3-nitrotyrosine and Sepsis

Topics: Asthma; Biomarkers; Carbon Monoxide; Chromatography, Gas; Chromatography, High Pressure Liquid; Clinical Enzyme Tests; Enzyme-Linked Immunosorbent Assay; Guanine; Infections; Neoplasms; Nitric Oxide; Pulmonary Disease, Chronic Obstructive; Reference Values; Sepsis; Tyrosine

Sepsis aggregates undesirable immune response causing depression of ventricular myocardium and diastolic dysfunction. This present study examined the effect of a plant-derived flavone tangeretin (TG) on autophagy and reduction in myocardial dysfunction. The sepsis was induced by cecum ligation and puncture (CLP) in male Sprague-Dawley rats. Abnormal changes were seen in the heart after the sepsis induction. These abnormalities were analyzed based on the cardiac markers, namely Cardiac myosin light chain-1 (cMLC1) and Cardiac troponin I (cTnl), echocardiography, and plasma parameters, like Lactate dehydrogenase (LDH) and Creatinine kinase (CK). Microanatomy of the heart was studied using hematoxylin and eosin stained histopathological samples of cardiac tissue. Western blot technique was used to detect the nature and extent of protein with the amount of a specific RNA (gene expression) in the cardiac homogenate. Oxidative damage was analyzed using redox marker, reduced glutathione. This study successfully showed that TG attenuated sepsis-induced myocardial dysfunction by inhibiting myocardial autophagy via silencing the Phosphatase and tensin homolog (PTEN) expression and acting on the AKT/mTOR pathway. The present findings supported that TG is a novel cardioprotective therapeutic target for sepsis induced myocardial dysfunction.

Topics: Animals; Autophagy; Cardiomyopathies; Cardiotonic Agents; Cecum; Cell Death; Disease Models, Animal; Flavones; Glutathione; Heart; Ligation; Male; Myocardium; NLR Family, Pyrin Domain-Containing 3 Protein; Protein Carbonylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Punctures; Rats, Sprague-Dawley; Sepsis; STAT3 Transcription Factor; TOR Serine-Threonine Kinases; Tyrosine

Sepsis is a leading cause of mortality among patients in intensive care units across the USA. Thioredoxin-1 (Trx-1) is an essential 12 kDa cytosolic protein that, apart from maintaining the cellular redox state, possesses multifunctional properties. In this study, we explored the possibility of controlling adverse myocardial depression by overexpression of Trx-1 in a mouse model of severe sepsis.. Adult C57BL/6J and Trx-1. Echocardiography analysis showed preserved cardiac function in the Trx-1. Our results indicate that overexpression of Trx-1 attenuates cardiac dysfunction during CLP. The mechanism of action may involve reduction of oxidative stress, apoptosis, and vascular permeability through activation of Trx-1/HO-1 and anti-apoptotic protein survivin.

Topics: Aldehydes; Animals; Apoptosis; Capillary Permeability; Cardiomyopathies; Carrier Proteins; Caspase 3; Disease Models, Animal; Echocardiography; Female; Heart; Heme Oxygenase-1; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardium; Oxidative Stress; Repressor Proteins; Sepsis; Survivin; Thioredoxins; Tyrosine

High FGF23 and low α-Klotho levels associate with systemic inflammation and reduced nitric oxide (NO) bioavailability, but the dynamics of this relationship in patients with CKD has not been investigated.. We sequentially measured serum intact FGF23 and carboxyl-terminal (iFGF23, cFGF23), the iFGF23/cFGF23 ratio, αKlotho, biomarkers of inflammation (hs-CRP, IL-6 and TNF-α) and sepsis (procalcitonin), nitrotyrosine (reflecting NO synthesis and oxidative stress), serum iron and ferritin and CKD-MBD biomarkers, PTH, 25(OH)VD, 1,25(OH)2 VD at peak of intercurrent sepsis and after complete resolution in a series of 17 patients with CKD.. At peak infection, biomarkers of inflammation/sepsis, ferritin and nitrotyrosine were all very high (all P < 0·01) and declined towards the normal range thereafter (P < 0·01). iFGF23 was 191 ± 10 pg/ml (geometric mean, SD) and doubled to 371 ± 8 pg/ml (P = 0·003) after the resolution of infection, while cFGF23 did not change (246 ± 5 pg/mL vs. 248 ± 5 pg/mL, P = 0·50). As a consequence, the iFGF23/cFGF23 ratio, an indicator of the proteolytic cleavage of the FGF23 molecule, was 0·78 ± 3·87 at peak infection and increased to 1·49 ± 3·00 after resolution of infection (P < 0·001). In contrast, serum α-Klotho levels were upregulated at peak infection (peak infection: 526 ± 4 pg/ml, postinfection: 447 ± 4 pg/ml, P = 0·001). The eGFR, PTH and vitamin D did not change significantly throughout.. Acute inflammation/sepsis suppresses the active form of FGF23 and activates α-Klotho, the latter effect being likely attributable to enhance proteolysis of FGF23 molecule. iFGF23 downregulation and α-Klotho upregulation during acute sepsis may participate into the counter-regulatory response to severe inflammation in CKD patients with sepsis.

Topics: Adult; Aged; Biomarkers; C-Reactive Protein; Calcitonin; Calcitriol; Female; Ferritins; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Glucuronidase; Humans; Inflammation; Interleukin-6; Iron; Klotho Proteins; Male; Middle Aged; Oxidative Stress; Parathyroid Hormone; Prospective Studies; Renal Insufficiency, Chronic; Sepsis; Tumor Necrosis Factor-alpha; Tyrosine; Vitamin D

This study investigated the effect of dietary fish oil on systemic inflammation and hepatic injury in mice with polymicrobial sepsis. Male ICR mice were assigned to a control group (C, n=30) and a fish oil group (FO, n=30). Mice in the C group were fed a semi-purified diet with 10% soybean oil, and those in the FO group were fed a fish oil diet (2.5% fish oil+7.5% soybean oil; w/w). Three weeks later, sepsis was induced by cecal ligation and puncture (CLP), and mice were sacrificed at 0, 6 and 24 h after CLP, respectively. Results showed that compared with C group, the FO group had lower plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and nitrite at 6 and 24 h after CLP. Also, peritoneal lavage fluid concentrations of TNF-α and prostaglandin (PG) E2 were significantly lower at 24 h in the FO than in the C group. The FO group had lower myeloperoxidase activities at 6 h after CLP in various organs. Plasma aminotransferase and alanine aminotransferase activities revealed significantly decreased in the FO group. The DNA-binding activity of peroxisome proliferators-activated receptor gamma (PPARγ) and mRNA expression of I kappaB alpha (IκBα) were up-regulated while nuclear factor (NF)-κB p65 DNA-binding activity, inducible nitric oxide synthase protein expression and the concentration of nitrotyrosine were significantly decreased in the FO group in liver after CLP. These results indicate that dietary fish oil administration may attenuate systemic inflammation and up-regulate hepatic PPARγ DNA-binding activity, which may consequently have ameliorated liver injury in these septic mice.

