3-nitrotyrosine and Chronic-Disease

Inhaled nitric oxide (iNO) is an effective adjunct in the treatment of infants with respiratory failure. Although there are clear benefits to this therapy, potential toxicity could result from reactive nitrosylated species.. To evaluate whether iNO therapy is associated with increased serum markers of oxidative stress.. Multiple markers were prospectively evaluated in the serum of term infants with severe respiratory failure treated with iNO for 1 to 72 hours. These were compared to those of patients exposed to greater than 80% oxygen for more than 6 hours and room air controls.. After 24 hours of exposure, the iNO-treated infants had increased serum lipid hydroperoxides (LPO), protein carbonyls and nitrotyrosine residues as well as increased serum total glutathione (GSH) content. The increase in LPO peaked at 24 hours and correlated with the cumulative dose of iNO whereas other markers did not. The presence of chronic lung disease (CLD) did not correlate with serum markers of oxidative injury.. In term infants with respiratory failure, prolonged iNO exposure is associated with a transient increase in markers of oxidative stress, but this finding does not appear to predict the development of CLD.

Topics: Administration, Inhalation; Biomarkers; Bronchodilator Agents; Chronic Disease; Female; Glutathione; Humans; Infant, Newborn; Lipid Peroxides; Male; Nitric Oxide; Oxidative Stress; Prospective Studies; Respiratory Distress Syndrome, Newborn; Tyrosine

Chronic heart failure (CHF) is accompanied by an inflammatory activation which occurs both systemically and in the skeletal muscle. Exercise training has been shown to reduce the local expression of cytokines and inducible nitric oxide synthase (iNOS) in muscle biopsies of CHF patients. INOS-derived NO can inhibit oxidative phosphorylation and contribute to skeletal muscle dysfunction in CHF.. To investigate the correlation between changes in local iNOS expression associated with regular exercise and changes in aerobic enzyme activities in the skeletal muscle of patients with CHF. Twenty male CHF patients [ejection fraction 25% (SE 2), age 54 (SE 2) years] were randomized to a training (n=10) or a control group (C, n=10).. At baseline and after 6 months skeletal muscle iNOS expression was measured by real-time polymerase chain reaction. INOS protein and protein nitrosylation were assessed by immunohistochemistry. Cytochrome c oxidase (COX) activity was quantified electrochemically using the Clark oxygen electrode.. Exercise training led to a 27% increase in cytochrome c oxidase activity [from 21.8 (SE 3.2) to 27.7 (SE 3.5) nmol O2/mg per min, P=0.02 versus baseline]. Changes in iNOS expression and iNOS protein content were inversely correlated with changes in COX-activity (r=-0.60, P=0.01; r=-0.71, P<0.001).. The inverse correlation between iNOS expression/iNOS protein content and COX-activity indicates that local anti-inflammatory effects may contribute to improved muscular oxidative metabolism.

Topics: Aged; Chronic Disease; Electron Transport Complex IV; Energy Metabolism; Exercise; Exercise Test; Exercise Tolerance; Follow-Up Studies; Heart Failure; Humans; Immunohistochemistry; Male; Middle Aged; Muscle, Skeletal; Nitric Oxide Synthase Type II; Oxidative Stress; Oxygen Consumption; Prospective Studies; Tyrosine

The present study was designed to investigate the effects of chronic predictable stress (CPS) and chronic unpredictable stress (CUS) on immunological responses in KLH-sensitized rats and involvement of NOergic signaling pathways mediating such responses. Male Wistar rats (200-250g) were exposed to either CPS or CUS for 14days and IgG antibody levels and delayed type hypersensitivity (DTH) response was determined to assess changes in adaptive immunity. To evaluate the role of nitric oxide during such immunomodulation, biochemical estimation of stable metabolite of nitric oxide (NOx) and 3-nitrotyrosine (3-NT, a marker of peroxynitrite formation) were done in both blood and brain. Chronic stress exposure resulted in suppression of IgG and DTH response and elevated NOx and 3-NT levels, with a difference in magnitude of response in CPS vs CUS. Pretreatment with aminoguanidine (iNOS inhibitor) caused further reduction of adaptive immune responses and attenuated the increased NOx and 3-NT levels in CPS or CUS exposed rats. On the other hand 7-NI (nNOS inhibitor) did not significantly affect these estimated parameters. The results suggest involvement of iNOS and lesser/no role of nNOS during modulation of adaptive immunity to stress. Thus, the result showed that predictability of stressors results in differential degree of modulation of immune responses and complex NO-mediated signaling mechanisms may be involved during responses.

Topics: Adaptive Immunity; Animals; Brain; Chronic Disease; Disease Models, Animal; Hypersensitivity, Delayed; Immunity, Humoral; Immunoglobulin G; Immunosuppression Therapy; Male; Nitric Oxide; Rats; Restraint, Physical; Stress, Psychological; Tyrosine

Augmented vasoconstrictor reactivity is thought to play an important role in the development of chronic hypoxia (CH)-induced neonatal pulmonary hypertension. However, whether this response to CH results from pulmonary endothelial dysfunction and reduced nitric oxide (NO)-mediated vasodilation is not well understood. We hypothesized that neonatal CH enhances basal tone and pulmonary vasoconstrictor sensitivity by limiting NO-dependent pulmonary vasodilation. To test this hypothesis, we assessed the effects of the NO synthase (NOS) inhibitor

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Chronic Disease; Enzyme Inhibitors; Free Radical Scavengers; Hypoxia; Lung; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Tyrosine; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

Genistein is a major isoflavonoid abundantly found in soy. Earlier genistein has been reported to possess protective effect against a multitude of disorders including cancer. Previously we demonstrated the protective effects of Genistein in d-Galactosamine (D-GalN) induced fulminant hepatic failure (FHF) in rats. In present study, we evaluated the hepatoprotective activity of Genistein in rat model of chronic liver damage and liver fibrosis.. Liver fibrosis was induced by intraperitoneal injection of D-GalN (250 mg/kg BW) twice a week for 12 weeks. Genistein (5 mg/kg BW) was given via intra-gastric route as co-treatment daily for 12 weeks.. Genistein co-treatment significantly attenuated D-GalN-induced chronic liver damage and liver fibrosis as evident from a significant amelioration in functional impairment, including inhibition of the activation of Hepatic stellate cells (HSC), decreased expression in alpha smooth muscle actin (α-SMA) and accumulation of collagen matrix, and an elevation in serum alanine transaminase (ALT) and aspartate transaminase (AST) level. In addition Genistein co-treatment was associated with elevated expression of hepatic Smad7, which ultimately blunts the expression of TGF-β and the activation of TGF-β/Smad signaling. Furthermore Genistein significantly prevented the histopathological changes induced by D-GalN.. Our results suggest that Genistein could be a novel therapeutic/nutraceutical agent in treating chronic liver damage and liver fibrosis. In addition our study also suggests a possible mechanism of action in which Smad7-induced inhibition of TGF-β/Smad2/3 can be a central mechanism by which Genistein protects liver from chronic injury.

Topics: Animals; Blotting, Western; Chronic Disease; Collagen; Galactosamine; Genistein; Hepatic Stellate Cells; In Situ Nick-End Labeling; Liver; Liver Cirrhosis; Male; Rats, Wistar; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tyrosine

Liver fibrosis and cirrhosis are leading causes of morbidity and mortality, with majority of preventable cases attributed to excessive alcohol consumption, viral hepatitis, or non-alcoholic fatty liver disease. We previously reported the hepatoprotective effect of Glycine propionyl-l-carnitine (GPLC) against the fulminant hepatic failure (FHF) induced by d-Galactosamine (D-GalN). In this study we evaluated the protective effect of GPLC against D-GalN induced chronic liver damage.. Animals received D-GalN twice a week for 12 weeks at a dose of 250 mg/kg body weight (BW). GPLC was given daily for 12 weeks as co-treatment along with D-GalN at a dose of 35 mg/kg BW.. D-GalN injection resulted in a considerable decrease in body weight, hepatocellular disintegration, necrosis and lipid peroxidation as evident from altered levels of SOD, CAT and MDA while GPLC significantly restored the reduced body weight and ameliorated hepatocellular damage and lipid peroxidation. D-GalN administration resulted in DNA damage as evident from TUNEL positive cells in disease control rats while; GPLC significantly alleviated the genotoxic effects of D-GalN. Further histopathological analysis revealed significant tissue and cellular damage, and increased collagen content in D-GalN challenged rats. GPLC however ameliorated the damage as evident from normal cellular and morphological architecture in GPLC co-treated rats. Hydroxyproline and nitrotyrosine (NTY) levels marked a significant decrease in GPLC co-treated rats relative to disease control. GPLC significantly blocked D-GalN induced pro-inflammatory cytokine (TNF-α, IL-6) production and at the same time inhibited the expression of α-smooth muscle actin (α-SMA), collagen-I (COL-I) and transforming growth factor-β (TGF-β) significantly.. Our results demonstrate significant protective activity of GPLC in chronic liver damage and other complications related to it. This study is a novel study to demonstrate the hepatoprotective effect of GPLC in chronic liver damage.

