhTrx, 0.7-20 mg kg(-1), i.p.) was given 10 min before reperfusion.. Treatment with 2 mg kg(-1) rhTrx significantly decreased myocardial apoptosis and reduced infarct size (P<0.01). Nitrotyrosine content of cardiomyocytes was markedly reduced in rhTrx-treated animals (P<0.01). To further identify the mechanisms by which rhTrx may exert its anti-nitrative effect, iNOS expression and production of NOx and superoxide were determined. Treatment with rhTrx had no significant effect on iNOS expression or NOx content in the ischemic/reperfused heart. However, it markedly upregulated mSOD and reduced tissue superoxide content. To further establish a causative link between the anti- peroxynitrite effect and the cardioprotective effect of rhTrx, cultured adult cardiomyocytes were incubated with SIN-1, a peroxynitrite donor, (50 microM for 3 h) resulting in a nitrotyrosine content comparable to that seen in the ischemic/reperfused heart and causing significant cardiomyocyte apoptosis (P<0.01). Treatment with rhTrx markedly decreased SIN-1 induced apoptosis (P<0.01).. These results demonstrate that Trx is a novel anti-apoptotic and cardioprotective molecule that exerts its cardioprotective effects by reducing ischemia/reperfusion-induced oxidative/nitrative stress.

Topics: Animals; Apoptosis; Male; Mice; Molsidomine; Myocardial Ischemia; Myocardium; Myocytes, Cardiac; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid; Superoxides; Thioredoxins; Tyrosine

Peroxynitrite, a potent oxidant generated by the reaction of NO with superoxide, has been implicated in the promotion of atherosclerosis. We designed this study to determine whether peroxynitrite induces its proatherogenic effects through induction of endoplasmic reticulum (ER) stress.. Human vascular endothelial cells treated with Sin-1, a peroxynitrite generator, induced the expression of the ER chaperones GRP78 and GRP94 and increased eIF2alpha phosphorylation. These effects were inhibited by the peroxynitrite scavenger uric acid. Sin-1 caused the depletion of ER-Ca2+, an effect known to induce ER stress, resulting in the elevation of cytosolic Ca2+ and programmed cell death (PCD). Sin-1 treatment was also found, via 3-nitrotyrosine and GRP78 colocalization, to act directly on the ER. Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented Sin-1-induced PCD. Consistent with these in vitro findings, 3-nitrotyrosine was observed and colocalized with GRP78 in endothelial cells of early atherosclerotic lesions from apolipoprotein E-deficient mice.. Peroxynitrite is an ER stress-inducing agent. Its effects include the depletion of ER-Ca2+, a known mechanism of ER stress induction. The observation that 3-nitrotyrosine-containing proteins colocalize with markers of ER stress within early atherosclerotic lesions suggests that peroxynitrite contributes to atherogenesis through a mechanism involving ER stress.

Topics: Animals; Aorta; Apolipoproteins E; Apoptosis; Atherosclerosis; Calcium; Cells, Cultured; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endothelium, Vascular; Female; Homeostasis; Humans; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Oxidative Stress; Peroxynitrous Acid; Superoxides; Tyrosine; Umbilical Veins

This paper describes the O(2)-dependent control of the reactivity of nitrogen oxide species for the production of biologically important nitrated and nitrosated compounds. In this study, the effects of O(2) on the reactivity of NO, NO(2), and ONOO(-)/ONOOH for nitration of tyrosine (Tyr) and nitrosation of glutathione (GSH) and morpholine (MOR) were examined. NO produced S-nitrosoglutathione (GSNO) and N-nitrosomorpholine (NMOR) through the formation of N(2)O(3) under aerobic conditions, and NO(2) produced 3-nitrotyrosine (3-NO(2)Tyr), GSNO, and NMOR. Transnitrosation from GSNO to MOR was observed only in the presence of O(2). Although preformed ONOO(-)/ONOOH produced all the products under aerobic conditions, the formation of 3-NO(2)Tyr and GSNO was markedly reduced and the formation of NMOR was enhanced under anaerobic conditions. The reactivity of the CO(2) adduct of ONOO(-) was similarly dependent on O(2). 3-NO(2)Tyr was produced effectively by reaction with ONOO(-)/ONOOH at the O(2) concentration of 270 microM and by reaction with its CO(2) adduct at O(2) concentrations greater than 5 microM. Generation of.OH from ONOO(-)/ONOOH was suppressed under anaerobic conditions. The reactivity of ONOO(-)/ONOOH and.OH generation from ONOO(-) were reversibly controlled by the O(2) concentration.

Topics: Anaerobiosis; Electron Spin Resonance Spectroscopy; Glutathione; Molsidomine; Morpholines; Nitric Oxide; Nitrogen Oxides; Nitrosamines; Nitrous Oxide; Oxygen; Peroxynitrous Acid; S-Nitrosoglutathione; Solutions; Tyrosine

1. This study was designed to investigate the effects of the nitric oxide (NO) donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) on N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP, 1 x 10(-7) M)-induced human eosinophil chemotaxis, cyclic guanosine-3',5'-monophosphate (cGMP) levels, protein nitration and cytotoxicity. 2. Human eosinophils were exposed to SNP, SIN-1 and SNAP (0.001-1.0 mM) for either short (10 min) or prolonged (90 min) time periods. Exposition of eosinophils with these NO donors significantly inhibited the eosinophil chemotaxis irrespective of whether cells were exposed to these agents for 10 or 90 min. No marked differences were detected among them regarding the profile of chemotaxis inhibition. 3. Exposition of eosinophils to SNP, SIN-1 and SNAP (0.001-1.0 mM) markedly elevated the cGMP levels above basal levels, but the 90-min exposition resulted in significantly higher levels compared with the 10-min protocols (5.3+/-0.6 and 2.6+/-0.2 nM 1.5 x 10(6) cells(-1), respectively). The cGMP levels achieved with SNAP were greater than SNP and SIN-1. 4. The NO donors did not induce cell toxicity in any experimental condition used. Additionally, eosinophils exposed to SNP, SIN-1 and SNAP (1.0 mM each) either for 10 or 90 min did not show any tyrosine nitration in conditions where a strong nitration of bovine serum albumin was observed. 5. Our findings show that inhibitory effects of fMLP-induced human eosinophil chemotaxis by NO donors at short or prolonged exposition time were accompanied by significant elevations of cGMP levels. However, additional elevations of cGMP levels do not change the functional profile (chemotaxis inhibition) of stimulated eosinophils.