Topics: Animals; Biomarkers; Fatty Acids, Omega-3; Fish Oils; Inflammation; Interleukin-10; Interleukin-6; Liver; Liver Diseases; Male; Mice; Mice, Inbred ICR; NF-kappa B; Nitric Oxide Synthase Type II; Peroxidase; PPAR gamma; Sepsis; Tumor Necrosis Factor-alpha; Tyrosine; Up-Regulation

Excessive free radical production by immune cells has been linked to cell death and tissue injury during sepsis. Peroxynitrite is a short-lived oxidant and a potent inducer of cell death that has been identified in several pathological conditions. Caffeic acid phenethyl ester (CAPE) is an active component of honeybee products and exhibits antioxidant, anti-inflammatory, and immunomodulatory activities. The present study examined the ability of CAPE to scavenge peroxynitrite in RAW 264.7 murine macrophages stimulated with lipopolysaccharide/interferon-γ that was used as an in vitro model. Conversion of 123-dihydrorhodamine to its oxidation product 123-rhodamine was used to measure peroxynitrite production. Two mouse models of sepsis (endotoxemia and cecal ligation and puncture) were used as in vivo models. The level of serum 3-nitrotyrosine was used as an in vivo marker of peroxynitrite. The results demonstrated that CAPE significantly improved the viability of lipopolysaccharide/interferon-γ-treated RAW 264.7 cells and significantly inhibited nitric oxide production, with effects similar to those observed with an inhibitor of inducible nitric oxide synthase (1400W). In addition, CAPE exclusively inhibited the synthesis of peroxynitrite from the artificial substrate SIN-1 and directly prevented the peroxynitrite-mediated conversion of dihydrorhodamine-123 to its fluorescent oxidation product rhodamine-123. In both sepsis models, CAPE inhibited cellular peroxynitrite synthesis, as evidenced by the absence of serum 3-nitrotyrosine, an in vivo marker of peroxynitrite. Thus, CAPE attenuates the inflammatory responses that lead to cell damage and, potentially, cell death through suppression of the production of cytotoxic molecules such as nitric oxide and peroxynitrite. These observations provide evidence of the therapeutic potential of CAPE treatment for a wide range of inflammatory disorders.

Topics: Animals; Caffeic Acids; Cells, Cultured; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Free Radical Scavengers; Interferon-gamma; Lipopolysaccharides; Macrophage Activation; Macrophages; Male; Mice, Inbred BALB C; Nitric Oxide; Peroxynitrous Acid; Phenylethyl Alcohol; Sepsis; Tyrosine

Balanced hydroxyethyl starch (HES) solutions with a molecular weight of 130 kDa (tetrastarches) are frequently used in clinical practice. These solutions are derived either from waxy maize or potato starch and they are not bioequivalent.. Investigation of the effects of waxy maize-derived and potato-derived starches on intestinal microcirculation and pulmonary inflammation in experimental sepsis.. A randomised (three groups), blinded animal study.. Animal experimental facility in a university hospital.. Twenty-one male Sprague-Dawley rats weighing 275 to 300 g.. Sepsis was induced by caecal ligation and puncture. Animals received balanced crystalloid infusion (6 ml kg h) for 23 h followed by randomised 1 h bolus infusion (30 ml kg h) of crystalloid: balanced crystalloid solution or waxy maize starch: 6% wt/vol HES 130/0.4 or potato starch: 6% wt/vol HES 130/0.42. Results are presented as median (interquartiles).. Using intravital microscopy, mucosal perfusion was assessed by intercapillary area (ICA) between all perfused capillaries (ICAtotal) and continuously perfused capillaries only (ICAcont). Mucosal blood flow was calculated from arteriolar diameter and red blood cell velocity. Intestinal wall 3-nitrotyrosine (3-NTint) content and exhaled nitric oxide (exNO), to indicate pulmonary inflammation, were measured.. Both tetrastarches improved capillary perfusion compared to the crystalloid group, as indicated by reduced ICAtotal [crystalloid 1054 (905 to 1211) μm; waxy maize starch 789 (744 to 940) μm, P <0.05; potato starch 674 (536 to 693) μm, P < 0.05] and reduced ICAcont [crystalloid 1060 (996 to 1340) μm; waxy maize starch 860 (793 to 975) μm, P <0.05; potato starch 701 (558 to 728) μm, P <0.05]. Mucosal blood flow and systemic blood pressure did not differ significantly between groups. 3-NTint was comparable among all groups. exNO was significantly reduced from 11.1 (5.0 to 16.5) ppb to 4.2 (4.0 to 4.8) ppb in the waxy maize group, whereas no significant difference was detected in the potato starch group 6.2 (4.8 to 10.5).. Bolus infusion of balanced 6% wt/vol tetrastarches augments mucosal capillary perfusion. Pulmonary inflammation in sepsis is differentially influenced by tetrastarches produced from different raw materials.