Topics: Actins; Animals; Body Weight; Carnitine; Chronic Disease; Collagen Type I; Galactosamine; Gene Expression Regulation; Glycine; Hydroxyproline; In Situ Nick-End Labeling; Interleukin-6; Liver; Liver Cirrhosis; Male; Oxidative Stress; Rats, Wistar; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Tyrosine

Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was upregulated and hippocampal neuronal apoptosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Activating Transcription Factor 4; Animals; Apoptosis; Chronic Disease; Copper Sulfate; Cyclic AMP Response Element-Binding Protein; Deoxyguanosine; Disks Large Homolog 4 Protein; DNA-Binding Proteins; eIF-2 Kinase; Guanylate Kinases; Hippocampus; Male; Membrane Proteins; Memory Disorders; Mice, Inbred C57BL; Neurons; Oxidative Stress; Random Allocation; Signal Transduction; Spatial Memory; Synapsins; Transcription Factor CHOP; Transcription Factors; Tyrosine

We have previously reported that treatment of rats subjected to permanent bilateral common carotid artery occlusion (pBCCAO), a model of chronic cerebral hypoperfusion (CCH), with S-nitrosoglutathione (GSNO), an endogenous nitric oxide carrier, improved cognitive functions and decreased amyloid-β accumulation in the brains. Since CCH has been implicated in tau hyperphosphorylation induced neurodegeneration, we investigated the role of GSNO in regulation of tau hyperphosphorylation in rat pBCCAO model. The rats subjected to pBCCAO had a significant increase in tau hyperphosphorylation with increased neuronal loss in hippocampal/cortical areas. GSNO treatment attenuated not only the tau hyperphosphorylation, but also the neurodegeneration in pBCCAO rat brains. The pBCCAO rat brains also showed increased activities of GSK-3β and Cdk5 (major tau kinases) and GSNO treatment significantly attenuated their activities. GSNO attenuated the increased calpain activities and calpain-mediated cleavage of p35 leading to production of p25 and aberrant Cdk5 activation. In in vitro studies using purified calpain protein, GSNO treatment inhibited calpain activities while 3-morpholinosydnonimine (a donor of peroxynitrite) treatment increased its activities, suggesting the opposing role of GSNO vs. peroxynitrite in regulation of calpain activities. In pBCCAO rat brains, GSNO treatment attenuated the expression of inducible nitric oxide synthase (iNOS) expression and also reduced the brain levels of nitro-tyrosine formation, thereby indicating the protective role of GSNO in iNOS/nitrosative-stress mediated calpain/tau pathologies under CCH conditions. Taken together with our previous report, these data support the therapeutic potential of GSNO, a biological NO carrier, as a neuro- and cognitive-protective agent under conditions of CCH.

Topics: Analysis of Variance; Animals; Brain; Brain Ischemia; Calpain; Chronic Disease; Cyclin-Dependent Kinase 5; Disease Models, Animal; Glycogen Synthase Kinase 3; Neuroprotective Agents; Phosphorylation; Rats; S-Nitrosoglutathione; Synaptosomes; tau Proteins; Tyrosine

Chronic hypoxia is one of the main causes of pulmonary hypertension (PH) associated with ROS production. Lectin-like oxidized low-density lipoprotein receptor (LOX)-1 is known to be an endothelial receptor of oxidized low-density lipoprotein, which is assumed to play a role in the initiation of ROS generation. We investigated the role of LOX-1 and ROS generation in PH and vascular remodeling in LOX-1 transgenic (TG) mice. We maintained 8- to 10-wk-old male LOX-1 TG mice and wild-type (WT) mice in normoxia (room air) or hypoxia (10% O2 chambers) for 3 wk. Right ventricular (RV) systolic pressure (RVSP) was comparable between the two groups under normoxic conditions; however, chronic hypoxia significantly increased RVSP and RV hypertrophy in LOX-1 TG mice compared with WT mice. Medial wall thickness of the pulmonary arteries was significantly greater in LOX-1 TG mice than in WT mice. Furthermore, hypoxia enhanced ROS production and nitrotyrosine expression in LOX-1 TG mice, supporting the observed pathological changes. Administration of the NADPH oxidase inhibitor apocynin caused a significant reduction in PH and vascular remodeling in LOX-1 TG mice. Our results suggest that LOX-1-ROS generation induces the development and progression of PH.

Topics: Animals; Antioxidants; Chronic Disease; Disease Models, Animal; Disease Progression; Enzyme Inhibitors; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lipoproteins, LDL; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; NADPH Oxidases; Oxidative Stress; Pulmonary Artery; Reactive Oxygen Species; Scavenger Receptors, Class E; Signal Transduction; Tyrosine; Up-Regulation; Ventricular Function, Right; Ventricular Pressure

Chronic venous insufficiency (CVI) represents a social and health care problem because it affects working age populations, particularly in jobs requiring orthostasis, has no effective pharmacologic treatment, and requires surgery. Oxidative stress is present in varicose veins, but whether this is reflected in the plasma is controversial. We aimed to quantify plasma oxidative stress biomarkers in the early stages of CVI and calculate a global index of oxidative stress representative of the disease.. Plasma was obtained from blood samples of nine patients with CEAP C2 stage CVI and 10 healthy controls. Biomarkers related to antioxidant defense systems (total thiols, reduced glutathione, uric acid, total antioxidant capacity, catalase), oxidative damage (malondialdehyde-bound protein, protein carbonyls, advanced oxidation products, and 3-nitrotyrosine), and activity of enzymes producing key free radicals (xanthine oxidase and myeloperoxidase) were assessed.. Compared with the controls, CVI patients exhibited decreased catalase activity and thiol levels and increased malondialdehyde-bound protein and protein carbonyls. These parameters were used to calculate the global index of oxidative stress in CVI, which was significantly different between groups.. It is possible to detect significant changes in plasma oxidative stress biomarkers in early stages of CVI and to calculate a global index representative of the oxidative status in an individual. This index, with the appropriate validation in a larger population, could be used for early detection or progression of CVI.

Topics: Adult; Advanced Oxidation Protein Products; Biomarkers; Case-Control Studies; Catalase; Chronic Disease; Disease Progression; Early Diagnosis; Female; Glutathione; Humans; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Peroxidase; Predictive Value of Tests; Protein Carbonylation; Sulfhydryl Compounds; Tyrosine; Ultrasonography, Doppler, Color; Uric Acid; Veins; Venous Insufficiency; Xanthine Oxidase

In spite of improvement in obstetrical care, pregnancy in women with type 1 diabetes mellitus is associated with increased perinatal morbidity and mortality. Hyperglycemia during pregnancy causes excessive fetal growth and chronic fetal hypoxia as reflected in increased erythropoietin (EPO) levels in amniotic fluid (AF).. We hypothesized that the degree of fetal hypoxia would correlate with fetal oxidative and nitrosative stress as evidenced ty the concentration of specific biomarkers in AF.. 19 pregnant women with type 1 or insulin-treated gestational diabetes mellitus were studied. AF samples were collected and processed for EPO, meta-tyrosine, nitro-tyrosine and 8-hydroxy-2-deoxiguanosine by chemiluminescent immunoassay and high-performance liquid chromatography coupled to tandem mass spectrometry methods, respectively.. The mean (SD) of the last HbA1c concentration before delivery was 7.7% (1.1). Median gestational age was 258 days (range 231-268). Birth weight was 3,868 ± 695 g with a z-score >2 SD in 47% of the cases. A significant correlation was found between the concentrations of AF EPO and meta-tyrosine/phenylalanine ratio (p < 0.001), nitro-tyrosine (p < 0.01) and 8-oxo-dG/2dG ratio (p < 0.001).. We confirmed that fetuses of type 1 diabetes or insulin-treated gestational diabetes pregnancies experience chronic hypoxia as reflected by increased EPO concentrations in AF near term. Moreover, EPO levels significantly correlated with the concentration of oxidative and nitrosative stress biomarkers in AF. This pro-oxidant status may predispose newborn infants to poor postnatal adaptation and early neonatal complications.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Amniocentesis; Amniotic Fluid; Biomarkers; Birth Weight; Chromatography, High Pressure Liquid; Chronic Disease; Deoxyguanosine; Diabetes Mellitus, Type 1; Diabetes, Gestational; Erythropoietin; Female; Fetal Hypoxia; Gestational Age; Glycated Hemoglobin; Humans; Hypoglycemic Agents; Immunoassay; Infant, Newborn; Insulin; Male; Nitrosation; Oxidative Stress; Pilot Projects; Pregnancy; Pregnancy in Diabetics; Tandem Mass Spectrometry; Tyrosine; Young Adult

Chronic liver diseases are commonly associated with tissue hypoxia that may cause inflammation, oxidative stress, liver cell injury and increased nuclear transcriptional regulation. The hepatic response to chronic hypoxia at the molecular level has not yet been clearly understood until now. The aim of this study is to investigate whether nuclear transcription factors [hypoxia-inducible factor-1 (HIF-1α), activator protein-1 (AP-1), nuclear factor-kappa B (NF-κB)] exhibit activity changes during hepatic response to chronic hypoxia. Blood and liver samples were collected from adult Sprague-Dawley rats living in atmospheric air or 10% oxygen for four weeks. Levels of serum alanine aminotransferase (ALT), 8-isoprostane and nitrotyrosine were measured. The activities of nuclear transcription factors and the expression of downstream genes (iNOS, eNOS, ET-1 and VEGF) were measured using RT-PCR, Western blotting and Gel shift analysis. Results showed that serum ALT level, 8-isoprostane level and formation of nitrotyrosine were within normal range at all time-points. In the hypoxic liver, DNA-binding activities of HIF-1α, NF-κB and AP-1 increased significantly. Expression levels of iNOS, VEGF and ET-1 progressively increased from day 7 to day 28. eNOS was also elevated in the hypoxic liver. In conclusion, our study suggests that increased activity of HIF-1α, AP-1 and NF-κB may partly play a significant role in the hepatic response to oxidative stress and liver injury under chronic hypoxia. The increased expression of VEGF, ET-1, iNOS and eNOS may be partly due to the compensatory mechanism in the vascular beds of the liver in response to chronic hypoxia.

Topics: Alanine Transaminase; Animals; Biomarkers; Blotting, Western; Chronic Disease; Dinoprost; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Endothelin-1; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Liver; Male; NF-kappa B; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcription Factor AP-1; Tyrosine; Vascular Endothelial Growth Factor A

Argininosuccinate synthase (ASS) is the rate-limiting enzyme in both the urea and the L-citrulline/nitric oxide (NO·) cycles regulating protein catabolism, ammonia levels, and NO· generation. Because a proteomics analysis identified ASS and nitric oxide synthase-2 (NOS2) as coinduced in rat hepatocytes by chronic ethanol consumption, which also occurred in alcoholic liver disease (ALD) and in cirrhosis patients, we hypothesized that ASS could play a role in ethanol binge and chronic ethanol-induced liver damage. To investigate the contribution of ASS to the pathophysiology of ALD, wildtype (WT) and Ass(+/-) mice (Ass(-/-) are lethal due to hyperammonemia) were exposed to an ethanol binge or to chronic ethanol drinking. Compared with WT, Ass(+/-) mice given an ethanol binge exhibited decreased steatosis, lower NOS2 induction, and less 3-nitrotyrosine (3-NT) protein residues, indicating that reducing nitrosative stress by way of the L-citrulline/NO· pathway plays a significant role in preventing liver damage. However, chronic ethanol-treated Ass(+/-) mice displayed enhanced liver injury compared with WT mice. This was due to hyperammonemia, lower phosphorylated AMP-activated protein kinase alpha (pAMPKα) to total AMPKα ratio, decreased sirtuin-1 (Sirt-1) and peroxisomal proliferator-activated receptor coactivator-1α (Pgc1α) messenger RNAs (mRNAs), lower fatty acid β-oxidation due to down-regulation of carnitine palmitoyl transferase-II (CPT-II), decreased antioxidant defense, and elevated lipid peroxidation end-products in spite of comparable nitrosative stress but likely reduced NOS3.. Partial Ass ablation protects only in acute ethanol-induced liver injury by decreasing nitrosative stress but not in a more chronic scenario where oxidative stress and impaired fatty acid β-oxidation are key events.