Topics: Adolescent; Adult; Blotting, Western; Cell Survival; Chemotaxis, Leukocyte; Cyclic GMP; Eosinophils; Female; Humans; In Vitro Techniques; Male; Middle Aged; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Nitric Oxide Donors; Nitroprusside; Penicillamine; Tetrazolium Salts; Thiazoles; Tyrosine

Based on evidence that thiol and tyrosine reagents inhibit some amino acid transporters, we tested the hypothesis that NO- and O2- -derived free radicals would impair nutrient uptake by the human placenta. Syncytiotrophoblast microvillous plasma membrane vesicles (MVM) and placental villous fragments were exposed to the drug SIN-1 in the presence or absence of superoxide dismutase (SOD) and hemoglobin (Hb). The uptake of [3H]arginine, [3H]taurine, and [3H]leucine; [14C]MeAIB; and 22Na was studied in MVM, whereas the uptake of [3H]taurine was examined in villous fragments. Nitrotyrosine formation was assessed by Western blotting and quantified by ELISA. In MVM, SIN-1 caused an inhibition of [3H]arginine, [3H]taurine, and [14C]MeAIB uptake but had no significant effect on equilibrium [3H]leucine uptake. These effects were prevented by SOD or Hb, implying that both NO and O2- radicals were essential. In contrast, 22Na+ uptake was significantly increased, and this effect was prevented by SOD. In villous fragments, SIN-1 impaired Na+-dependent [3H]taurine uptake, with no effect on Na+-independent uptake. Increased nitrotyrosine formation was observed in MVM after SIN-1 treatment. Endogenous NO- and O2- -derived free radicals may alter human placental nutrient transfer in vivo, with implications for fetal growth.

Topics: Amino Acids; Biological Transport; Female; Fetal Growth Retardation; Free Radicals; Humans; Infant, Newborn; Microvilli; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Placenta; Pregnancy; Sodium; Subcellular Fractions; Superoxides; Time Factors; Trophoblasts; Tyrosine

Coagulopathy has been associated with clinical scenarios that involve reactive nitrogen species such as peroxynitrite (OONO-). Further, OONO- decreases tissue factor and fibrinogen function in vitro. Thus, we hypothesized that exposure of plasma to the OONO- generated with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide, would result in a decrease in hemostatic function via diminished coagulation protein activity. Hemostatic function of plasma exposed to SIN-1 (0, 1, 5, and 10 mM for 60 min at 37 degrees C) was assessed with thrombelastography, activated partial thromboplastin time, and prothrombin time in the presence or absence of superoxide dismutase (SOD) or an OONO- scavenger. SIN-1 exposure resulted in a significant (P < 0.05), dose-dependent decrease in plasma hemostatic function and concurrent significant (P < 0.05) decreases in activities of factor VII, factor VIII complex, and factor X. Fibrinogen concentration was not affected by SIN-1. Antithrombin and protein C activity also decreased significantly (P < 0.05). Coincubation with SOD or an OONO- scavenger significantly (P < 0.05) attenuated SIN-1 mediated changes in hemostasis and procoagulant/ anticoagulant activity. We conclude that OONO- may decrease hemostatic function in human plasma by nitration of key procoagulants and that OONO- may play a significant role in hemorrhagic states.

Topics: Blood Coagulation Factors; Blotting, Western; Coagulants; Dose-Response Relationship, Drug; Factor X; Hemostasis; Humans; In Vitro Techniques; Molsidomine; Nitric Oxide Donors; Partial Thromboplastin Time; Peroxynitrous Acid; Plasma; Protein C; Prothrombin Time; Superoxide Dismutase; Thrombelastography; Tyrosine

To determine whether peroxynitrite at the concentration and duration present after spinal cord injury induces protein oxidation and nitration in vivo, the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) was administered into the gray matter of the rat spinal cord for 5 hr. The cords were removed at 6, 12, 24, and 48 hr after SIN-1 exposure, immunohistochemically stained with antibodies to dinitrophenyl (DNP) and nitrotyrosine (Ntyr), markers of protein oxidation and nitration, respectively, and the immunostained neurons were counted. The percentages of DNP-positive (P = 0.023-0.002) and Ntyr-positive (P < 0.001 for all) neurons were significantly higher in the SIN-1-exposed groups than in the ACSF controls at each time, suggesting that peroxynitrite induced intracellular oxidation and nitration of proteins. The percentages of DNP- and Ntyr-positive neurons were not significantly different over time in either SIN-1- or ACSF-exposed groups (P = 0.20-1.00). The percentage of DNP-positive neurons was 7.6 +/- 3% to 12 +/- 4.2% at 6-24 hr, and it was 14 +/- 2% to 19 +/- 2% at 6-24 hr for Ntyr-positive neurons after SIN-1-exposure, whereas both ranged over 2-3% in ACSF controls. Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP, a broad-spectrum scavenger of reactive species) significantly reduced the percentages of DNP- and Ntyr-positive neurons (P = 0.04 and 0.002, respectively) compared to a SIN-1-exposed, untreated group at 24 hr after SIN-1 exposure. There were no significant differences between MnTBAP-treated and ACSF controls (P = 0.7 for DNP and 0.2 for Ntyr). These results further demonstrate peroxynitrite-induced protein oxidation and nitration and the efficiency of MnTBAP in scavenging peroxynitrite.

Topics: 2,4-Dinitrophenol; Animals; Drug Interactions; Enzyme Inhibitors; Free Radical Scavengers; Immunohistochemistry; Male; Metalloporphyrins; Microdialysis; Molsidomine; Neurons; Oxidation-Reduction; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Time Factors; Tyrosine

Endogenous sources of reactive nitrogen species (RNS) act as second messengers in a variety of cell signaling events, whereas environmental sources of RNS like nitrogen dioxide (NO2) inhibit cell survival and growth through covalent modification of cellular macromolecules. To examine the effects of RNS on cell cycle progression, murine type II alveolar C10 cells arrested in G0 by serum deprivation were exposed to either NO2 or SIN-1, a generator of RNS, during cell cycle re-entry. In serum-stimulated cells, RNS did not prevent the immediate early gene response by AP-1, but rather blocked cyclin D1 gene expression, resulting cell cycle arrest at the boundary between G0 and G1. Dichlorofluorescin diacetate (DCF) fluorescence indicated that RNS induced sustained production of intracellular hydrogen peroxide (H2O2), which normally is produced only transiently in response to serum growth factors. Loading cells with catalase did not diminish the formation of 3-nitrotyrosine on the cell surface, but rather prevented enhanced DCF fluorescence and rescued cyclin D1 expression and S phase entry. These studies indicate environmental RNS interfere with cell cycle re-entry through an H2O2-dependent mechanism that influences expression of cyclin D1 and progression from G0 to the G1 phase of the cell cycle.