Topics: Animals; Arterioles; Capillaries; Disease Models, Animal; Erythrocytes; Hydroxyethyl Starch Derivatives; Inflammation; Intestines; Male; Microcirculation; Nitric Oxide; Oxidative Stress; Plasma Substitutes; Random Allocation; Rats; Rats, Sprague-Dawley; Sepsis; Solanum tuberosum; Tyrosine; Zea mays

This experimental animal study investigates the effects of combined recombinant human activated protein C (rhAPC) and ceftazidime on cardiopulmonary function in acute lung injury and severe sepsis. Twenty-one sheep (37 ± 2 kg) were operatively prepared and randomly allocated to either the sham, control, or treatment group (n = 7 each). Single treatments of rhAPC or ceftazidime were published previously; therefore, control groups were dispensed in the present study, what may be considered a study limitation. Acute lung injury and sepsis were induced according to an established protocol. The sham group received only the vehicle. The sheep were studied in awake state for 24 h and mechanically ventilated. Recombinant human APC (continuous infusion 24 μg/kg per hour) and ceftazidime (3-g bolus at 1 and 13 h) were intravenously administered. The animals were fluid resuscitated with Ringer's lactate to maintain hematocrit at baseline. Compared with injured controls, the treatment group had a significantly higher PaO₂/FIO₂ ratio, and the onset of acute respiratory distress syndrome was prevented. The increase in pulmonary microvascular shunt fraction and airway obstruction in bronchi and bronchiole, as well as lung 3-nitrotyrosine, lung myeloperoxidase, cardiac 3-nitrotyrosine, and cardiac malondialdehyde levels, was significantly reduced as compared with controls (P < 0.05 each). Treated sheep had significantly improved hemodynamics as reflected by mean arterial pressure, heart rate, cardiac index, and systemic vascular resistance index (P < 0.05 each). In addition, plasma oncotic pressure and urine output were significantly improved (P < 0.05 each). Combined rhAPC and ceftazidime significantly improved cardiopulmonary function, reduced pulmonary and cardiac tissue injury, and prevented the onset of acute respiratory distress syndrome in ovine severe sepsis without obvious adverse effects.

Topics: Animals; Blood Gas Analysis; Ceftazidime; Female; Hemodynamics; Humans; Lung; Male; Myocardium; Oxygen; Peroxidase; Protein C; Recombinant Proteins; Respiratory Distress Syndrome; Sepsis; Sheep; Treatment Outcome; Tyrosine

Vascular leakage in multiple organs is a characteristic pathological change in sepsis. Our recent study revealed that ascorbate protects endothelial barrier function in microvascular endothelial cell monolayers through inhibiting serine/threonine protein phosphatase 2A (PP2A) activation (Han M, Pendem S, Teh SL, Sukumaran DK, Wu F, Wilson JX. Free Radic Biol Med 48: 128-135, 2010). The present study addressed the mechanism of protection by ascorbate against vascular leakage in cecal ligation and puncture (CLP)-induced septic peritonitis in mice. CLP caused NADPH oxidase activation and endothelial nitric oxide synthase (eNOS) uncoupling to produce superoxide, increased NO production by inducible NOS (iNOS) and neuronal NOS (nNOS) activity, and elevated 3-nitrotyrosine (a product of peroxynitrite) formation and PP2A activity in the hindlimb skeletal muscles at 12 h after CLP. The increase in PP2A activity was associated with decreased levels of phosphorylated serine and threonine in occludin, which was immunoprecipitated from freshly harvested endothelial cells of the septic skeletal muscles. Moreover, CLP increased the vascular permeability to fluorescent dextran and Evans blue dye in skeletal muscles. An intravenous bolus injection of ascorbate (200 mg/kg body wt), given 30 min prior to CLP, prevented eNOS uncoupling, attenuated the increases in iNOS and nNOS activity, decreased 3-nitrotyrosine formation and PP2A activity, preserved the phosphorylation state of occludin, and completely inhibited the vascular leakage of dextran and Evans blue. A delayed ascorbate injection, given 3 h after CLP, also prevented the vascular permeability increase. We conclude that ascorbate injection protects against vascular leakage in sepsis by sequentially inhibiting excessive production of NO and superoxide, formation of peroxynitrite, PP2A activation, and occludin dephosphorylation. Our study provides a scientific basis for injection of ascorbate as an adjunct treatment for vascular leakage in sepsis.

Topics: Animals; Antioxidants; Ascorbic Acid; Capillary Permeability; Cecum; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Muscle, Skeletal; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Occludin; Peritonitis; Protein Phosphatase 2; Sepsis; Superoxides; Tyrosine

Acute lung injury (ALI) and sepsis are major contributors to the morbidity and mortality of critically ill patients. The current study was designed further evaluate the mechanism of pulmonary vascular hyperpermeability in sheep with these injuries.. Sheep were randomized to a sham-injured control group (n=6) or ALI/sepsis group (n=7). The sheep in the ALI/sepsis group received inhalation injury followed by instillation of Pseudomonas aeruginosa into the lungs. These groups were monitored for 24 h. Additional sheep (n=16) received the injury and lung tissue was harvested at different time points to measure lung wet/dry weight ratio, vascular endothelial growth factor (VEGF) mRNA and protein expression as well as 3-nitrotyrosine protein expression in lung homogenates.. The injury induced severe deterioration in pulmonary gas exchange, increases in lung lymph flow and protein content, and lung water content (P<0.01 each). These alterations were associated with elevated lung and plasma nitrite/nitrate concentrations, increased tracheal blood flow, and enhanced VEGF mRNA and protein expression in lung tissue as well as enhanced 3-nitrotyrosine protein expression (P<0.05 each).. This study describes the time course of pulmonary microvascular hyperpermeability in a clinical relevant large animal model and may improve the experimental design of future studies.