Topics: Acute Disease; Alcohol Drinking; Alcoholism; Animals; Argininosuccinate Synthase; Chronic Disease; Cytochrome P-450 CYP2E1; Disease Models, Animal; Down-Regulation; Ethanol; Female; Hepatocytes; Immunohistochemistry; Lipid Peroxidation; Liver Diseases, Alcoholic; Male; Mice; Nitric Oxide; Oxidative Stress; Random Allocation; Rats; Tyrosine

Peroxynitrite (PN, ONOO(-)) is a potent oxidant and nitrating agent that contributes to pain through peripheral and spinal mechanisms, but its supraspinal role is unknown. We present evidence here that PN in the rostral ventromedial medulla (RVM) is essential for descending nociceptive modulation in rats during inflammatory and neuropathic pain through PN-mediated suppression of opioid signaling. Carrageenan-induced thermal hyperalgesia was associated with increased 3-nitrotyrosine (NT), a PN biomarker, in the RVM. Furthermore, intra-RVM microinjections of the PN decomposition catalyst Fe(III)-5,10,15,20-tetrakis(N-methyl-pyridinium-4-yl)porphyrin (FeTMPyP(5+)) dose-dependently reversed this thermal hyperalgesia. These effects of FeTMPyP(5+) were abrogated by intra-RVM naloxone, implicating potential interplay between PN and opioids. In support, we identified NT colocalization with the endogenous opioid enkephalin (ENK) in the RVM during thermal hyperalgesia, suggesting potential in situ interactions. To address the functional significance of such interactions, we exposed methionine-enkephalin (MENK) to PN and identified the major metabolite, 3-nitrotyrosine-methionine-sulfoxide (NSO)-MENK, using liquid chromatography-mass spectrometry. Next, we isolated, purified, and tested NSO-MENK for opioid receptor binding affinity and analgesic effects. Compared to MENK, this NSO-MENK metabolite lacked appreciable binding affinity for δ, μ, and κ opioid receptors. Intrathecal injection of NSO-MENK in rats did not evoke antinociception, suggesting that PN-mediated chemical modifications of ENK suppress opioid signaling. When extended to chronic pain, intra-RVM FeTMPyP(5+) produced naloxone-sensitive reversal of mechanical allodynia in rats following chronic constriction injury of the sciatic nerve. Collectively, our data reveal the central role of PN in RVM descending facilitation during inflammatory and neuropathic pain potentially through anti-opioid activity.

Topics: Analysis of Variance; Animals; Carrageenan; CD11b Antigen; Cell Line, Transformed; Chromatography, Liquid; Chronic Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Electrochemical Techniques; Enkephalin, Methionine; Glial Fibrillary Acidic Protein; Humans; Hyperalgesia; Injections, Spinal; Male; Medulla Oblongata; Metalloporphyrins; Microinjections; Neuroglia; Neurons; Opioid Peptides; Pain; Pain Measurement; Peroxynitrous Acid; Phosphopyruvate Hydratase; Protein Binding; Radioligand Assay; Rats; Rats, Sprague-Dawley; Signal Transduction; Tyrosine

3'-Nitrotyrosine (3NT) is a post-translational modification (PTM) of body fluids and tissues that is sustained by chronic inflammation and oxidative stress, two main clinical traits of chronic kidney disease (CKD). Despite this background, protein targets and their differential susceptibility to in vivo nitration remain almost completely unexplored in CKD. This study reports a first investigation of plasma nitroproteome in these patients, carried out by both immunorecognition and LC-MS/MS techniques. Plasma proteins of chronic and end-stage KD patients showed a higher burden of nitration than in healthy controls, but main nitration targets appeared to be the same in these populations. Immunoblotting data showed that uremic albumin is largely represented in the uremic nitroproteome together with fibrinogen chains (A, B and C), transferrin, α1-antitrypsin, complement factor D, haptoglobin, and IgG light and heavy chains. However, immunopurification and affinity chromatography experiments demonstrated that the relative content of 3NT on the albumin molecule was very low when compared with that of the remaining plasma proteins. The uremic nitroproteome was investigated using also plasma proteins obtained by in vivo ultrafiltration from patients treated with protein leaking or standard high-flux hemodialyzers. The study of these samples revealed the possibility to selectively remove protein nitration products during hemodialysis. Identification of intramolecular sites of nitration was preliminarily obtained in IgG chains isolated by 2D PAGE and assessed by bidimensional tandem mass spectrometry after chemoselective tagging. Further studies are needed to confirm at the molecular level the presence of nitrated Tyr residues in other proteins tentatively identified as nitration targets in this study and to explore the biological meaning of such a selective modification of plasma proteins by reactive nitrogen species in uremia and dialysis patients.

Topics: Aged; Aged, 80 and over; Amino Acid Sequence; Blood Proteins; Case-Control Studies; Chronic Disease; Electrophoresis, Gel, Two-Dimensional; Female; Humans; Kidney Diseases; Male; Middle Aged; Molecular Sequence Data; Protein Processing, Post-Translational; Proteome; Tandem Mass Spectrometry; Tyrosine

Topics: Adult; Asthma; Chronic Disease; Eosinophils; Female; Humans; Immunohistochemistry; Lung; Macrophages, Alveolar; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type II; Pulmonary Eosinophilia; Tyrosine

The ageing kidney exhibits slowly developing chronic kidney disease (CKD) and is associated with nitric oxide (NO) deficiency and increased oxidative stress. The impact of exercise on the ageing kidney is not well understood. Here, we determined whether 12 weeks of treadmill exercise can influence age-dependent CKD in old (22-24 months) Fisher 344 (F344) male rats by comparing sedentary (SED) and exercise (EX) trained rats; young (3 months) rats were also studied. In addition to renal structure and function, we assessed protein levels of various isoforms of the NO synthases (NOS) and superoxide dismutase (SOD) enzymes as well as markers of oxidative stress, in kidney cortex and medulla. Renal function as determined by plasma creatinine, proteinuria, and glomerular structural injury worsened with age and was unaffected by exercise. Ageing also increased the protein abundance of neuronal NOSβ and p22phox while decreasing extracellular (EC) and copper/zinc (CuZn) SOD, in kidney cortex and medulla. H(2)O(2) content and nitrotyrosine abundance also increased in the kidney with age. None of these age-related changes were altered with exercise. However, exercise did increase renal cortical endothelial (e)NOS and EC SOD in young rats. Data indicate that exercise-induced increases in eNOS and EC SOD seen in young rats are lost with age. We conclude that chronic exercise is ineffective in reversing age-dependent CKD in the male F344 rat.

Topics: Aging; Animals; Aorta; Chronic Disease; Hydrogen Peroxide; Kidney; Kidney Diseases; Male; NADPH Oxidases; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Oxidative Stress; Physical Conditioning, Animal; Rats; Rats, Inbred F344; Superoxide Dismutase; Tyrosine

We previously demonstrated that chronic exposure to intermittent hypoxia (CIH) impairs endothelium-dependent vasodilation in rats. To determine the time course of this response, rats were exposed to CIH for 3, 14, 28, or 56 days. Then, we measured acetylcholine- and nitroprusside-induced vasodilation in isolated gracilis arteries. Also, we measured endothelial and inducible nitric oxide synthase, nitrotyrosine, and collagen in the arterial wall and urinary isoprostanes. Endothelium-dependent vasodilation was impaired after 2 weeks of CIH. Three days of CIH was not sufficient to produce this impairment and longer exposures (i.e. 4 and 8 weeks) did not exacerbate it. Impaired vasodilation was accompanied by increased collagen deposition. CIH elevated urinary isoprostane excretion, whereas there was no consistent effect on either isoform of nitric oxide synthase or nitrotyrosine. Exposure to CIH produces functional and structural deficits in skeletal muscle resistance arteries. These impairments develop within 2 weeks after initiation of exposure and they are accompanied by systemic evidence of oxidant stress.

Topics: Acetylcholine; Analysis of Variance; Animals; Arteries; Chronic Disease; Collagen; Endothelium, Vascular; Hypoxia; Immunohistochemistry; Isoprostanes; Male; Muscle, Skeletal; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroprusside; Oxygen; Partial Pressure; Rats; Rats, Sprague-Dawley; Time Factors; Tyrosine; Vascular Resistance; Vasodilation; Vasodilator Agents

The presence of a reciprocal link between inflammation and oxidative/nitrosative stress has been postulated in chronic heart failure (CHF). We aimed to determine signs of nitrosative stress in serum/plasma of CHF patients. ELISA tests were used for quantification of serum/plasma levels of Nitrotyrosine (NT), H(2)O(2), total NO, nitrite (NO(2)(-)), myeloperoxidase (MPO), Tumor Necrosis Factor-alpha (TNFalpha) and pro-Brain Natriuretic Peptide (proBNP) in 66 CHF patients (9 in NYHA I, 34 NYHA II, 23 NYHA III) and in 14 age-matched healthy subjects. NT levels were higher in NYHA III CHF patients compared to NYHA II (p<0.05), NYHA I (p<0.03) and controls (p<0.02), whereas NO(2)(-) and total NO were higher in NYHA III compared to I (p<0.05 and p<0.04, respectively) and controls (p<0.004 and 0.002) and in NYHA II compared to controls (p<0.04 and p<0.009). NT levels correlated significantly with MPO (r=0.37, p<0.003), TNFalpha (r=0.32, p<0.01) and proBNP (r=0.32, p<0.01). These data demonstrate an increased NT plasma level in patients with moderate/severe CHF which is associated to increased levels of markers of systemic inflammation.