Topics: Animals; Catalase; Cell Cycle; Cells, Cultured; Cyclin D1; Epithelial Cells; Fluoresceins; Gene Expression Regulation; Hydrogen Peroxide; Mice; Molsidomine; Nitric Oxide Donors; Nitrogen Dioxide; Oxidants; Pulmonary Alveoli; Signal Transduction; Superoxide Dismutase; Transcription Factor AP-1; Tyrosine

Peroxynitrite, a potent oxidant generated in inflammatory tissues, can nitrate tyrosine residues on a variety of proteins. Based on previous studies suggesting that actin might be a potential target for peroxynitrite-mediated nitration in neutrophils, we investigated the effects of peroxynitrite on actin function. We show here that peroxynitrite and the peroxynitrite generator (SIN-1) modified actin in a concentration-dependent manner, resulting in an inhibition of globular-actin polymerization and filamentous-actin depolymerization in vitro. The effects of peroxynitrite were inhibited by the pyrrolopyrimidine antioxidant PNU-101033E, which has been shown previously to specifically block peroxynitrite-mediated tyrosine nitration. Furthermore, spectrophotometric and immunoblot analysis of peroxynitrite-treated actin demonstrated a concentration-dependent increase in nitrotyrosine, which was also blocked by PNU-101033E. Activation of neutrophils in the presence of a nitric oxide donor (S-nitroso-N-acetylpenicillamine) resulted in nitration of exogenously added actin. Nitrated actin was also found in peroxynitrite-treated neutrophils, suggesting that actin may be an important intracellular target during inflammation. To investigate this issue, we analyzed the effect of peroxynitrite treatment on a number of actin-dependent neutrophil processes. Indeed, neutrophil actin polymerization, migration, phagocytosis, and respiratory burst activity were all inhibited by SIN-1 treatment in a concentration-dependent manner. Therefore, the ability of peroxynitrite to inhibit actin dynamics has a significant effect on actin-dependent, cellular processes in phagocytic cells and may modulate their host defense function.

Topics: Actins; Chemotaxis; Dimerization; Dose-Response Relationship, Drug; Humans; Molsidomine; Neutrophil Activation; Neutrophils; Nitric Oxide Donors; Peroxynitrous Acid; Phagocytosis; Reactive Oxygen Species; Respiratory Burst; Tyrosine

The protective effect of (-)-epicatechin 3-O-galate (ECg) against peroxynitrite (ONOO-)-mediated damage was examined using an animal model and a cell culture system. In rats subjected to lipopolysaccharide (LPS) administration plus ischemia-reperfusion, the plasma 3-nitrotyrosine level an indicator of ONOO- production in vivo, was elevated, whereas it declined significantly and dose-dependently after the oral administration of ECg at doses of 10 and 20 micromoles/kg body weight/day for 20 days prior to the process. Moreover, oral administration of ECg significantly enhanced the activities of the antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, and the antioxidant glutathione, showing enhancement of the biological defense system against the damage induced by ONOO-. In addition, the significant increase in the renal mitochondrial thiobarbituric acid-reactive substance level of LPS and ischemic-reperfused control rats was attenuated in rats given ECg. Furthermore, the elevations in the plasma urea nitrogen and creatinine (Cr) levels and the urinary methylguanidine/Cr ratio induced by the procedure were attenuated markedly after oral administration of ECg, implying amelioration of renal impairment. The addition of ECg (25 or 125 microM) prior to 3-morpholinosydnonimine (SIN-1, 800 microM) exposure reduced ONOO- formation and increased the viability of cultured renal epithelial (LLC-PK1) cells in a dose-dependent manner. In particular, ECg inhibited ONOO(-)-mediated apoptotic cell death, which was confirmed by decreases in the DNA fragmentation rate and the presence of apoptotic morphological changes, i.e. small nuclei and nuclear fragmentation. Furthermore, adding ECg before SIN-1 treatment regulated the cell cycle by enhancing G2/M phase arrest. This study provides evidence that ECg has protective activity against the renal damage induced by excessive ONOO- in cellular and in vivo systems.

Topics: Animals; Antioxidants; Caspases; Catechin; Cell Cycle; Cell Line; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Glutathione; Glutathione Peroxidase; Kidney; Lipid Peroxides; Lipopolysaccharides; LLC-PK1 Cells; Male; Molsidomine; Peroxynitrous Acid; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Rhodamines; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Time Factors; Tyrosine

Peroxynitrite (ONOO(-)), formed from a reaction of superoxide and nitric oxide, is one of the most potent cytotoxic species that are known to oxidize cellular constituents including essential proteins, lipids, and DNA. In this study, the ability of sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid), isolated from Brassica juncea, to scavenge ONOO(-) was investigated. The data obtained show that sinapic acid can efficiently scavenge native ONOO(-) as well as ONOO(-) derived from the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1). Spectrophotometric analyses revealed that sinapic acid suppressed the formation of ONOO(-)-mediated tyrosine nitration through an electron donation mechanism. In further studies, sinapic acid also showed a significant ability of inhibiting nitration of bovine serum albumin and low-density lipoprotein (LDL) in a dose-dependent manner. Sinapic acid decreased the LDL peroxidation induced by SIN-1-derived ONOO(-). The present study suggests that sinapic acid has an efficient ONOO(-) scavenging ability, which may well be a potent ONOO(-) oxidant scavenger for the protection of the cellular defense activity against the ONOO(-)-involved diseases.

Topics: Brassica; Coumaric Acids; Free Radical Scavengers; Lipid Peroxidation; Lipoproteins, LDL; Molsidomine; Nitrates; Nitric Oxide; Oxidation-Reduction; Peroxynitrous Acid; Serum Albumin, Bovine; Superoxides; Tyrosine

Serum levels of uric acid (UA), an inhibitor of peroxynitrite- (ONOO-) related chemical reactions, became elevated approximately 30 million years ago in hominid evolution. During a similar time frame, higher mammals lost the ability to synthesize another important radical scavenger, ascorbic acid (AA), leading to the suggestion that UA may have replaced AA as an antioxidant. However, in vivo treatment with AA does not protect against the development of experimental allergic encephalomyelitis (EAE), a disease that has been associated with the activity of ONOO- and is inhibited by UA. When compared in vitro, UA and AA were found to have similar capacities to inhibit the nitrating properties of ONOO-. However UA and AA had different capacities to prevent ONOO- -mediated oxidation, especially in the presence of iron ion (Fe3+). While UA at physiological concentrations effectively blocked dihydrorhodamine-123 oxidation in the presence of Fe3+, AA did not, regardless of whether the source of ONOO- was synthetic ONOO-, SIN-1, or RAW 264.7 cells. AA also potentiated lipid peroxidation in vivo and in vitro. In conclusion, the superior protective properties of UA in EAE may be related to its ability to neutralize the oxidative properties of ONOO- in the presence of free iron ions.