Topics: Acute Lung Injury; Animals; Capillary Permeability; Disease Models, Animal; Female; Lung; Microcirculation; Nitric Oxide; Pneumonia; Pseudomonas aeruginosa; Pseudomonas Infections; Pulmonary Circulation; Pulmonary Edema; Pulmonary Gas Exchange; RNA, Messenger; Sepsis; Sheep; Smoke Inhalation Injury; Time Factors; Tyrosine; Vascular Endothelial Growth Factor A

Vasopressin analogs are used as a supplement to norepinephrine in septic shock. The isolated effects of vasopressin agonists on sepsis-induced vascular dysfunction, however, remain controversial. Because V(2)-receptor stimulation induces vasodilation and procoagulant effects, a higher V(1a)- versus V(2)-receptor selectivity might be advantageous. We therefore hypothesized that a sole, titrated infusion of the selective V(1a)-agonist Phe(2)-Orn(8)-Vasotocin (POV) is more effective than the mixed V(1a)-/V(2)-agonist AVP for the treatment of vascular and cardiopulmonary dysfunction in methicillin resistant staphylococcus aureus pneumonia-induced, ovine sepsis. After the onset of hemodynamic instability, awake, chronically instrumented, mechanically ventilated, and fluid resuscitated sheep were randomly assigned to receive continuous infusions of either POV, AVP, or saline solution (control; each n = 6). AVP and POV were titrated to maintain mean arterial pressure above baseline - 10 mmHg. When compared with that of control animals, AVP and POV reduced neutrophil migration (myeloperoxidase activity, alveolar neutrophils) and plasma levels of nitric oxide, resulting in higher mean arterial pressures and a reduced vascular leakage (net fluid balance, chest and abdominal fluid, pulmonary bloodless wet-to-dry-weight ratio, alveolar and septal edema). Notably, POV stabilized hemodynamics at lower doses than AVP. In addition, POV, but not AVP, reduced myocardial and pulmonary tissue concentrations of 3-nitrotyrosine, VEGF, and angiopoietin-2, thereby leading to an abolishment of cumulative fluid accumulation (POV, 9 ± 15 ml/kg vs. AVP, 110 ± 13 ml/kg vs. control, 213 ± 16 ml/kg; P < 0.001 each) and an attenuated cardiopulmonary dysfunction (left ventricular stroke work index, PaO(2)-to-FiO(2) ratio) versus control animals. Highly selective V(1a)-agonism appears to be superior to unselective vasopressin analogs for the treatment of sepsis-induced vascular dysfunction.

Topics: Angiopoietin-2; Animals; Arginine Vasopressin; Arterial Pressure; Blood Vessels; Capillary Permeability; Disease Models, Animal; Female; Hemodynamics; Infusions, Intravenous; Methicillin-Resistant Staphylococcus aureus; Neutrophil Infiltration; Nitric Oxide; Pneumonia, Staphylococcal; Receptors, Vasopressin; Sepsis; Sheep; Smoke Inhalation Injury; Time Factors; Tyrosine; Vascular Endothelial Growth Factor A; Vasoconstriction; Vasoconstrictor Agents; Vasotocin; Ventricular Function, Left

Infection is known to impair the growth of developing lungs. It is known that plasma free nitrotyrosine (NT) levels can reach 150 μM during sepsis. Free NT incorporates into microtubules and impairs cell function. We hypothesize that free NT perturbs the angiogenic activity of pulmonary artery endothelial cells (PAEC) in developing lungs. PAEC from fetal lamb lungs were incubated with NT (1-100 μM). We examined the effects of NT on tube formation, cell proliferation, apoptosis, and α-tubulin assembly in PAEC. We assessed superoxide anion (O2) and NO levels in PAEC during NT exposure. Effects of NT on endothelial NO synthase (eNOS) were examined with respect to eNOS-dimer formation and the association of eNOS chaperone, heat-shock-protein-90 (hsp90). NT decreased tube formation and increased apoptosis in PAEC. NT also decreased NO levels, increased NOS-dependent O2 generation, and promoted α-tubulin depolymerization. Although NT increased eNOS homodimer formation, it decreased the hsp90 association with eNOS. Our data suggest that increased NT formation during sepsis may uncouple eNOS activity and increase oxidative stress. Because NO plays an important role in angiogenesis and vasodilation, these observations suggest a mechanism for the impaired vasodilation and angiogenesis during sepsis in the developing lung.

Topics: Animals; Apoptosis; Cell Proliferation; Cells, Cultured; Cesarean Section; Endothelial Cells; Gestational Age; HSP90 Heat-Shock Proteins; Lung; Microtubules; Neovascularization, Physiologic; Nitric Oxide; Nitric Oxide Synthase Type III; Premature Birth; Protein Multimerization; Pulmonary Artery; Sepsis; Sheep; Superoxide Dismutase; Superoxides; Tubulin; Tyrosine

Formation of nitric oxide and its derivative reactive nitrogen species during endotoxemia has been implicated in the pathogenesis of the associated cardiovascular dysfunction. This stress can promote nitrosative post-translational modifications of proteins that may alter their activity and contribute to dysregulation. We utilized the ascorbate-dependent biotin-switch method to assay protein S-nitrosylation and immunoblotted for tyrosine nitration to monitor changes in nitrosative protein oxidation during endotoxemia. Hearts from lipopolysaccharide (LPS)-treated rats showed no apparent variation in global protein S-nitrosylation, but this may be due to the poor sensitivity of the biotin-switch method. To sensitize our monitoring of protein S-nitrosylation we exposed isolated hearts to the efficient trans-nitrosylating agent nitrosocysteine (which generated a robust biotin-switch signal) and then identified a number of target proteins using mass spectrometry. We were then able to probe for these target proteins in affinity-capture preparations of S-nitrosylated proteins prepared from vehicle- or LPS-treated animals. Unexpectedly this showed a time-dependent loss in S-nitrosylation during sepsis, which we hypothesized, may be due to concomitant superoxide formation that may lower nitric oxide but simultaneously generate the tyrosine-nitrating agent peroxynitrite. Indeed, this was confirmed by immunoblotting for global tyrosine nitration, which increased time-dependently and temporally correlated with a decrease in mean arterial pressure. We assessed if tyrosine nitration was causative in lowering blood pressure using the putative peroxynitrite scavenger FeTPPS. However, FeTPPS was ineffective in reducing global protein nitration and actually exacerbated LPS-induced hypotension.