Topics: Aged; Biomarkers; Chronic Disease; Cohort Studies; Female; Heart Failure; Humans; Inflammation Mediators; Male; Middle Aged; Peroxidase; Tyrosine

A dyshomeostasis of extra- and intracellular Ca(2+) and Zn(2+) occurs in rats receiving chronic aldosterone/salt treatment (ALDOST). Herein, we hypothesized that the dyshomeostasis of intracellular Ca(2+) and Zn(2+) is intrinsically coupled that alters the redox state of cardiac myocytes and mitochondria, with Ca(2+) serving as a pro-oxidant and Zn(2+) as an antioxidant. Toward this end, we harvested hearts from rats receiving 4 weeks of ALDOST alone or cotreatment with either spironolactone (Spiro), an aldosterone receptor antagonist, or amlodipine (Amlod), an L-type Ca(2+) channel blocker, and from age/sex-matched untreated controls. In each group, we monitored cardiomyocyte [Ca(2+)]i and [Zn(2+)]i and mitochondrial [Ca(2+)]m and [Zn(2+)]m; biomarkers of oxidative stress and antioxidant defenses; expression of Zn transporters, Zip1 and ZnT-1; metallothionein-1, a Zn(2+)-binding protein; and metal response element transcription factor-1, a [Zn(2+)]i sensor and regulator of antioxidant defenses. Compared with controls, at 4-week ALDOST, we found the following: (a) increased [Ca(2+)]i and [Zn(2+)]i, together with increased [Ca(2+)]m and [Zn(2+)]m, each of which could be prevented by Spiro and attenuated with Amlod; (b) increased levels of 3-nitrotyrosine and 4-hydroxy-2-nonenal in cardiomyocytes, together with increased H(2)O(2) production, malondialdehyde, and oxidized glutathione in mitochondria that were coincident with increased activities of Cu/Zn superoxide dismutase and glutathione peroxidase; and (c) increased expression of metallothionein-1, Zip1 and ZnT-1, and metal response element transcription factor-1, attenuated by Spiro. Thus, an intrinsically coupled dyshomeostasis of intracellular Ca(2+) and Zn(2+) occurs in cardiac myocytes and mitochondria in rats receiving ALDOST, where it serves to alter their redox state through a respective induction of oxidative stress and generation of antioxidant defenses. The importance of therapeutic strategies that can uncouple these two divalent cations and modulate their ratio in favor of sustained antioxidant defenses is therefore suggested.

Topics: Aldehydes; Aldosterone; Amlodipine; Animals; Calcium; Calcium Channel Blockers; Chronic Disease; Disease Models, Animal; Glutathione Peroxidase; Homeostasis; Hydrogen Peroxide; Hyperaldosteronism; Male; Metallothionein; Mineralocorticoid Receptor Antagonists; Mitochondria, Heart; Myocytes, Cardiac; Oxidative Stress; Rats; Rats, Sprague-Dawley; Spironolactone; Superoxide Dismutase; Tyrosine; Zinc

Adjuvant arthritis (AA) is a condition that involves systemic oxidative stress. Unexpectedly, it was found that sarcoplasmic reticulum Ca(2 +)-ATPase (SERCA) activity was elevated in muscles of rats with AA compared to controls, suggesting possible conformational changes in the enzyme. There was no alteration in the nucleotide binding site but rather in the transmembrane domain according to the tryptophan polar/non-polar fluorescence ratio. Higher relative expression of SERCA, higher content of nitrotyrosine but no increase in phospholipid oxidation in AA SR was found. In vitro treatments of SR with HOCl showed that in AA animals SERCA activity was more susceptible to oxidative stress, but SR phospholipids were more resistant and SERCA could also be activated by phosphatidic acid. It was concluded that increased SERCA activity in AA was due to increased levels of SERCA protein and structural changes to the protein, probably induced by direct and specific oxidation involving reactive nitrogen species.

Topics: Adaptation, Physiological; Animals; Arthritis, Experimental; Calcium; Calsequestrin; Chronic Disease; Kinetics; Lipid Peroxidation; Muscle, Skeletal; Mycobacterium; Oxidation-Reduction; Oxidative Stress; Phosphatidic Acids; Protein Carbonylation; Protein Conformation; Rats; Rats, Inbred Lew; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tyrosine; Up-Regulation

Chronic lung hypoxia results in hypoxic pulmonary hypertension. Concomitant chronic hypercapnia partly inhibits the effect of hypoxia on pulmonary vasculature. Adult male rats exposed to 3 weeks hypoxia (Fi(02)=0.1) combined with hypercapnia (Fi(C02)=0.04-0.05) had lower pulmonary arterial blood pressure, increased weight of the right heart ventricle, and less pronounced structural remodeling of the peripheral pulmonary arteries compared with rats exposed only to chronic hypoxia (Fi(02)=0.1). According to our hypothesis, hypoxic pulmonary hypertension is triggered by hypoxic injury to the walls of the peripheral pulmonary arteries. Hypercapnia inhibits release of both oxygen radicals and nitric oxide at the beginning of exposure to the hypoxic environment. The plasma concentration of nitrotyrosine, the marker of peroxynitrite activity, is lower in hypoxic rats exposed to hypercapnia than in those exposed to hypoxia alone. Hypercapnia blunts hypoxia-induced collagenolysis in the walls of prealveolar pulmonary arteries. We conclude that hypercapnia inhibits the development of hypoxic pulmonary hypertension by the inhibition of radical injury to the walls of peripheral pulmonary arteries.

Topics: Animals; Blood Pressure; Chronic Disease; Disease Models, Animal; Hypercapnia; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lung Injury; Male; Oxidative Stress; Pulmonary Artery; Rats; Rats, Wistar; Time Factors; Tyrosine

Nitric oxide (NO) has been implicated as a potential causative factor for endogenous p53 mutations in gastrointestinal malignancy. To investigate the role of NO in esophageal adenocarcinoma (EADC), we studied patterns of p53 mutations, expression of inducible nitric oxide synthase (iNOS) and the tissue accumulation of nitrotyrosine (NTS), a stable reaction product of NO and a marker for cellular protein damage, in human premalignant and malignant esophageal epithelia. Tissues were obtained from patients with gastroesophageal reflux disease (GERD)-induced esophagitis (n = 76), Barrett's esophagus (BE; n = 119) and primary EADC (n = 54). DNA sequencing was used to characterize p53 mutations, RT-PCR to study iNOS mRNA expression, and immunohistochemistry to study NTS. Relative to self-matched normal epithelia, a progressive increase in iNOS mRNA expression was seen in GERD (30%; 23/76), BE (48%; 57/119), and EADC (63%; 34/54) tissues (P < 0.001). Among patients with EADC, elevated levels of NTS immunoreactivity were more frequent in tumors with p53 mutations (11/21; 52%) compared with tumors with wild-type p53 (9/33; 27%; P = 0.063), and specifically in tumors with p53 mutations at CpG dinucleotides (10/12; 83%) compared with non-CpG p53 mutations (1/9; 11%; P = 0.008). The increasing frequency of iNOS (mRNA) overexpression in GERD, BE and EADC supports the hypothesis that an active inflammatory process, most likely a consequence of GERD, underlies molecular progression to EADC. The highly significant association between NTS, reflecting chronic NO-induced cellular protein damage, and endogenous p53 mutations at CpG dinucleotides, provides further evidence for a molecular link between chronic inflammation and esophageal malignancy.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Aged, 80 and over; Barrett Esophagus; Chronic Disease; Esophageal Neoplasms; Female; Genes, p53; Humans; Inflammation Mediators; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type II; Point Mutation; Tumor Suppressor Protein p53; Tyrosine

In liver cirrhosis, elevated levels of NO and ROS (reactive oxygen species) might greatly favour the generation of peroxynitrite. Peroxynitrite is a highly reactive oxidant and it can potentially alter the vascular reactivity and the function of different organs. In the present study, we evaluated whether peroxynitrite levels are related to the progression of renal vascular and excretory dysfunction during experimental cirrhosis induced by chronic BDL (bile-duct ligation) in rats. Experiments were performed at 7, 15 and 21 days after BDL in rats and in rats 21 days post-BDL chronically treated with L-NAME (N(G)-nitro-L-arginine methyl ester). Sodium balance, BP (blood pressure), basal RPP (renal perfusion pressure) and the renal vascular response to PHE (phenylephrine) and ACh (acetylcholine) in isolated perfused kidneys were measured. NO levels were calculated as 24-h urinary excretion of nitrites, ROS as TBARS (thiobarbituric acid-reacting substances), and peroxynitrite formation as the renal expression of nitrotyrosine. BDL rats had progressive sodium retention, and decreased BP, RPP and renal vascular responses to PHE and ACh in the time following BDL. They also had increasing levels of NO and ROS, and renal nitrotyrosine accumulation,especially in the medulla. All of these changes were either prevented or significantly decreased by chronic L-NAME administration. In conclusion, these results suggest that the increasing levels of peroxynitrite might contribute to the altered renal vascular response and sodium retention in the development of the experimental biliary cirrhosis. Moreover, the beneficial effects of decreasing NO synthesis are, at least in part, mediated by anti-peroxinitrite-related effects.

Topics: Animals; Body Weight; Chronic Disease; Kidney; Liver Cirrhosis, Experimental; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Organ Size; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Renal Circulation; Spleen; Tyrosine

Pathogenesis of hypoxic pulmonary hypertension is initiated by oxidative injury to the pulmonary vascular wall. Because nitric oxide (NO) can contribute to oxidative stress and because the inducible isoform of NO synthase (iNOS) is often upregulated in association with tissue injury, we hypothesized that iNOS-derived NO participates in the pulmonary vascular wall injury at the onset of hypoxic pulmonary hypertension. An effective and selective dose of an iNOS inhibitor, L-N6-(1-iminoethyl)lysine (L-NIL), for chronic peroral treatment was first determined (8 mg/l in drinking water) by measuring exhaled NO concentration and systemic arterial pressure after LPS injection under ketamine+xylazine anesthesia. A separate batch of rats was then exposed to hypoxia (10% O2) and given L-NIL or a nonselective inhibitor of all NO synthases, N(G)-nitro-L-arginine methyl ester (L-NAME, 500 mg/l), in drinking water. Both inhibitors, applied just before and during 1-wk hypoxia, equally reduced pulmonary arterial pressure (PAP) measured under ketamine+xylazine anesthesia. If hypoxia continued for 2 more wk after L-NIL treatment was discontinued, PAP was still lower than in untreated hypoxic controls. Immunostaining of lung vessels showed negligible iNOS presence in control rats, striking iNOS expression after 4 days of hypoxia, and return of iNOS immunostaining toward normally low levels after 20 days of hypoxia. Lung NO production, measured as NO concentration in exhaled air, was markedly elevated as early as on the first day of hypoxia. We conclude that transient iNOS induction in the pulmonary vascular wall at the beginning of chronic hypoxia participates in the pathogenesis of pulmonary hypertension.