Topics: Albumins; Animals; Antioxidants; Ascorbic Acid; Blood-Brain Barrier; Cell Line; Encephalomyelitis, Autoimmune, Experimental; Free Radicals; Immunohistochemistry; Iron; Lipid Peroxidation; Mice; Molsidomine; Myelin Sheath; Nitric Oxide; Oxygen; Peroxynitrous Acid; Rhodamines; Time Factors; Tyrosine; Uric Acid

3-Nitrotyrosine (3-NT) is a specific marker of protein nitration by peroxynitrite (ONOO-) produced from nitric oxide and superoxide. Increase in 3-NT containing protein (3-NT protein) was reported in brains from patients with some neurodegenerative disorders and aging. In this paper, intracellular localization of 3-NT protein was examined in dopaminergic SH-SY5Y cells using the selective antibody against protein-bound 3-NT. 3-NT protein was detected in plasma membrane/nucleus and mitochondria fractions, and interestingly in polypeptide composition of mitochondrial complex I. ONOO--generating SIN-1 induced apoptotic cell death with concomitant increase in 3-NT protein and reduction in mitochondrial ATP synthesis. In addition, an inhibitor of proteasomes, carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal, enhanced the effects of ONOO-. These results suggest that ONOO- may induce mitochondrial dysfunction and cell death in neurons through nitration of mitochondrial complex I subunits.

Topics: Adenosine Triphosphate; Apoptosis; Cell Death; Cysteine Proteinase Inhibitors; Dopamine; Electron Transport Complex I; Humans; Mitochondria; Molsidomine; NADH, NADPH Oxidoreductases; Nitric Oxide Donors; Oligopeptides; Peroxynitrous Acid; Subcellular Fractions; Tumor Cells, Cultured; Tyrosine

There is increasing evidence for the involvement of reactive nitrogen species like peroxynitrite (ONOO-) in airway pathology, for example during allergic airway inflammation. Therefore, the effect of peroxynitrite exposure on airway responsiveness and inflammation was studied.. Male BALB/c mice were treated intra-tracheally with authentic peroxynitrite and the peroxynitrite donor 3-morpholinosydnonimine (SIN-1). Control animals received decomposed solutions of peroxynitrite and SIN- 1.. Airway inflammation was monitored by bronchoalveolar lavage, three and seven days after administration. Airway responsiveness to methacholine and acetylcholine was measured on day 1, 2, 3 and 7 post administration using whole body plethysmography.. Intra-tracheal administration of peroxynitrite 200 microM in 50 microl phosphate buffered saline (PBS) induced a significant increase in macrophages (>35%, p < 0.05) in the airway lumen three days after administration. In contrast, neither intra-tracheal administration of authentic peroxynitrite (up to 5 mM) nor the peroxynitrite donor SIN-1 (1 mM, both intra-tracheal and nebulized) changed airway responsiveness to methacholine. Moreover, peroxynitrite (5 mM) did not alter responsiveness to acetylcholine.. Administration of peroxynitrite directly into the airways of BALB/c mice, induces airway inflammation, but not airway hyperresponsiveness. It is suggested that antioxidants in the epithelial lining fluid and/or the epithelium itself form an efficient barrier, which prevents peroxynitrite from reaching putative targets in the airway interstitium.

Topics: Acetylcholine; Animals; Bronchi; Bronchial Hyperreactivity; Inflammation; Male; Methacholine Chloride; Mice; Mice, Inbred BALB C; Molsidomine; Peroxynitrous Acid; Trachea; Tyrosine

Nitration of proteins by peroxynitrite may alter protein function. We hypothesized that reactive nitrogen species modulate fibronectin-induced fibroblast migration. To test this hypothesis, we evaluated fibroblast migration induced by fibronectin incubated with and without peroxynitrite. Peroxynitrite attenuated fibronectin-induced fibroblast migration in a dose-dependent manner but did not attenuate complement-activated serum-induced fibroblast migration. The reducing agents, deferoxamine and dithiothreitol (DTT), and L-tyrosine reversed the inhibition by peroxynitrite. PAPA-NONOate, a nitric oxide (NO) donor, and superoxide generated by the action of xanthine oxidase on lumazine or xanthine, also showed an inhibitory effect on fibroblast migration. The peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), caused a concentration-dependent inhibition of fibroblast migration. Peroxynitrite reduced fibronectin binding to fibroblasts and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of fibronectin binding to fibroblasts and suggest that peroxynitrite may play a role in regulation of fibroblast migration.

Topics: Blood; Cell Line; Cell Movement; Complement Activation; Deferoxamine; Dithiothreitol; Embryo, Mammalian; Fibroblasts; Fibronectins; Humans; Lung; Molsidomine; Nitrates; Nitric Oxide Donors; Nitrogen; Reactive Oxygen Species; Reducing Agents; Superoxides; Tyrosine; Xanthine Oxidase

To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers.. Polymorphonuclear neutrophils from 3 lactating cows.. Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots.. Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner.. Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.

Topics: Aniline Compounds; Animals; Antioxidants; Blotting, Western; Cattle; Cell Death; Enzyme Inhibitors; Epithelial Cells; Female; Free Radical Scavengers; Histidine; L-Lactate Dehydrogenase; Lipopolysaccharides; Mammary Glands, Animal; Molsidomine; Neutrophil Activation; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Oxidants; Peroxidase; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Tyrosine

Peroxynitrite, formed by nitric oxide (NO) and superoxide, can alter protein function by nitrating amino acids such as tyrosine, cysteine, trytophan, or methionine. Inducible nitric oxide synthase (Type 2 NOS or iNOS) converts arginine to citrulline, releasing NO. We hypothesized that peroxynitrite could function as a negative feedback modulator of NO production by nitration of iNOS. Confluent cultures of the murine lung epithelial cell line, LA-4 were stimulated with cytokines to express iNOS, peroxynitrite was added, and the flasks sealed. After 3 h, NO in the headspace above the culture was sampled. Peroxynitrite caused a concentration-dependent decrease in NO. Similar results were obtained when 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, was added to the flasks. PAPA-NONOate, the NO generator, did not affect the headspace NO. Nitration of the iNOS was confirmed by detection of 3-nitrotyrosine by Western blotting. These data suggest a mechanism for inhibition of NO synthesis at inflammatory sites where iNOS, NO, and superoxide would be expected.