Topics: Animals; Biotin; Blood Pressure; Cysteine; Endotoxemia; Escherichia coli; Heart; HEK293 Cells; Humans; Hypotension; Immunoblotting; In Vitro Techniques; Lipopolysaccharides; Mass Spectrometry; Metalloporphyrins; Mice; Mice, Inbred C57BL; Models, Animal; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Peroxynitrous Acid; Protein Processing, Post-Translational; Proteins; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Sensitivity and Specificity; Sepsis; Signal Transduction; Telemetry; Tyrosine

Hepatic energy depletion has been described in severe sepsis, and lipopolysaccharide (LPS) has been shown to cause mitochondrial DNA (mtDNA) damage. To clarify the mechanisms of LPS-induced mtDNA damage and mitochondrial alterations, we treated wild-type (WT) or transgenic manganese superoxide dismutase-overerexpressing (MnSOD(+++)) mice with a single dose of LPS (5 mg/kg). In WT mice, LPS increased mitochondrial reactive oxygen species formation, hepatic inducible nitric oxide synthase (NOS) mRNA and protein, tumor necrosis factor-alpha, interleukin-1 beta, and high-mobility group protein B1 concentrations. Six to 48 h after LPS administration (5 mg/kg), liver mtDNA levels, respiratory complex I activity, and adenosine triphosphate (ATP) contents were decreased. In addition, LPS increased interferon-β concentration and decreased mitochondrial transcription factor A (Tfam) mRNA, Tfam protein, and mtDNA-encoded mRNAs. Morphological studies showed mild hepatic inflammation. The LPS (5 mg/kg)-induced mtDNA depletion, complex I inactivation, ATP depletion, and alanine aminotransferase increase were prevented in MnSOD(+++) mice or in WT mice cotreated with 1400W (a NOS inhibitor), (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride, monohydrate (a superoxide scavenger) or uric acid (a peroxynitrite scavenger). The MnSOD overexpression delayed death in mice challenged by a higher, lethal dose of LPS (25 mg/kg). In conclusion, LPS administration damages mtDNA and alters mitochondrial function. The protective effects of MnSOD, NOS inhibitors, and superoxide or peroxynitrite scavengers point out a role of the superoxide anion reacting with NO to form mtDNA- and protein-damaging peroxynitrite. In addition to the acute damage caused by reactive species, decreased levels of mitochondrial transcripts contribute to mitochondrial dysfunction.

Topics: Aconitate Hydratase; Adenosine Triphosphate; Alanine Transaminase; Animals; ATP Synthetase Complexes; DNA-Binding Proteins; DNA, Mitochondrial; Electron Transport Complex I; Electron Transport Complex III; Electron Transport Complex IV; Hep G2 Cells; High Mobility Group Proteins; Humans; Interferon-beta; Iron; Lipopolysaccharides; Liver; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Reactive Oxygen Species; Sepsis; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Toll-Like Receptor 4; Transcription Factors; Transcription, Genetic; Tumor Necrosis Factor-alpha; Tyrosine

Different isoforms of nitric oxide synthases (NOS) and determinants of oxidative/nitrosative stress play important roles in the pathophysiology of pulmonary dysfunction induced by acute lung injury (ALI) and sepsis. However, the time changes of these pathogenic factors are largely undetermined.. Twenty-four chronically instrumented sheep were subjected to inhalation of 48 breaths of cotton smoke and instillation of live Pseudomonas aeruginosa into both lungs and were euthanized at 4, 8, 12, 18, and 24 hours post-injury. Additional sheep received sham injury and were euthanized after 24 hrs (control). All animals were mechanically ventilated and fluid resuscitated. Lung tissue was obtained at the respective time points for the measurement of neuronal, endothelial, and inducible NOS (nNOS, eNOS, iNOS) mRNA and their protein expression, calcium-dependent and -independent NOS activity, 3-nitrotyrosine (3-NT), and poly(ADP-ribose) (PAR) protein expression.. The injury induced severe pulmonary dysfunction as indicated by a progressive decline in oxygenation index and concomitant increase in pulmonary shunt fraction. These changes were associated with an early and transient increase in eNOS and an early and profound increase in iNOS expression, while expression of nNOS remained unchanged. Both 3-NT, a marker of protein nitration, and PAR, an indicator of DNA damage, increased early but only transiently.. Identification of the time course of the described pathogenetic factors provides important additional information on the pulmonary response to ALI and sepsis in the ovine model. This information may be crucial for future studies, especially when considering the timing of novel treatment strategies including selective inhibition of NOS isoforms, modulation of peroxynitrite, and PARP.

Topics: Acute Lung Injury; Animals; Disease Models, Animal; Interleukin-8; Lung; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Poly Adenosine Diphosphate Ribose; Reverse Transcriptase Polymerase Chain Reaction; Sepsis; Sheep; Time Factors; Tyrosine

Methicillin-resistant Staphylococcus aureus (MRSA)-related pneumonia and/or sepsis are a frequent serious menace. The aim of the study was to establish a standardized and reproducible model of MRSA-induced septic pneumonia to evaluate new therapies. Sheep were operatively prepared for chronic study. After 5 days' recovery, tracheostomy was performed under anesthesia, and smoke injury was induced by inhalation of cotton smoke (48 breaths, <40 degrees C). Methicillin-resistant S. aureus (AW6) (approximately 2.5x10(11) colony-forming units) was instilled into the airway by a bronchoscope. After the injury, animals were awakened and maintained on mechanical ventilation by 100% oxygen for first 3 h, and thereafter, oxygen concentration was adjusted according to blood gases. The sheep were resuscitated by lactated Ringer solution with an initial rate of 2 mL kg(-1) h(-1) that was further adjusted according to hematocrit. Study groups include (1) sham (noninjured, nontreated; n=6), (2) S+MRSA (exposed to smoke inhalation and MRSA, nontreated; n=6), and (3) smoke (exposed to smoke inhalation alone; n=6). Injured (S+MRSA) animals showed the signs of severe sepsis-related multiple organ failure 3 h after insult. Cardiovascular morbidity was evidenced by severe hypotension, with increased heart rate, cardiac output, left atrial pressure and severely decreased systemic vascular resistance index, and left ventricle stroke work index. Pulmonary dysfunction was characterized by deteriorated gas exchange (PaO2/FIO2 and pulmonary shunt) and increased ventilatory pressures. The S+MRSA group showed significantly greater lung tissue water content, myeloperoxidase activity, and cytokine production compared with uninjured sham animals. Microvascular hyperpermeability was evidenced by marked fluid retention (fluid net balance), decreased plasma protein with decreased plasma oncotic pressure, and increased pulmonary microvascular pressure. All these changes were accompanied by 6- to 7-fold increase in plasma nitrite/nitrate and increased production of reactive nitrogen species in lung. The smoke inhalation alone had a little or no effect on these variables. This model closely mimics hyperdynamic human sepsis. The excessive production of NO may be extensively involved in the pathogenic process.