Topics: Administration, Oral; Animals; Chronic Disease; Dose-Response Relationship, Drug; Enzyme Inhibitors; Exhalation; Hypertension, Pulmonary; Hypoxia; Lung; Lysine; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type II; Pulmonary Artery; Rats; Rats, Wistar; Time Factors; Tyrosine

Chronic infection of the lungs with Pseudomonas aeruginosa complicates many long-term lung diseases including cystic fibrosis, bronchiectasis, chronic obstructive lung disease, and mechanical ventilation. In acute inflammatory lung diseases, increased nitric oxide synthase (NOS-2) expression leads to excess nitric oxide (NO) production, resulting in the production of reactive nitrogen intermediates, which contribute to tissue damage. In contrast, the contribution of NO to pulmonary damage in chronic Pseudomonas infection of the lung has not been directly examined and is unclear. Although NOS-2 expression is increased in this condition, NO production is not abnormally elevated. It was hypothesized that chronic infection of the airways does not cause increased NO production but, in contrast, leads to inappropriately low NO concentrations that are pro-inflammatory. A rodent model of chronic airway infection was used to examine the effects on lung damage of augmenting or inhibiting NO production after airway infection with P. aeruginosa was well established. Three days post-infection, L-arginine, which augments NO production, or L-NAME, an inhibitor of NO production, was administered in drinking water. Lung damage was assessed 12 days later. L-arginine treatment reduced tissue damage, inhibited neutrophil recruitment, and reduced the pro-inflammatory cytokine interleukin (IL)-1beta. Treatment with L-NAME caused loss of alveolar walls, greater vascular damage, and increased levels of the pro-inflammatory cytokine IL-6. Thus, in chronic airway infection, inhibition of NO production worsened lung damage, whereas augmenting NO ameliorated this damage. This is the first demonstration that augmenting endogenous NO production in chronic infective lung disease caused by P. aeruginosa is anti-inflammatory. Given that infection with this organism complicates many chronic lung diseases, most notoriously cystic fibrosis, these findings have important clinical implications.

Topics: Animals; Arginine; Bronchoalveolar Lavage Fluid; Cell Count; Chronic Disease; Disease Models, Animal; Enzyme Inhibitors; Interleukin-1; Interleukin-6; Lung; Lung Diseases; Male; Neutrophils; NG-Nitroarginine Methyl Ester; Nitric Oxide; Pseudomonas Infections; Rats; Rats, Inbred WKY; Tyrosine; Vascular Endothelial Growth Factor A

Nitric oxide mediates and modulates pulmonary transition from fetal to postnatal life. NO is synthesized by 3 nitric oxide synthase isoforms. One key pathway of nitric oxide metabolism results in nitrotyrosine, a stable, measurable marker of nitric oxide production.. The purpose of this study was to assess, by semiquantitative immunohistochemistry, nitric oxide synthase isoforms and nitrotyrosine at different airway and vascular tree levels in the lungs of neonates at different gestational ages and to compare results in control groups to those in infants with chronic lung disease.. Formalin-fixed, paraffin-embedded, postmortem lung blocks were prepared for immunohistochemistry using antibodies to each nitric oxide synthase isoform and to nitrotyrosine. Blinded observers evaluated the airway and vascular trees for staining intensity (0-3 scale) at 5 levels and 3 levels, respectively. The control population consisted of infants from 22 to 42 weeks' gestation who died in < 48 hours. Results were compared with gestation-matched infants with varying severity of chronic lung disease.. In control and chronic lung disease groups, 22 to 42 weeks' gestation, staining for all 3 of the nitric oxide synthase isoforms was found in the airway epithelium from the bronchus to the alveolus or distal-most airspace. The abundance or distribution of nitric oxide synthase-3 staining in the airways did not show significant correlation with gestational age or severity of chronic lung disease. In the vascular tree, intense nitric oxide synthase-3 and moderate nitric oxide synthase-2 staining was found; nitric oxide synthase-1 was not consistently stained. Nitrotyrosine did stain in the pulmonary tree. Compared with controls where nitrotyrosine staining was minimal, regardless of gestation, in infants with chronic lung disease there was more than fourfold increase between severe chronic lung disease (n = 12) and either mild chronic lung disease or control infants (n = 16).. All 3 of the nitric oxide synthase isoforms and nitrotyrosine are detectable by immunohistochemistry early in lung development. Nitric oxide synthase ontogeny shows no significant changes in abundance or distribution with advancing gestational age nor with chronic lung disease. Nitrotyrosine is significantly increased in severe chronic lung disease.

Topics: Age Factors; Autopsy; Chronic Disease; Humans; Immunohistochemistry; Infant, Newborn; Infant, Premature; Lung; Lung Diseases; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Severity of Illness Index; Tyrosine

Murine leprosy is a chronic disease of the mouse, the most popular animal model used in biomedical investigation, which is caused by Mycobacterium lepraemurium (MLM) whose characteristic lesion is the macrophage-made granuloma. From onset to the end of the disease, the granuloma undergoes changes that gradually transform the environment into a more appropriate milieu for the growth of M. lepraemurium. The mechanisms that participate in the formation and maturation of the murine leprosy granulomas are not completely understood; however, microbial and host-factors are believed to participate in their formation. In this study, we analysed the role of various pro-inflammatory and anti-inflammatory proteins in granulomas of murine leprosy after 21 weeks of infection. We assessed the expression of cyclooxygenase-2 (COX-2), alpha acid-glycoprotein (AGP), and inducible nitric oxide synthase (iNOS) at sequential stages of infection. We also looked for the nitric-oxide nitrosylation product, nitrotyrosine (NT) in the granulomatous lesions of murine leprosy. We found that a pro-inflammatory environment predominates in the early granulomas while an anti-inflammatory environment predominates in late granulomas. No obvious signs of bacillary destruction were observed during the entire period of infection, but nitrosylation products and cell alterations were observed in granulomas in the advanced stages of disease. The change from a pro-inflammatory to an anti-inflammatory environment, which is probably driven by the bacillus itself, results in a more conducive environment for both bacillus replication and the disease progression.

Topics: Animals; Chronic Disease; Cyclooxygenase 2; Female; Granuloma; Immunohistochemistry; Leprosy; Mice; Mice, Inbred Strains; Models, Animal; Mycobacterium lepraemurium; Nitric Oxide Synthase Type II; Orosomucoid; Staining and Labeling; Tyrosine

Although aldosterone, acting via mineralocorticoid receptors, causes left ventricular (LV) hypertrophy in experimental models of high-aldosterone hypertension, little is known about the role of aldosterone or mineralocorticoid receptors in mediating adverse remodeling in response to chronic pressure overload.. We used the mineralocorticoid receptor-selective antagonist eplerenone (EPL) to test the role of mineralocorticoid receptors in mediating the transition from hypertrophy to failure in mice with chronic pressure overload caused by ascending aortic constriction (AAC). One week after AAC, mice were randomized to regular chow or chow containing EPL (200 mg/kg per day) for an additional 7 weeks. EPL had no significant effect on systolic blood pressure after AAC. Eight weeks after AAC, EPL treatment improved survival (94% versus 65%), attenuated the increases in LV end-diastolic (3.4+/-0.1 versus 3.7+/-0.1 mm) and end-systolic (2.0+/-0.1 versus 2.5+/-0.2 mm) dimensions, and ameliorated the decrease in fractional shortening (42+/-2% versus 34+/-4%). EPL also decreased myocardial fibrosis, myocyte apoptosis, and the ratio of matrix metalloproteinase-2/tissue inhibitor of matrix metalloproteinase-2. These beneficial effects of EPL were associated with less myocardial oxidative stress, as assessed by 3-nitrotyrosine staining, reduced expression of the adhesion molecule intercellular adhesion molecule-1, and reduced infiltration by macrophages.. Mineralocorticoid receptors play an important role in mediating the transition from LV hypertrophy to failure with chronic pressure overload. The effects of mineralocorticoid receptor stimulation are associated with alterations in the interstitial matrix and myocyte apoptosis and may be mediated, at least in part, by oxidative stress and inflammation.

Topics: Animals; Aorta; Apoptosis; Blood Pressure; Cell Size; Chronic Disease; Constriction, Pathologic; Drug Evaluation, Preclinical; Eplerenone; Fibrosis; Heart Failure; Hypertrophy, Left Ventricular; Intercellular Adhesion Molecule-1; Ligation; Male; Matrix Metalloproteinases; Mice; Mineralocorticoid Receptor Antagonists; Myocarditis; Myocardium; Myocytes, Cardiac; Oxidative Stress; Pressure; Random Allocation; Receptors, Mineralocorticoid; Spironolactone; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tyrosine

The gp91phox-containing NADPH oxidase is the major source of reactive oxygen species (ROS) in the cardiovascular system and inactivation of gp91phox has been reported to blunt hypertension and cardiac hypertrophy seen in angiotensin (Ang) II-infused animals. In the current study, we sought to determine the role of gp91phox-derived ROS on cardiovascular outcomes of chronic exposure to Ang II. The gp91phox-deficient mice were crossed with transgenic mice expressing active human renin in the liver (TTRhRen). TTRhRen mice exhibit chronic Ang II-dependent hypertension and frank cardiac hypertrophy by age 10 to 12 weeks. Four genotypes of mice were generated: control, TTRhRen trangenics (TTRhRen), gp91phox-deficient (gp91-), and TTRhRen transgenic gp91phox-deficient (TTRhRen/gp91-). Eight to 10 mice/group were studied. ROS levels were significantly reduced (P<0.05) in the heart and aorta of TTRhRen/gp91- and gp91-mice compared with control counterparts, and this was associated with reduced cardiac, aortic, and renal NADPH oxidase activity (P<0.05). Systolic blood pressure (SBP), cardiac mass, and cardiac fibrosis were increased in TTRhRen versus controls. In contrast to its action on ROS generation, gp91phox inactivation had no effect on development of hypertension or cardiac hypertrophy in TTRhRen mice, although interstitial fibrosis was reduced. Cardiac and renal expression of gp91phox homologues, Nox1 and Nox4, was not different between groups. Thus, although eliminating gp91phox-associated ROS production may be important in cardiovascular consequences in acute insult models, it does not prevent the development of hypertension and cardiac hypertrophy in a model in which the endogenous renin-angiotensin system is chronically upregulated.