Topics: Animals; Base Sequence; Cell Line; DNA Primers; Enzyme Inhibitors; Epithelial Cells; Gene Expression; Hydrazines; Lung; Mice; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidants; RNA, Messenger; Tyrosine

Most thymocytes are deleted by thymic selection. The mechanisms of cell death are far from being clear. Peroxynitrite is a powerful oxidant produced in vivo by the reaction of superoxide (O2*-) with nitric oxide (NO*) and is able to mediate apoptosis. The aim of this study was to analyze whether NO and peroxynitrite could play a role in human thymocyte apoptosis. The results indicate that 3-(4-morpholinyl)-sydnonimine (SIN-1, an O2*- and NO* donor) and chemically synthesized peroxynitrite, but not S-nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO* donor), have a strong apoptotic effect on human thymocytes (annexin V staining and TUNEL reaction). This effect was inhibited by exogenous superoxide dismutase (SOD), which interacts with O2*- and inhibits the formation of peroxynitrite. Because peroxynitrite formation requires NO*, thymic stromal cells were investigated to determine if they produced NO*. Inducible NOS was synthesized in cultured thymic epithelial cells in certain conditions of cytokine stimulation, as shown by messenger RNA levels, protein analysis, and nitrite production in the supernatants. SIN-1-treated thymocytes had high levels of tyrosine nitration, abolished by the addition of exogenous SOD. Tyrosine nitration was also detected in thymus extracts and sections, suggesting the presence of peroxynitrite in situ. In thymus sections, clusters of nitrotyrosine-positive cells were found in the cortex and corticomedullary areas colocalized with cells positive in the TUNEL reaction. These data indicate an association between human thymocyte apoptosis and nitrotyrosine formation. Thus, the results support the notion of a physiologic role for peroxynitrite in human thymocyte apoptosis. (Blood. 2001;97:3521-3530)

Topics: Annexin A5; Apoptosis; Cells, Cultured; Child, Preschool; Enzyme Inhibitors; Epithelial Cells; Gene Expression; Humans; In Situ Nick-End Labeling; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Stromal Cells; Superoxide Dismutase; Superoxides; T-Lymphocytes; Thymus Gland; Tyrosine

The administration of bacterial lipopolysaccharide (LPS; endotoxin) can stimulate the development of the systemic inflammatory response syndrome, which can compromise the function of many organ systems, resulting in multiple organ failure. Activation of macrophages and cytokines by endotoxin and the subsequent formation of reactive oxygen and nitrogen species are of central pathogenic importance in various inflammatory diseases including sepsis. However, whether different tissues behave the same in pathological changes produced by LPS and what factors may affect pathological processes and protein tyrosine nitration in different organs, still remain to be evaluated. In the present study, we investigated the distribution of nitrotyrosine and other pathological changes induced by LPS in rat liver, spleen, and lung, all of which are rich in macrophages and endothelial cells. Our study revealed two important findings: first, a denitration activity in spleen white pulp might play a key role to protect the areas from nitration. Similar activity might also exist in endothelial cells of sinusoids and capillaries. Second, protein nitration might not induce significant tissue damage as shown in liver and spleen. However, inflammatory infiltration with increased formation NO* and other reactive species may result in severe tissue injury, as demonstrated in lung after LPS administration.

Topics: Animals; Endothelium, Vascular; Immunoblotting; Immunoenzyme Techniques; Lipopolysaccharides; Liver; Lung; Macrophages; Male; Molsidomine; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Peroxidase; Rats; Rats, Sprague-Dawley; Spleen; Superoxides; Tyrosine

Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated barrier dysfunction in cultured porcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD, Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am J Physiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,.NO rapidly combines with superoxide (O) to form the powerful oxidant peroxynitrite (ONOO(-)), which we hypothesized would cause PAEC monolayer barrier dysfunction. To test this hypothesis, we treated PAEC with ONOO(-) (500 microM) or 3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 microM). SIN-1-mediated ONOO(-) formation was confirmed by monitoring the oxidation of dihydrorhodamine 123 to rhodamine. Both ONOO(-) and SIN-1 increased albumin clearance (P < 0.05) in the absence of cytotoxicity and altered the architecture of the cytoskeletal proteins actin and beta-catenin as detected by immunofluorescent confocal imaging. ONOO(-)-induced barrier dysfunction was partially reversible and was attenuated by cysteine. Both ONOO(-) and SIN-1 nitrated tyrosine residues, including those on beta-catenin and actin, and oxidized proteins in PAEC. The introduction of actin treated with ONOO(-) into PAEC monolayers via liposomes also resulted in barrier dysfunction. These results indicate that ONOO(-) directly alters endothelial cytoskeletal proteins, leading to barrier dysfunction.

Topics: Actins; Animals; beta Catenin; Cell Survival; Cells, Cultured; Cytoskeletal Proteins; Endothelium, Vascular; Kinetics; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Oxidants; Pulmonary Artery; Reactive Oxygen Species; Superoxide Dismutase; Swine; Trans-Activators; Tyrosine

Evidence of an overload of reactive oxygen species and peroxynitrite, a derivative of nitric oxide, in sporadic amyotrophic lateral sclerosis suggests that peroxynitrite could impair cholinergic functions. Because of the impossibility of obtaining synaptosomes from vertebrate neuromuscular junctions, we used cholinergic synaptosomes purified from Torpedo marmorata electroneurons to characterize the defects triggered by peroxynitrite in more detail. Addition of peroxynitrite or its donor 3-morpholinosydnonimine abolished high-affinity choline uptake and synthesis of acetylcholine from acetate. T. marmorata choline acetyltransferase (ChAT) was impaired to the same extent as bovine brain ChAT. A hallmark of peroxynitrite action is the nitration of tyrosine residues in proteins. Peroxynitrite induced a concentration-dependent appearance of nitrotyrosines in several neuronal proteins from synaptosomes and, more readily, from synaptic vesicles. Peroxynitrite also triggered tyrosine nitrations in purified ChAT. Peroxynitrite-dependent nitrations were impaired when synaptosomes were pretreated with thioreductants (glutathione, N-acetyl cysteine, dithiothreitol) or antioxidants (uric acid, melatonin, bovine serum albumin, desferrioxamine). Deleterious effects of peroxynitrite on choline transport and ChAT activity were prevented by the thioreductants but only partially by the antioxidants, suggesting a mechanism other than tyrosine nitration, which may involve cysteine oxidation. Further development of protective agents acting on choline transport and on ChAT activity may offer interesting therapeutic possibilities with respect to cholinergic dysfunction occurring in neurodegenerative diseases.