Topics: Animals; Disease Models, Animal; Female; Hematocrit; Hemodynamics; Lung; Methicillin; Methicillin Resistance; Pneumonia; Sepsis; Sheep; Smoke Inhalation Injury; Staphylococcus aureus; Time Factors; Tyrosine

The mechanisms responsible for myocardial dysfunction in the setting of sepsis remain undefined. Fas ligation with its cognate ligand (FasL) induces apoptosis and activates cellular inflammatory responses associated with tissue injury. We determined whether interruption of Fas/FasL interaction by cardiac-specific expression of soluble Fas (sFas), a competitive inhibitor of FasL, would improve myocardial dysfunction and inflammation in a lipopolysaccharide (LPS)-induced mouse model of sepsis. Wild-type (WT) and sFas transgenic mice were injected intraperitoneally with 10 mg/kg LPS or with an equivalent volume of saline. At 18 h after LPS administration, echocardiographic evaluation revealed a significant decrease in left ventricular fractional shortening in the WT mice, whereas the fractional shortening was preserved in the sFas mice. Activation of nuclear factor-kappa B (NF-kappaB) and the increase in the transcript levels of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 resulting from LPS treatment were attenuated in the myocardium of sFas mice. sFas expression also inhibited LPS-induced upregulation of Toll-like receptor 4 (TLR-4) and inducible nitric oxide synthase (iNOS), and formation of peroxynitrite in the myocardium. LPS-induced increase in caspase-3/7 activity and apoptotic cell death were suppressed in sFas mice compared with WT mice. LPS-induced lung injury and increase in lung water content were also significantly reduced in sFas mice. These data indicate that neutralization of FasL by expression of sFas significantly preserves cardiac function and reduces inflammatory responses in the heart, suggesting that Fas/FasL signaling pathway is important in mediating the deleterious effects of LPS on myocardial function.

Topics: Animals; Caspase 3; Caspase 7; Cell Death; Cytokines; Echocardiography; Enzyme Activation; fas Receptor; Gene Expression Regulation; Heart; Lipopolysaccharides; Male; Mice; Myocardium; NF-kappa B; Nitric Oxide Synthase Type II; Organ Specificity; Sepsis; Solubility; Toll-Like Receptor 4; Tyrosine

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO(-/-) mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO(-/-) mice unexpectedly had improved survival compared with wild-type (WT) mice within 5-12 h after intraperitoneal E. coli challenge. Lungs of MPO(-/-) mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO(-/-) mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO(-/-) mice. Inhibition of iNOS in MPO(-/-) mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO(-/-) mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.

Topics: Animals; Escherichia coli; Escherichia coli Infections; Gene Expression Regulation, Enzymologic; Lung; Lung Injury; Mice; Mice, Knockout; Neutrophils; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidants; Peroxidase; Pulmonary Edema; Sepsis; Tyrosine

To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge.. Prospective, randomized, controlled, experimental animal study with repeated measurements.. Investigational intensive care unit at a university hospital.. Eighteen sheep (37.2 +/- 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2-5 x 10(11) colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 mug/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringer's lactate solution to maintain constant pulmonary artery occlusion pressure.. In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in Pao2/Fio2 ratio (control group values were 521 +/- 22 at baseline [BL], 72 +/- 5 at 12 hrs, and 74 +/- 7 at 24 hrs, vs. rhAPC group values of 541 +/- 12 at BL, 151 +/- 29 at 12 hours [p < .05 vs. control], and 118 +/- 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 +/- 0.02, and at 24 hrs, 0.65 +/- 0.08; rhAPC group at BL, 0.24 +/- 0.04, and at 24 hrs, 0.45 +/- 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 +/- 1, and at 24 hrs, 36 +/- 4; rhAPC group at BL, 21 +/- 1, and at 24 hrs, 28 +/- 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 +/- 2; control, 17 +/- 1; rhAPC, 12 +/- 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals).. Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

Topics: Airway Obstruction; Animals; Blood Pressure; Body Temperature; Disease Models, Animal; Female; Fibrinolytic Agents; Infusions, Intravenous; Lung; Nitrates; Nitrites; Organ Size; Prospective Studies; Protein C; Pulmonary Edema; Random Allocation; Recombinant Proteins; Respiratory Function Tests; Sepsis; Sheep; Smoke Inhalation Injury; Tyrosine

We investigated the role of oxidative stress in the pathogenesis of septic ileus. Sepsis was induced by intraperitoneal (i.p.) injection of lipopolysaccharides (LPS, 20 mg kg(-1)) in mice. The effect of two i.p. injections of superoxide dismutase [polyethylene glycol (PEG)-SOD, 4000 U kg(-1)] and catalase (PEG-CAT, 15,000 U kg(-1)) was investigated on gastric emptying, intestinal transit and total nitrite plasma concentrations. We also performed immunohistochemical experiments on gastric and ileal tissue. LPS significantly delayed gastric emptying and intestinal transit while plasma nitrite levels increased. Polyethylene glycol (PEG)-SOD reversed the endotoxin-induced delay in gastric emptying and improved the delay in intestinal transit without effect on plasma nitrite levels. PEG-CAT slightly improved the delay in gastric emptying without effect on intestinal transit. Immunohistochemistry showed the presence of nitrotyrosine (NT) and 4-hydroxy-2-nonenal (HNE) in the gastric and ileal mucosa of LPS-treated mice. Treatment with PEG-SOD or PEG-CAT of LPS mice diminished the presence of NT or HNE in both tissues. In addition, LPS induced a significant increase in inducible nitric oxide synthase (iNOS)-positive residential macrophages in the external musculature of stomach and ileum, which significantly decreased after PEG-SOD or PEG-CAT treatment. The present results support a role for oxidative and nitrosative stress in the pathogenesis of septic ileus in mice.