Topics: Angiotensin II; Animals; Blood Pressure; Cardiomegaly; Chronic Disease; Collagen; Humans; Hypertension; Kidney; Membrane Glycoproteins; Mice; Mice, Transgenic; Myocardium; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; Reactive Oxygen Species; Renin; Tyrosine

Equine pituitary pars intermedia dysfunction (PPID) is a spontaneously occurring progressive disease affecting aged horses and ponies. The pathogenesis of PPID is poorly understood, but the available evidence supports a loss of dopaminergic inhibition of the melanotropes of the pars intermedia. Horses with PPID have increased plasma concentrations of pars intermedia pro-opiomelanocortin-derived peptides that decrease in response to dopamine or dopamine agonist administration. Dopamine and dopamine metabolite concentrations are decreased in the pars intermedia of affected horses compared to age-matched control horses. Horses with disease that are treated with the dopamine agonist pergolide show improvement in clinical signs and normalisation of diagnostic test results. In the present study, immunohistochemical evaluation of pituitary and hypothalamic tissue demonstrated reduced tyrosine hydroxylase immunoreactivity in affected horses compared to age-matched and young controls, supporting the role of dopaminergic neurodegeneration in PPID. In addition, immunohistochemical evaluation revealed an increase in the oxidative stress marker, 3-nitrotyrosine and in nerve terminal protein, alpha-synuclein that colocalised in the pars intermedia of horses with disease. These findings suggest a role for nitration of overexpressed alpha-synuclein in the pathogenesis of neurodegeneration in PPID.

Topics: alpha-Synuclein; Animals; Blotting, Western; Chronic Disease; Dopamine; Horse Diseases; Horses; Immunohistochemistry; Nerve Degeneration; Nerve Tissue Proteins; Nitrogen; Oxidative Stress; Pituitary ACTH Hypersecretion; Pituitary Gland; Synucleins; Tyrosine; Tyrosine 3-Monooxygenase

We report on developmental changes of pulmonary and systemic nitric oxide (NO) metabolites in a baboon model of chronic lung disease with or without exposure to inhaled NO. The plasma levels of nitrite and nitrate, staining for S-nitrosothiols and 3-nitrotyrosine in the large airways, increased between 125 d and 140 d of gestation (term 185 d) in animals developing in utero. The developmental increase in NO-mediated protein modifications was not interrupted by delivery at 125 d of gestation and mechanical ventilation for 14 d, whereas plasma nitrite and nitrate levels increased in this model. Exposure to inhaled NO resulted in a further increase in plasma nitrite and nitrate and an increase in plasma S-nitrosothiol without altering lung NO synthase expression. These data demonstrate a developmental progression in levels of pulmonary NO metabolites that parallel known maturational increases in total NO synthase activity in the lung. Despite known suppression of total pulmonary NO synthase activity in the chronic lung disease model, pulmonary and systemic NO metabolite levels are higher than in the developmental control animals. Thus, a deficiency in NO production and biological function in the premature baboon was not apparent by the detection and quantification of these surrogate markers of NO production.

Topics: Administration, Inhalation; Animals; Animals, Newborn; Chronic Disease; Cysteine; Disease Models, Animal; Female; Fetus; Free Radical Scavengers; Lung; Lung Diseases; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Nitrites; Papio; Pregnancy; S-Nitrosothiols; Tyrosine

High levels of inducible nitric oxide synthase and nitrotyrosine in Helicobacter pylori-infected gastric mucosa may contribute to development of gastric cancer. We investigated the relation between expression of inducible nitric oxide synthase and proinflammatory cytokines in gastric mucosa and serum markers of gastritis.. The study included 103 patients with H. pylori infection. We examined levels of interleukin-1beta, interleukin-6 and interleukin-8 by enzyme-linked immunosorbent assay and evaluated expression of inducible nitric oxide synthase and nitrotyrosine by immunohistochemical staining. Furthermore, we assessed serum levels of pepsinogens, gastrin, anti-parietal cell antibody, nitrite and nitrate, as markers of gastritis.. Thirty-seven of 103 (35.6%) gastric mucosa specimens showed simultaneous expression of inducible nitric oxide synthase and nitrotyrosine. In these patients (inducible nitric oxide synthase-positive group), the serum level of gastrin was significantly higher than that of the inducible nitric oxide synthase-negative group (509.5 +/- 141.5 pg/mL vs. 210.0 +/- 227.2 pg/mL; P < 0.01), whereas there were no significant differences in serum levels of pepsinogen, anti-parietal cell antibody, and nitrate and nitrite or in scores of histological gastritis. Interleukin-6 levels were significantly higher in the inducible nitric oxide synthase-positive group than in the inducible nitric oxide synthase-negative group (25.9 +/- 7.0 pg/mg protein vs. 10.6 +/- 4.9 pg/mg protein; P < 0.05).. Inducible nitric oxide synthase-producing gastritis was correlated with high levels of interleukin-6. Patients with hypergastrinaemia should be carefully followed on a long-term basis to ensure that the development of any malignancy is detected early.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Chronic Disease; Female; Gastric Mucosa; Gastrins; Gastritis; Helicobacter Infections; Helicobacter pylori; Humans; Interleukin-6; Male; Middle Aged; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Tyrosine

To investigate the possible role of eosinophil peroxidase (EPO) in 3-nitrotyrosine (3NT) formation.. Observational study employing immunocytochemistry to assess the presence of 3NT, inducible nitric oxide synthase (iNOS), eosinophils, mast cells, neutrophils, and lymphocytes in ethmoid sinus mucosal biopsies from normal controls and subjects with allergic and nonallergic chronic sinusitis and nasal polyposis.. 3NT was more evident in biopsies from sinusitis patients (2.67 +/- 0.14, n = 21) than in healthy mucosa (0.43 +/- 0.2, n = 7, P < 0.01), but scores in atopic and nonatopic subjects were similar. Colocalization studies confirmed that 3NT was largely confined to eosinophils. No relationship was found between 3NT and other immune cells. 3NT detection was not correlated with the amount of immunostaining for iNOS.. Chronic sinusitis is accompanied by 3NT formation, which is largely restricted to the eosinophils, and likely driven by the action of eosinophil peroxidase, rather than by nitric oxide levels.. B-2.

Topics: Adult; Aged; Chronic Disease; Eosinophil Peroxidase; Eosinophils; Female; Humans; Male; Middle Aged; Nasal Polyps; Sinusitis; Tyrosine

Inflammation in the lung can lead to increased expression of inducible nitric oxide synthase (iNOS) and enhanced NO production. It has been postulated that the resultant highly reactive NO metabolites may have an important role in host defence, although they might also contribute to tissue damage. However, in a number of inflammatory lung diseases, including bronchiectasis, iNOS expression is increased but no elevation of airway NO can be detected. A potential explanation for this finding is that NO is rapidly scavenged by reaction with superoxide radicals, forming peroxynitrite, which is preferentially metabolized via nitration and nitrosation reactions. To test this hypothesis, anaesthetized, specific pathogen-free rats were inoculated with Pseudomonas aeruginosa incorporated into agar beads (chronically infected group) or sterile agar beads (control group). Ten to 15 days later, the lungs were isolated and fixed. Pseudomonas organisms were isolated from the lungs of the chronically infected group. These lungs showed extensive inflammatory cell infiltration and tissue damage, which were not observed in control lungs. Expression of iNOS was increased in the chronically infected group when compared with the control group. However, the mean number of cells staining for nitrotyrosine in the chronically infected group was not significantly different from that in the controls, nor was there an excess of nitrotyrosine, nitrate, nitrite or nitrosothiol concentrations in the infected lungs. Thus, no evidence was found of increased NO metabolites in chronically infected lungs, including products of the peroxynitrite pathway. These findings suggest that chronic infection does not cause increased iNOS activity in the lung, despite increased expression of iNOS.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Count; Chronic Disease; Fluorescent Antibody Technique; Lung; Lung Diseases; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pseudomonas Infections; Rats; Rats, Sprague-Dawley; Tyrosine

Experimental allergic encephalomyelitis (EAE) is the animal model for multiple sclerosis. The present study underlines the importance of sodium phenylacetate (NaPA), a drug approved for urea cycle disorders, in inhibiting the disease process of adoptively transferred EAE in female SJL/J mice at multiple steps. Myelin basic protein (MBP)-primed T cells alone induced the expression of NO synthase (iNOS) and the activation of NF-kappaB in mouse microglial cells through cell-cell contact. However, pretreatment of MBP-primed T cells with NaPA markedly inhibited its ability to induce microglial expression of iNOS and activation of NF-kappaB. Consistently, adoptive transfer of MBP-primed T cells, but not that of NaPA-pretreated MBP-primed T cells, induced the clinical symptoms of EAE in female SJL/J mice. Furthermore, MBP-primed T cells isolated from NaPA-treated donor mice were also less efficient than MBP-primed T cells isolated from normal donor mice in inducing iNOS in microglial cells and transferring EAE to recipient mice. Interestingly, clinical symptoms of EAE were much less in mice receiving NaPA through drinking water than those without NaPA. Similar to NaPA, sodium phenylbutyrate, a chemically synthesized precursor of NaPA, also inhibited the disease process of EAE. Histological and immunocytochemical analysis showed that NaPA inhibited EAE-induced spinal cord mononuclear cell invasion and normalized iNOS, nitrotyrosine, and p65 (the RelA subunit of NF-kappaB) expression within the spinal cord. Taken together, our results raise the possibility that NaPA or sodium phenylbutyrate taken through drinking water or milk may reduce the observed neuroinflammation and disease process in multiple sclerosis patients.