Topics: Acetates; Acetylcholine; Animals; Antioxidants; Biological Transport; Carbon Radioisotopes; Choline; Choline O-Acetyltransferase; Drug Interactions; Molsidomine; Nitrates; Oxidants; Reducing Agents; Torpedo; Tyrosine; Uric Acid

3-Morpholinosyndnomine (SIN-1) has been reported to be a peroxynitrite (OONO(-)) donor because it produces both nitric oxide (NO) and superoxide (O(2)(-).) upon decomposition in aqueous solution. However, SIN-1 can decompose to primarily NO in the presence of electron acceptors, including those found in biological tissues, making it necessary to determine the release product(s) formed in any given biological system. In a mixed cortical cell culture system, SIN-1 caused a concentration-dependent increase in cortical cell injury with a parallel increase in the release of cellular proteins containing 3-nitrotyrosine into the culture medium. The increase in 3-nitrotyrosine immunoreactivity, a footprint of OONO(-) production, was specific for SIN-1 as exposure to neurotoxic concentrations of an NO donor (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (DETA/NO), or NMDA did not result in the nitration of protein tyrosine residues. Both SIN-1-induced injury and 3-nitrotyrosine staining were prevented by the addition of either 5,10,15,20-Tetrakis (4-sulfonatophenyl) prophyrinato iron (III) [FeTPPS], an OONO(-) decomposition catalyst, or uric acid, an OONO(-) scavenger. Removal of NO alone was sufficient to inhibit the formation of OONO(-) from SIN-1 as well as its cytotoxicity. Removal of O(2)(-). and the subsequently formed H(2)O(2) by superoxide dismutase (SOD) plus catalase likewise prevented the nitration of protein-bound tyrosine but actually enhanced the cytotoxicity of SIN-1, indicating that cortical cells can cope with the oxidative but not the nitrosative stress generated. Finally, neural injury induced by SIN-1 in unadulterated cortical cells was prevented by antagonism of AMPA/kainate receptors, while blockade of the NMDA receptor was without effect. In contrast, activation of both NMDA and non-NMDA receptors contributed to the SIN-1-mediated neurotoxicity when cultures were exposed in the presence of SOD plus catalase. Thus, whether SIN-1 initiates neural cell death in an OONO(-)-dependent or -independent manner is determined by the antioxidant status of the cells. Further, the mode of excitotoxicity by which injury progresses is determined by the NO-related species generated.

Topics: Animals; Astrocytes; Catalase; Cell Death; Cells, Cultured; Cerebral Cortex; Free Radical Scavengers; Mice; Molsidomine; Neurons; Neurotoxins; Peroxynitrous Acid; Superoxide Dismutase; Tyrosine

Taurine is actively transported at the retinal pigment epithelial (RPE) apical membrane in an Na(+)- and Cl(-)-dependent manner. Diabetes may alter the function of the taurine transporter. Because nitric oxide (NO) is a molecule implicated in the pathogenesis of diabetes, we asked whether NO would alter the activity of the taurine transporter in cultured ARPE-19 cells. The activity of the transporter was stimulated in the presence of the NO donor 3-morpholinosydnonimine. The stimulatory effects of 3-morpholinosydnonimine were not observed during the initial 16-h treatment; however, stimulation of taurine uptake was elevated dramatically above control values with 20- and 24-h treatments. Kinetic analysis revealed that the stimulation was associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study provides the first evidence of an increase in taurine transporter gene expression in human RPE cells cultured under conditions of elevated levels of NO.

Topics: Animals; Antioxidants; Ascorbic Acid; Carrier Proteins; Cell Line; Diabetes Mellitus; Dose-Response Relationship, Drug; Glutathione; Humans; Immunohistochemistry; Membrane Glycoproteins; Membrane Transport Proteins; Methylene Blue; Mice; Mice, Inbred ICR; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Pigment Epithelium of Eye; Protein Synthesis Inhibitors; Taurine; Time Factors; Tyrosine

Pretreatment of interferon-gamma and lipopolysaccharides made C6 glioma cells highly vulnerable to glucose deprivation. Neither 12 h of glucose deprivation nor 2-day treatment with interferon-gamma (100 U/ml) and lipopolysaccharides (1 microg/ml) altered the viability of C6 glioma cells. However, significant death of immunostimulated C6 glioma cells was observed after 5 h of glucose deprivation. The augmented death was prevented by dehydroepiandrosterone (DHEA) treatment during immunostimulation, but not by DHEA treatment during glucose deprivation. DHEA reduced the rise in nitrotyrosine immunoreactivity, a marker of peroxynitrite, and superoxide production in glucose-deprived immunostimulated C6 glioma cells. DHEA, however, did not protect glucose-deprived C6 glioma cells from the exogenously produced peroxynitrite by 3-morpholinosydnonimine. Further, DHEA did not alter the production of total reactive oxygen species and nitric oxide in immunostimulated C6 glioma cells. Superoxide dismutase (SOD) and the synthetic SOD mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin inhibited the death of glucose-deprived immunostimulated C6 glioma cells. In addition, a superoxide anion generator paraquat reversed the protective effect of DHEA on the augmented death. The data indicate that DHEA prevents the glucose deprivation-evoked augmented death by inhibiting the production of superoxide anion in immunostimulated C6 glioma cells.