Topics: Aldehydes; Animals; Antioxidants; Catalase; Disease Models, Animal; Gastric Emptying; Gastrointestinal Transit; Ileus; Immunohistochemistry; Intestinal Mucosa; Lipopolysaccharides; Macrophages; Male; Mice; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Oxidative Stress; Sepsis; Superoxide Dismutase; Tyrosine

Pulmonary oxidant stress is an important pathophysiologic feature of acute lung injury. It is unclear whether nitric oxide contributes to this oxidant stress. Thus, we examined the role of inducible nitric oxide synthase (iNOS) in pulmonary oxidant stress in murine sepsis and the differential contribution of different cellular sources of iNOS.. Randomized, controlled animal study.. Research laboratory of an academic institution.. Male iNOS+/+, iNOS-/- C57Bl/6 mice, and bone-marrow transplanted iNOS chimeric mice: +to- (wild-type iNOS+/+ donor bone-marrow transplanted into iNOS-/- recipient mice) and the reciprocal -to+ chimeras.. Animals were randomized to sepsis (n = 264), induced by cecal ligation and perforation, vs. naive groups (n = 138).. In septic iNOS-/- vs. wild-type iNOS+/+ mice, sepsis-induced pulmonary oxidant stress (33 +/- 11 [mean +/- sem] vs. 365 +/- 48 pg 8-isoprostane/mg protein, p < .01) and nitrosative stress (0.0 +/- 0.0 vs. 0.9 +/- 0.4 micromol 3-nitrotyrosine/mmol para-tyrosine, p < .05) were abolished, despite similar septic increases in pulmonary myeloperoxidase activity in both (86 +/- 20 vs. 83 +/- 12 mU/mg protein, p = .78). In +to- iNOS chimeric mice (iNOS localized only to donor bone-marrow-derived inflammatory cells), cecal ligation and perforation resulted in significant pulmonary oxidant stress (368 +/- 81 pg 8-isoprostane/mg protein) and nitrosative stress (0.6 +/- 0.2 micromol 3-nitrotyrosine/mmol para-tyrosine), similar in degree to septic wild-type mice. In contrast, pulmonary oxidant and nitrosative stresses were absent in septic -to+ iNOS chimeras (iNOS localized only to recipient parenchymal cells), similar to iNOS-/- mice.. In murine sepsis-induced acute lung injury, pulmonary oxidant stress is completely iNOS dependent and is associated with tyrosine nitration. Moreover, pulmonary oxidant stress and nitrosative stress were uniquely dependent on the presence of iNOS in inflammatory cells (e.g., macrophages and neutrophils), with no apparent contribution of iNOS in pulmonary parenchymal cells. iNOS inhibition targeted specifically to inflammatory cells may be an effective therapeutic approach in sepsis and acute lung injury.

Topics: Analysis of Variance; Animals; Bone Marrow Transplantation; Chimera; Dinoprost; Lung; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Random Allocation; Respiratory Distress Syndrome; Sepsis; Tyrosine

It is known that in various pathophysiological conditions, reactive oxidants cause DNA strand breakage and subsequent activation of the nuclear enzyme poly(ADP ribose) polymerase (PARP). Activation of PARP results in cellular dysfunction. We hypothesized that pharmacological inhibition of PARP reduces the damage in the ovine model of acute lung injury (ALI). After smoke inhalation, Pseudomonas aeruginosa (5 x 109 cfu/kg) was instilled into both lungs. All of the animals were mechanically ventilated with 100% O2. The infusion of the PARP inhibitor (INO-1001, n = 6) began 1 h after the injury and thereafter through 24 h (3 mg bolus + 0.3 mg/kg/h, i.v.). Control animals (n = 6) were treated with saline. Sham injury animals (n = 8) received sham smoke and were mechanically ventilated in the same fashion. One-half of those sham animals (n = 4) were given the same dose of INO-1001. PaO2/FiO2 ratio at 24 h in saline and in the INO-1001-treated groups were 95 +/- 22 and 181 +/- 22, respectively (P < 0.05). Peak airway pressure at 24 h in the saline- and INO-1001-treated groups was 32.6 +/- 3.0 and 24.4 +/- 2.2, respectively (P < 0.05). Pulmonary shunt fraction was also significantly attenuated. INO-1001 treatment reduced pulmonary histological injury and attenuated poly (ADP-ribose) accumulation in the lung. In conclusion, inhibition of PARP improved the ALI after smoke inhalation and pneumonia. The results suggest that the activation of PARP plays a role in the pathophysiology of ALI in sheep.

Topics: Animals; Antithrombins; DNA Damage; Enzyme Inhibitors; Female; Hematocrit; Hemoglobins; Immunohistochemistry; Indoles; Lipid Peroxidation; Lung; Malondialdehyde; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Poly(ADP-ribose) Polymerase Inhibitors; Pseudomonas aeruginosa; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sepsis; Sheep; Time Factors; Tyrosine

Peroxisome proliferator activator receptor-gamma (PPARgamma) is a nuclear receptor that controls the expression of several genes involved in metabolic homeostasis. We investigated the role of PPARgamma during the inflammatory response in sepsis by the use of the PPARgamma ligands, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and ciglitazone. Polymicrobial sepsis was induced by cecal ligation and puncture in rats and was associated with hypotension, multiple organ failure, and 50% mortality. PPARgamma expression was markedly reduced in lung and thoracic aorta after sepsis. Immunohistochemistry showed positive staining for nitrotyrosine and poly(ADP-ribose) synthetase in thoracic aortas. Plasma levels of TNF-alpha, IL-6, and IL-10 were increased. Elevated activity of myeloperoxidase was found in lung, colon, and liver, indicating a massive infiltration of neutrophils. These events were preceded by degradation of inhibitor kappaBalpha (IkappaBalpha), activation of IkappaB kinase complex, and c-Jun NH(2)-terminal kinase and, subsequently, activation of NF-kappaB and AP-1 in the lung. In vivo treatment with ciglitazone or 15d-PGJ(2) ameliorated hypotension and survival, blunted cytokine production, and reduced neutrophil infiltration in lung, colon, and liver. These beneficial effects of the PPARgamma ligands were associated with the reduction of IkappaB kinase complex and c-Jun NH(2)-terminal kinase activation and the reduction of NF-kappaB and AP-1 DNA binding in the lung. Furthermore, treatment with ciglitazone or 15d-PGJ(2) up-regulated the expression of PPARgamma in lung and thoracic aorta and abolished nitrotyrosine formation and poly(ADP-ribose) expression in aorta. Our data suggest that PPARgamma ligands attenuate the inflammatory response in sepsis through regulation of the NF-kappaB and AP-1 pathways.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Aorta, Thoracic; Bacteremia; Blood Glucose; Blood Pressure; Down-Regulation; I-kappa B Kinase; I-kappa B Proteins; Interleukin-10; Interleukin-6; Leukocyte Count; Ligands; Lung; Male; Microbodies; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Neutrophil Infiltration; NF-kappa B; NF-KappaB Inhibitor alpha; Poly(ADP-ribose) Polymerases; Prostaglandin D2; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Sepsis; Signal Transduction; Survival Rate; Thiazolidinediones; Transcription Factor AP-1; Transcription Factors; Tumor Necrosis Factor-alpha; Tyrosine