Topics: Acute Disease; Administration, Oral; Adoptive Transfer; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Differentiation; Cell Line; Cell Movement; Chronic Disease; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; Female; Growth Inhibitors; Immunosuppressive Agents; Injections, Intraperitoneal; Lymphocyte Activation; Mice; Mice, Inbred Strains; Microglia; Myelin Basic Protein; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phenylacetates; Phenylbutyrates; Severity of Illness Index; Spinal Cord; T-Lymphocyte Subsets; Transcription Factor RelA; Tyrosine

Chronic alcohol consumption is a well-known risk factor for hepatic injury, and mitochondrial damage plays a significant role in this process. Nitric oxide (NO) is an important modulator of mitochondrial function and is known to inhibit mitochondrial respiration. However, the impact of chronic alcohol consumption on NO-dependent control of liver mitochondrial function is unknown. This study examines the effect of alcohol exposure on liver mitochondria in a rat model and explores the interaction of NO and mitochondrial respiration in this context. Mitochondria were isolated from the liver of both control and ethanol-fed rats after 5 to 6 weeks of alcohol consumption. Mitochondria isolated from ethanol-treated rats showed a significant decrease in state 3 respiration and respiratory control ratio that was accompanied by an increased sensitivity to NO-dependent inhibition of respiration. In conclusion, we show that chronic alcohol consumption leads to increased sensitivity to the inhibition of respiration by NO. We propose that this results in a greater vulnerability to hypoxia and the development of alcohol-induced hepatotoxicity.

Topics: Alcohol Drinking; Animals; Cell Hypoxia; Cell Respiration; Central Nervous System Depressants; Chronic Disease; Ethanol; Liver; Male; Mitochondria, Liver; Nitric Oxide; Oxygen Consumption; Oxyhemoglobins; Rats; Rats, Sprague-Dawley; Tyrosine

Helicobacter pylori is the main cause of gastritis and a primary carcinogen. The aim of this study was to assess oxidative damage in mucosal compartments of gastric mucosa in H. pylori positive and negative atrophic and nonatrophic gastritis.. Five groups of 10 patients each were identified according to H. pylori positive or negative chronic atrophic (Hp-CAG and CAG, respectively) and nonatrophic gastritis (Hp-CG and CG, respectively), and H. pylori negative normal mucosa (controls). Oxidative damage was evaluated by nitrotyrosine immunohistochemistry in the whole mucosa and in each compartment at baseline and at 2 and 12 months after eradication. Types of intestinal metaplasia were classified by histochemistry.. Total nitrotyrosine levels appeared significantly higher in H. pylori positive than in negative patients, and in Hp-CAG than in Hp-CG (p <.001); no differences were found between H. pylori negative gastritis and normal mucosa. Nitrotyrosine were found in foveolae and intestinal metaplasia only in Hp-CAG. At 12 months after H. pylori eradication, total nitrotyrosine levels showed a trend toward a decrease in Hp-CG and decreased significantly in Hp-CAG (p =.002), disappearing from the foveolae (p =.002), but remaining unchanged in intestinal metaplasia. Type I and II of intestinal metaplasia were present with the same prevalence in Hp-CAG and CAG, and did not change after H. pylori eradication.. Oxidative damage of the gastric mucosa increases from Hp-CG to Hp-CAG, involving the foveolae and intestinal metaplasia. H. pylori eradication induces a complete healing of foveolae but not of intestinal metaplasia, reducing the overall oxidative damage in the mucosa.

Topics: Adult; Aged; Chronic Disease; Female; Gastric Mucosa; Gastritis; Gastritis, Atrophic; Helicobacter Infections; Helicobacter pylori; Humans; Immunohistochemistry; Male; Metaplasia; Middle Aged; Tyrosine

Nitrosative stress resulting from increased nitric oxide (NO) synthesis contributes to the pathogenesis of chronic inflammatory diseases, including chronic viral hepatitis. Our goal was to assess the expression of inducible nitric oxide synthase (iNOS) and the formation of nitrotyrosine (NTY), as a marker of nitrosative stress, in liver biopsies from primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) patients.. Intrahepatic expression of iNOS and NTY was measured immunohistochemically and compared to histological scores of the severity of liver disease.. Hepatocellular iNOS expression was observed in liver sections from PBC patients (with a diffuse lobular distribution) and from AIH patients (marked staining in areas of pronounced inflammation and necrosis), but not in control liver sections, including non-autoimmune cholestatic liver disease. Liver samples from PBC and AIH patients, but not from controls, showed NTY accumulation in clusters of hepatocytes and Kupffer cells. Increased iNOS expression and NTY accumulation correlated with the histological severity of PBC or AIH, especially with the degree of inflammation.. Patients with PBC and AIH showed an enhanced intrahepatic iNOS expression and NTY accumulation, related to the histological severity of liver disease, consistent with NO-mediated nitration of hepatocellular proteins contributing to liver damage in both diseases.

Topics: Adult; Chronic Disease; Female; Hepatitis, Autoimmune; Humans; Liver; Liver Cirrhosis, Biliary; Liver Diseases; Male; Middle Aged; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Severity of Illness Index; Tyrosine

Limited availability of endothelial tissue is a major constraint when investigating the cellular mechanisms of endothelial dysfunction in patients with metabolic and cardiovascular diseases. We propose a novel approach that combines collection of 200-1,000 endothelial cells from a superficial forearm vein or the radial artery, with reliable measurements of protein expression by quantitative immunofluorescence analysis. This method was validated against immunoblot analysis in cultured endothelial cells. Levels of vascular endothelial cell activation, oxidative stress, and nitric oxide synthase expression were measured and compared in five patients with severe chronic heart failure and in four healthy age-matched subjects. In summary, vascular endothelial biopsy coupled with measurement of protein expression by quantitative immunofluorescence analysis provides a novel approach to the study of the vascular endothelium in humans.

Topics: Aged; Arteries; Biopsy; Cardiac Output, Low; Cells, Cultured; Chronic Disease; Cyclooxygenase 2; Endothelium, Vascular; Fluorescent Antibody Technique; Humans; Isoenzymes; Male; Membrane Proteins; Middle Aged; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Prostaglandin-Endoperoxide Synthases; Reference Values; Reproducibility of Results; Tyrosine; Veins

Helicobacter pylori infection causes gastric diseases, but the responsible mechanisms are not completely understood. They can involve DNA and tissue damage induced by reactive oxygen and nitrogen species. Our aim is to investigate the effects of bacterial eradication on oxidative stress by measuring changes of relevant markers.. Antral biopsies were obtained from 34 patients with chronic atrophic gastritis and peptic ulcer disease before and after bacterial eradication. The expression of inducible nitric oxide synthase (iNOS) and levels of nitrotyrosine (NTYR) and 8-hydroxy-2'-deoxyguanosine were assessed immunohistochemically as markers of nitric oxide (NO) production and of damage to proteins and DNA, respectively.. Before treatment, the percentages of patients with staining were: 56 for iNOS in inflammatory cells, 79 and 61 for NTYR and 8-hydroxy-2'-deoxyguanosine in foveolar cells, respectively, and 82 for 8-hydroxy-2'-deoxyguanosine in lymphoid follicles. NTYR staining was associated with the intensity of inflammation (p = 0.04) and gastritis activity (p = 0.07). The prevalence of 8-hydroxy-2'-deoxyguanosine tended to be associated with that of NTYR. After successful H. pylori eradication, the prevalence of iNOS and NTYR (in mild gastritis) staining decreased (p < 0.001 and p < 0.06, respectively). 8-Hydroxy-2'-deoxyguanosine staining disappeared in 24% of cases but appeared in 18% of previously negative cases despite eradication.. Targets of oxidative stress associated with H. pylori infection are inflammatory and deep foveolar cells and lymphoid follicles. This is the first report of 8-hydroxy-2'-deoxyguanosine localization in gastric mucosa. Oxidative stress is reduced by bacterial eradication in the first stages of mild gastritis. Moderate-severe gastritis may be a step that is reversible for iNOS, but partly irreversible for NTYR and 8-hydroxy-2'-deoxyguanosine.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Biomarkers; Chronic Disease; Deoxyguanosine; Female; Gastric Mucosa; Gastritis; Helicobacter Infections; Helicobacter pylori; Humans; Immunohistochemistry; Male; Middle Aged; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Peptic Ulcer; Tyrosine

Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice.. For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry.. After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice.. Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.

Topics: Animals; Cell Adhesion Molecules; Chronic Disease; Colitis; Dextran Sulfate; Immunoglobulins; Immunohistochemistry; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucoproteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Tyrosine

Atrial fibrillation (AF) is associated with severe contractile dysfunction and structural and electrophysiological remodeling. Mechanisms responsible for impaired contractility are undefined, and current therapies do not address this dysfunction. We have found that myofibrillar creatine kinase (MM-CK), an important controller of myocyte contractility, is highly sensitive to oxidative injury, and we hypothesized that increased oxidative stress and energetic impairment during AF could contribute to contractile dysfunction. Methods and Results-- Right atrial appendages were obtained from AF patients undergoing the Maze procedure and from control patients who were in normal sinus rhythm and undergoing cardiac surgery. MM-CK activity was reduced in AF patients compared with controls (25.4+/-3.4 versus 18.2+/-3.8 micromol/mg of myofibrillar protein per minute; control versus AF; P<0.05). No reduction in total CK activity or myosin ATPase activity was detected. This selective reduction in MM-CK activity was associated with increased relative expression of the beta-myosin isoform (25+/-6 versus 63+/-5%beta, CTRL versus AF; P<0.05). Western blotting of AF myofibrillar isolates demonstrated no changes in protein composition but showed increased prevalence of protein oxidation as detected by Western blotting for 3-nitrotyrosine (peroxynitrite biomarker) and protein carbonyls (hydroxyl radical biomarker; P<0.05). Patterns of these oxidative markers were distinct, which suggests discrete chemical events and differential protein vulnerabilities in vivo. MM-CK inhibition was statistically correlated to extent of nitration (P<0.01) but not to carbonyl presence.. The present results provide novel evidence of oxidative damage in human AF that altered myofibrillar energetics may contribute to atrial contractile dysfunction and that protein nitration may be an important participant in this condition.