Topics: Adjuvants, Immunologic; Animals; Astrocytes; Brain; Brain Ischemia; Cell Death; Cytokines; Dehydroepiandrosterone; Enzyme Inhibitors; Free Radical Scavengers; Glioma; Glucose; Herbicides; Humans; Interferon-gamma; Lipopolysaccharides; Metalloporphyrins; Molsidomine; Neurons; Nitric Oxide; Oxidative Stress; Paraquat; Peroxynitrous Acid; Superoxide Dismutase; Tumor Cells, Cultured; Tyrosine

Peroxynitrite, formed by the reaction between nitric oxide and superoxide, has been shown to induce protein nitration, which compromises protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the neutrophil chemotactic activity (NCA) of interleukin-8 (IL-8) incubated with peroxynitrite was evaluated. Peroxynitrite attenuated IL-8 NCA in a dose-dependent manner (p < 0.01) but did not significantly reduce NCA induced by leukotriene B(4) or complement-activated serum. The reducing agents, dithionite, deferoxamine, and dithiothreitol, reversed and exogenous L-tyrosine abrogated the peroxynitrite-induced NCA inhibition. Papa-NONOate [N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1, 2-dialase or sodium nitroprusside, NO donors, or a combination of xanthine and xanthine oxidase to generate superoxide did not show an inhibitory effect on NCA induced by IL-8. In contrast, small amounts of SIN-1, a peroxynitrite generator, caused a concentration-dependent inhibition of NCA by IL-8. Consistent with its capacity to reduce NCA, peroxynitrite treatment reduced IL-8 binding to neutrophils. Nitrotyrosine was detected in the IL-8 incubated with peroxynitrite by enzyme-linked immunosorbent assay. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of IL-8 binding to neutrophils and a reduction in NCA and suggest that oxidants may play an important role in regulation of IL-8-induced neutrophil chemotaxis.

Topics: Chemotaxis, Leukocyte; Humans; Interleukin-8; Leukotriene B4; Molsidomine; Neutrophils; Nitrates; Nitric Oxide Donors; Tyrosine

We assessed whether reactive oxygen-nitrogen intermediates generated by alveolar macrophages (AMs) oxidized and nitrated human surfactant protein (SP) A. SP-A was exposed to lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4) for 30 min in the presence and absence of 1.2 mM CO(2). In the presence of CO(2), lipopolysaccharide-stimulated AMs had significantly higher nitric oxide synthase (NOS) activity (as quantified by the conversion of L-[U-(14)C]arginine to L-[U-(14)C]citrulline) and secreted threefold higher levels of nitrate plus nitrite in the medium [28 +/- 3 vs. 6 +/- 1 (SE) nmol. 6.5 h(-1). 10(6) AMs(-1)]. Western blotting studies of immunoprecipitated SP-A indicated that CO(2) enhanced SP-A nitration by AMs and decreased carbonyl formation. CO(2) (0-1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ability of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO(2). Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr(164) and Tyr(166)) in the absence of CO(2) and three tyrosines (Tyr(164), Tyr(166), and Tyr(161)) in the presence of 1.2 mM CO(2). These findings indicate that physiological levels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO(2) increased nitration, at least partially, by enhancing enzymatic nitric oxide production.

Topics: Carbon Dioxide; Cells, Cultured; Enzyme Activation; Epithelial Cells; Humans; Hypercapnia; Lipid Metabolism; Lipopolysaccharides; Macrophages, Alveolar; Mass Spectrometry; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrites; Oxidation-Reduction; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Respiratory Distress Syndrome; Tyrosine

Peroxynitrite (PN) is likely to be generated in vivo from nitric oxide and superoxide. We have previously shown that prostacyclin synthase, a heme-thiolate enzyme essential for regulation of vascular tone, is nitrated and inactivated by submicromolar concentrations of PN [Zou, M.-H. & Ullrich, V. (1996) FEBS Lett. 382, 101-104] and we have studied the effect of heme proteins on the PN-mediated nitration of phenolic compounds in model systems [Mehl, M., Daiber, A. & Ullrich, V. (1999) Nitric Oxide: Biol. Chem. 2, 259-269]. In the present work we show that bolus additions of PN or PN-generating systems, such as SIN-1, can induce the nitration of P450BM-3 (wild-type and F87Y variant), for which we suggest an autocatalytic mechanism. HPLC and MS-analysis revealed that the wild-type protein is selectively nitrated at Y334, which was found at the entrance of a water channel connected to the active site iron center. In the F87Y variant, Y87, which is directly located at the active site, was nitrated in addition to Y334. According to Western blots stained with a nitrotyrosine antibody, this nitration started at 0.5 microM of PN and was half-maximal between 100 and 150 microM of PN. Furthermore, PN caused inactivation of the P450BM-3 monooxygenase as well as the reductase activity with an IC50 value of 2-3 microM. As two thiol residues/protein molecule were oxidized by PN and the inactivation was prevented by GSH or dithiothreitol, but not by uric acid (a powerful inhibitor of the nitration), our data strongly indicate that the inactivation is due to thiol oxidation at the reductase domain rather then to nitration of Y residues. Stopped-flow data presented here support our previous hypothesis that ferryl-species are involved as intermediates during the reactions of P450 enzymes with PN.

Topics: Animals; Bacillus; Bacterial Proteins; Binding Sites; Blotting, Western; Cattle; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Dithiothreitol; Dose-Response Relationship, Drug; Erythrocytes; Glutathione; Inhibitory Concentration 50; Iron; Mass Spectrometry; Mixed Function Oxygenases; Models, Chemical; Models, Molecular; Molsidomine; NADPH-Ferrihemoprotein Reductase; Nitrates; Nitric Oxide Donors; Nitrogen; Oxygen; Superoxide Dismutase; Temperature; Time Factors; Tyrosine; Uric Acid

Nitric oxide reacts rapidly with superoxide to form the strong nitrating agent peroxynitrite, which is responsible for much of the tissue damage associated with diverse pathophysiological conditions such as inflammation. The occurrence of free or protein-bound nitrotyrosine (NTYR) has been considered as evidence for in vivo formation of peroxynitrite. However, various agents can nitrate tyrosine, and their relative significance in vivo has not been determined due to lack of a sensitive method to analyze NTYR in tissue proteins and biological fluids. We have developed a new HPLC-electrochemical detection method to analyze NTYR in protein hydrolyzates or biological fluids. The sample is injected directly into a reversed-phase HPLC column and NTYR is subsequently reduced by a platinum column to 3-aminotyrosine, which is quantified with an electrochemical detector. The method is simple, selective, and sensitive (detection limit, 0.1 pmol per 20-microl injection). We have applied this method to compare in vitro the ability of various nitrating agents to form NTYR in bovine serum albumin and human plasma. Yields of NTYR formed in human plasma proteins incubated with 1 or 10 mM nitrating agent decreased in the following order: synthetic peroxynitrite > 3-morpholinosydonimine, a generator of both NO and superoxide > Angeli's salt, which forms nitroxyl anion (NO-) > spermine-NONOate, which releases NO > sodium nitrite plus hypochlorite, which forms the nitrating agent nitryl chloride (NO2Cl). A simple purification method using a C18 Sep-Pak cartridge is also described for analysis of free NTYR in human plasma.

Topics: Chromatography, High Pressure Liquid; Electrochemistry; Humans; Hypochlorous Acid; Indicators and Reagents; Molsidomine; Nitrates; Nitrites; Nitrogen Oxides; Serum Albumin, Bovine; Sodium Nitrite; Spectrophotometry, Ultraviolet; Spermine; Tyrosine

We found that the exposure of human neuroblastoma SH-SY5Y cells to the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) induced tyrosine nitration of a 130-kDa protein, and prevented tyrosine phosphorylation of the 130-kDa protein. The focal adhesion protein p130cas was identified as a component of the 130-kDa protein using specific antibody. These results suggest that p130cas is a new target protein for nitration induced by SIN-1.

Topics: Crk-Associated Substrate Protein; Humans; Molsidomine; Neuroblastoma; Nitrates; Nitric Oxide Donors; Oxidants; Phosphoproteins; Phosphorylation; Proteins; Retinoblastoma-Like Protein p130; Tumor Cells, Cultured; Tyrosine

Eosinophils and increased production of nitric oxide (NO) and superoxide, components of peroxynitrite, have been implicated in the pathogenesis of a number of allergic disorders including asthma. Peroxynitrite induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may modulate eosinophil migration by modulating chemotactic cytokines. To test this hypothesis, the eosinophil chemotactic responses of regulated on activation, normal T cell expressed and secreted (RANTES) and interleukin (IL)-5 incubated with and without peroxynitrite were evaluated. Peroxynitrite-attenuated RANTES and IL-5 induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum ECA. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, a NO donor, or superoxide generated by lumazine or xanthine and xanthine oxidase, did not show an inhibitory effect on ECA. The peroxynitrite generator, 3-morpholinosydnonimine, caused a concentration-dependent inhibition of ECA. Peroxynitrite reduced RANTES and IL-5 binding to eosinophils and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of RANTES and IL-5 binding to eosinophils and suggest that peroxynitrite may play a role in regulation of eosinophil chemotaxis.

Topics: Chemokine CCL5; Chemotaxis; Deferoxamine; Dithiothreitol; Dose-Response Relationship, Drug; Eosinophils; Humans; Hydrazines; Interleukin-5; Leukotriene B4; Molsidomine; Nitrates; Nitric Oxide; Nitrogen; Pteridines; Reactive Oxygen Species; Superoxides; Tyrosine; Xanthine

Carboxy-PTIO reacts rapidly with NO to yield NO2 and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1, Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC50 = 36 +/- 5 microM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC50 of 0.12 +/- 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC50 of 0.11 +/- 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger.

Topics: Animals; Benzoates; Cattle; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Free Radical Scavengers; Imidazoles; Kinetics; Molsidomine; Nitrates; Nitric Oxide; Nitrites; Serum Albumin, Bovine; Swine; Tyrosine

The cytotoxic effects of 3-morpholinosydnonimine (Sin-1) and S-nitroso-N-acetylpenicillamine-amine (SNAP) on replicatively active human hepatocyte cells in culture was determined as a function of oxidant type. Both Sin-1 which yields nitric oxide and peroxynitrite following the generation of superoxide anion plus nitric oxide, and SNAP which generates nitric oxide, induced dose dependent decreases in the colony forming capabilities of the human hepatocytes. Sin-1 was much more cytotoxic (LD50 = 400 microM) than SNAP (LD50 = 1250 microM). Comparatively, both compounds were much less cytotoxic than H2O2 (LD50 = 96 microM). Sin-1 induced 4-fold higher levels of cellular nitrite than that generated by the chemical in cell free medium. Nitrotyrosine, a marker of peroxynitrite formation in cells, was immunohistochemically detected in hepatocytes treated with both Sin-1 and SNAP. The formation of 3-nitrotyrosine by hepatocytes incubated with SNAP, suggests that hepatocytes generate intracellular superoxide which reacts with the exogenous nitric oxide derived from SNAP to produce intracellular peroxynitrite, resulting in the SNAP cytotoxicity. The enhanced levels of Sin-1 cytotoxicity on the hepatocytes is suggested to be due both to the chemical generation of peroxynitrite and superoxide anion by Sin-1. These data indicate that peroxynitrite is formed in cultured human hepatocytes inhibiting their replication, and that peroxynitirite may play a significant role in the pathogenesis of liver disease.

Topics: Cells, Cultured; Enzyme Inhibitors; Humans; In Vitro Techniques; Liver; Molsidomine; Nitrites; Nitrogen; Penicillamine; S-Nitroso-N-Acetylpenicillamine; Sodium-Potassium-Exchanging ATPase; Tyrosine

Experimental evidence has implicated oxidative stress in the development of Parkinson's disease, amyotrophic lateral sclerosis, and other degenerative neuronal disorders. Recently, peroxynitrite, which is formed by the nearly diffusion-limited reaction of nitric oxide with superoxide, has been suggested to be a mediator of oxidant-induced cellular injury. The potential role of peroxynitrite in the pathology associated with Parkinson's disease was evaluated by examining its effect on DOPA synthesis in PC12 pheochromocytoma cells. Peroxynitrite was generated from the compound 3-morpholinosydnonimine (SIN-1), which releases superoxide and nitric oxide simultaneously. Exposure of PC12 cells to peroxynitrite for 60 min greatly diminished their ability to synthesize DOPA without apparent cell death. The inhibition was due neither to the formation of free nitrotyrosine nor the oxidation of DOPA by peroxynitrite. The inhibition in DOPA synthesis by SIN-1 was abolished when superoxide was scavenged by the addition of superoxide dismutase. These data indicated that neither nitric oxide nor hydrogen peroxide generated by the dismutation of superoxide is responsible for the SIN-1-mediated inhibition of DOPA production. The inhibition of DOPA synthesis at high concentration of SIN-1 persisted even after removal of SIN-1. The inactivation of the tyrosine hydroxylase may be responsible for the significant decline in DOPA formation by peroxynitrite. Inactivation of tyrosine hydroxylase may be part of the initial insult in oxidative damage that eventually leads to cell death.

Topics: Animals; Dihydroxyphenylalanine; Molsidomine; Nitrates; Oxidation-Reduction; PC12 Cells; Rats; Tyrosine