Topics: Aged; Atrial Appendage; Atrial Fibrillation; Biomarkers; Blotting, Western; Chronic Disease; Creatine Kinase; Creatine Kinase, MB Form; DNA; Electrophoresis, Polyacrylamide Gel; Energy Metabolism; Female; Humans; Hydroxyl Radical; Isoenzymes; Male; Middle Aged; Myocardial Contraction; Myocardium; Myofibrils; Myosins; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; Protein Isoforms; Proteins; Tyrosine

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

Topics: Administration, Oral; Adult; Antioxidants; Ascorbic Acid; Biological Availability; Blood Platelets; Case-Control Studies; Chronic Disease; Cyclic GMP; Free Radical Scavengers; Glutathione; Humans; Male; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; Peroxynitrous Acid; Platelet Aggregation; Smoking; Time Factors; Tyrosine

Extremely high rates of squamous cell carcinoma of the esophagus (SCCE) are observed in Iran, reflecting unknown, genetic and/or epidemiological risk factors. Among genetic alterations in SCCE, TP53 mutations are the most frequent, vary among populations, and may provide clues on etiological mechanisms. We have analysed mutations in TP53 (exons 5-8) in 98 SCCE from Iran by temporal temperature gel electrophoresis and direct sequencing. We found 58 mutations in 49 patients (50%), with a high prevalence of C to T transitions at CpG dinucleotides (29.3%). The TP53 mutation pattern in Iran was significantly different from that observed in SCCEs from high incidence areas of China and Western Europe (P=0.007). Moreover, the prevalence of mutations at A : T base pairs (transitions and transversions) was higher in men than in women (38.7% vs 11.1%, P=0.033). COX-2 overexpression was detected in 69% of the cases evaluated (24/35), without significant association with TP53 mutation. Accumulation of nitrotyrosine, a marker of protein damage by excess levels of nitric oxide, was observed in tumor cells in six of 18 [corrected] cases analysed. These results are consistent with the hypothesis that several factors are involved in TP53 mutagenesis in Iran. These factors include a baseline of chronic inflammatory stress, which may have a multiplicative impact on the sensitivity of esophageal cells to exogenous factors of risk.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; China; Chronic Disease; Codon; Codon, Nonsense; CpG Islands; Cyclooxygenase 2; DNA Mutational Analysis; DNA, Neoplasm; Esophageal Neoplasms; Esophagitis; Europe; Exons; Female; Frameshift Mutation; Genes, p53; Humans; Incidence; Iran; Isoenzymes; Male; Membrane Proteins; Middle Aged; Mutation; Neoplasm Proteins; Nitric Oxide; Point Mutation; Prostaglandin-Endoperoxide Synthases; Risk Factors; Sequence Analysis, DNA; Tyrosine

Long-term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still unknown. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium-independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO2+ NO3- (NOx-) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NOx- accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and support a possible neuroprotective role for specific iNOS inhibitors in this situation.

Topics: Animals; Blotting, Western; Calcium; Cerebral Cortex; Chronic Disease; Glutamic Acid; Immunohistochemistry; L-Lactate Dehydrogenase; Male; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Rats; Rats, Wistar; Restraint, Physical; Stress, Physiological; Synaptosomes; Tyrosine

Crohn's disease is a chronic disease characterized by oxidant-induced tissue injury and increased intestinal permeability. A consequence of oxidative damage is the accumulation of DNA strand breaks and activation of poly(ADP-ribose) polymerase (PARP), which subsequently catalyzes ADP-ribosylation of target proteins. In this study, we assessed the role of PARP in the colitis seen in interleukin (IL)-10 gene-deficient mice. IL-10 gene-deficient mice demonstrated significant alterations in colonic cellular energy status in conjunction with increased permeability, proinflammatory cytokine release, and nitrosative stress. After 14 days of treatment with the PARP inhibitor 3-aminobenzamide, IL-10 gene-deficient mice demonstrated normalized colonic permeability; reduced tumor necrosis factor-alpha and interferon-gamma secretion, inducible nitric oxide synthase expression, and nitrotyrosine levels; and significantly attenuated inflammation. Time course studies demonstrated that 3-aminobenzamide rapidly altered cellular metabolic activity and decreased cellular lactate levels. This was associated with normalization of colonic permeability and followed by a downregulation of proinflammatory cytokine release. Our data demonstrate that inhibition of PARP activity results in a marked improvement of colonic inflammatory disease and a normalization of cellular metabolic function and intestinal permeability.

Topics: Animals; Benzamides; Chronic Disease; Colitis; Disease Models, Animal; Energy Metabolism; Enzyme Inhibitors; Inflammatory Bowel Diseases; Interferon-gamma; Interleukin-10; Intestinal Absorption; Intestinal Mucosa; Mice; Mice, Inbred Strains; Mice, Knockout; Neutrophils; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Tumor Necrosis Factor-alpha; Tyrosine

There is limited evidence that inhibition of the activity of the protease calpain I reduces inflammation. Here we investigate the effects of calpain inhibitor I in animal models of acute and chronic inflammation (carrageenan-induced pleurisy and collagen-induced arthritis). We report here for the first time that calpain inhibitor I (given at 5, 10, or 20 mg/kg i.p. in the pleurisy model or at 5 mg/kg i.p every 48 hours in the arthritis model) exerts potent anti-inflammatory effects (eg, inhibition of pleural exudate formation, mononuclear cell infiltration, delayed the development of the clinical signs and histological injury) in vivo. Furthermore, calpain inhibitor I reduced (1) the staining for nitrotyrosine and poly (ADP-ribose) polymerase (immunohistochemistry) and (2) the expression of inducible nitric oxide synthase and cyclooxygenase-2 in the lungs of carrageenan-treated rats and in joints from collagen-treated rats. Thus, prevention of the activation of calpain I reduces the development of acute and chronic inflammation. Inhibition of calpain I activity may represent a novel therapeutic approach for the therapy of inflammation.

Topics: Acute Disease; Animals; Arthritis; Carrageenan; Chronic Disease; Collagen; Cyclooxygenase 2; Cysteine Proteinase Inhibitors; Glycoproteins; Isoenzymes; Lung; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pleurisy; Poly Adenosine Diphosphate Ribose; Prostaglandin-Endoperoxide Synthases; Radiography; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Tarsus, Animal; Tyrosine

We have examined the localization of inducible nitric oxide synthase (iNOS) and nitrotyrosine (the product of nitration of tyrosine by peroxynitrite, a highly reactive derivative of nitric oxide [NO]) in demyelinating lesions from (i) two young adult patients with acute multiple sclerosis (MS), (ii) a child with MS (consistent with diffuse sclerosis), and (iii) five adult patients with chronic MS. Previous reports have suggested a possible correlation between iNOS, peroxynitrite, related nitrogen-derived oxidants, and the demyelinating processes in MS. We have demonstrated iNOS-immunoreactive cells in both acute-MS and diffuse-sclerosis-type lesions. In acute-MS lesions, iNOS was localized in both monocytes/macrophages and reactive astrocytes. However, foamy (myelin-laden) macrophages and the majority of reactive astrocytes were iNOS negative. In specimens from the childhood MS patient, iNOS protein was present only in a subpopulation of reactive or hypertrophic astrocytes. In contrast, no iNOS staining was detected in chronic-MS lesions. Immunohistochemical staining of acute-MS lesions with an antibody to nitrotyrosine revealed codistribution of iNOS- and nitrotyrosine-positive cells, although nitrotyrosine staining was more widespread in cells of the monocyte/macrophage lineage. In diffuse-sclerosis-type lesions, nitrotyrosine staining was present in hypertrophic astrocytes, whereas it was absent in chronic-MS lesions. These results suggest that NO and nitrogen-derived oxidants may play a role in the initiation of demyelination in acute-MS lesions but not in the later phase of the disease.

Topics: Acute Disease; Adolescent; Adult; Aged; Aged, 80 and over; Antibodies; Antibodies, Monoclonal; Astrocytes; Brain; Chronic Disease; Diffuse Cerebral Sclerosis of Schilder; Female; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Macrophages; Male; Middle Aged; Monocytes; Multiple Sclerosis; Myelin Sheath; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Tyrosine

Peroxynitrite, which is generated by the reaction of nitric oxide (NO) with superoxide, is a strong oxidant that can damage subcellular organelles, membranes and enzymes through its actions on proteins, lipids, and DNA, including the nitration of tyrosine residues of proteins. Detection of nitrotyrosine (NT) serves as a biochemical marker of peroxynitrite-induced damage. In the present studies, NT was detected by immunohistochemistry in CNS tissues from mice with acute experimental autoimmune encephalomyelitis (EAE). NT immunoreactivity was displayed by many mononuclear inflammatory cells, including CD4+ cells. It was also observed in astrocytes near EAE lesions. Immunostaining for the inducible isoform of NO synthase (iNOS) was also observed, particularly during acute EAE. These data strongly suggest that peroxynitrite formation is a major consequence of NO produced via iNOS, and implicate this powerful oxidant in the pathogenesis of EAE.

Topics: Acute Disease; Animals; Chronic Disease; Encephalomyelitis, Autoimmune, Experimental; Female; Immunohistochemistry; Mice; Mice, Inbred Strains; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Recurrence; Remission Induction; Serum Albumin, Bovine; Spinal Cord; Tyrosine

Reaction of nitric oxide (NO.) with superoxide radical generates peroxy-nitrite, which can decompose to products that nitrate aromatic amino acids. Such nitro-aromatics may be 'markers' of NO.-dependent oxidative damage. Blood serum and synovial fluid from patients with the inflammatory joint disease rheumatoid arthritis contain 3-nitrotyrosine. By contrast, body fluids from normal subjects and patients with osteoarthritis contain no detectable 3-nitrotyrosine; much lower levels were found in serum from patients in the early stages of rheumatoid arthritis. This is evidence that NO. plays a role in joint damage in rheumatoid arthritis.

Topics: Arthritis, Rheumatoid; Chromatography, High Pressure Liquid; Chronic Disease; Humans; Nitric Oxide; Spectrophotometry, Ultraviolet; Synovial Fluid; Tyrosine