Free radicals have been implicated in the etiology of cardiac dysfunction during sepsis, but the actual species responsible remains unclear. We studied the alterations in myocardial nitric oxide (NO), superoxide, and peroxynitrite generation along with cardiac mechanical function and efficiency in hearts from lipopolysaccharide (LPS)-treated rats. Six hours after LPS (4 mg/kg ip) or saline (control) treatment, hearts were isolated and perfused for 1 h with recirculating Krebs-Henseleit buffer and paced at 300 beats/min. Cardiac work, O(2) consumption, and cardiac efficiency were markedly depressed in LPS hearts compared with controls. Plasma nitrate/nitrite level was elevated in LPS rats, and ventricular NO production was enhanced as measured by electron spin resonance spectroscopy, Ca(2+)-independent NO synthase (NOS) activity, and inducible NOS immunohistochemistry. Ventricular superoxide production was also enhanced in LPS-treated hearts as seen by lucigenin chemiluminescence and xanthine oxidase activity. Increased nitrotyrosine staining (immunohistochemistry) and higher lipid hydroperoxides levels were also detected in LPS-treated hearts, indicating oxygen radical-induced stress. Enhanced generation of both NO and superoxide, and thus peroxynitrite, occur in dysfunctional hearts from endotoxemic rats.

Topics: Animals; Endotoxemia; Heart Diseases; Hydrogen Peroxide; Lipopolysaccharides; Male; Myocardial Contraction; Myocardium; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Oxygen Consumption; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Sepsis; Superoxides; Tyrosine; Xanthine Oxidase

Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity.

Topics: Amino Acid Sequence; Animals; Cell Line; Humans; Mice; Models, Molecular; Molecular Sequence Data; Muscle, Skeletal; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Conformation; Rats; Recombinant Proteins; Sepsis; Spodoptera; Transfection; Tyrosine

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) mediates hypotension and metabolic derangements in sepsis. We hypothesized that selective iNOS-inhibition would prevent hypotension in septic rats without inhibiting endothelium-dependent vasodilation caused by the physiologically important endothelial NOS. Rats were exposed to lipopolysaccharide (LPS) for 6 h and the selective iNOS-inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL), the nonselective NOS-inhibitor N:(G)-nitro-L-arginine methyl ester (L-NAME), or control. Mean arterial pressure (MAP) and vasodilation to acetylcholine (ACh, endothelium-dependent), sodium nitroprusside (SNP, endothelium-independent), and isoproterenol (ISO, endothelium-independent beta agonist) were determined. Exhaled NO, nitrate/nitrite-(NOx) levels, metabolic data, and immunohistochemical staining for nitrotyrosine, a tracer of peroxynitrite-formation were also determined. In control rats, L-NAME increased MAP, decreased the response to ACh, and increased the response to SNP, whereas L-NIL did not alter these variables. LPS decreased MAP by 18% +/- 1%, decreased vasodilation (ACh, SNP, and ISO), increased exhaled NO, NOx, nitrotyrosine staining, and caused acidosis and hypoglycemia. L-NIL restored MAP and vasodilation (ACh, SNP, and ISO) to baseline and prevented the changes in exhaled NO, NOx, pH, and glucose levels. In contrast, L-NAME restored MAP and SNP vasodilation, but did not alter the decreased response to ACh and ISO or prevent the changes in exhaled NO and glucose levels. Finally, L-NIL but not L-NAME decreased nitrotyrosine staining in LPS rats. In conclusion, L-NIL prevents hypotension and metabolic derangements in septic rats without affecting endothelium-dependent vasodilation whereas L-NAME does not.. Sepsis causes hypotension and metabolic derangements partly because of increased nitric oxide. Selective inhibition of nitric oxide produced by the inducible nitric oxide synthase enzyme prevents hypotension and attenuates metabolic derangements while preserving the important vascular function associated with endothelium-dependent vasodilation in septic rats.

Topics: Acetylcholine; Animals; Blood Pressure; Endothelium, Vascular; Enzyme Inhibitors; Hypotension; Lipopolysaccharides; Lysine; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Sepsis; Survival Rate; Tyrosine; Vasodilation

Sepsis causes impairment of diaphragmatic contractility and endurance capacity. Nitric oxide (NO) produced via inducible NO synthase (iNOS) has been implicated in the pathogenesis. Peroxynitrite, a NO-derived powerful oxidant, may be responsible for infection-induced diaphragmatic muscle failure. Therefore, we examined whether ONO1714, a new selective iNOS inhibitor, prevents sepsis-induced diaphragmatic dysfunction. Fifty male Golden-Syrian hamsters were randomly divided into five groups: hamsters that underwent sham laparotomy alone and received saline injection (Group Sham), those that underwent cecal ligation with puncture (CLP) and received saline injection (Group Sepsis), those that underwent sham laparotomy and received injection of ONO1714 0.3 mg/kg (Group Sham-ONO1714high), those that underwent CLP and received ONO1714 0.1 mg/kg (Group Sepsis-ONO1714low), and those that underwent CLP and received ONO1714 0.3 mg/kg (Group Sepsis-ONO1714high). ONO1714 or saline was intraperitoneally injected 10 min before surgery. Diaphragmatic contractility was assessed in vitro using diaphragm muscle strips excised 24 h after operation. Diaphragm fatigability was assessed by time until tension decreased to 50% of the initial value (T50%) during fatigue trials. Twitch, tetanic tensions, and T50% during fatigue trials were reduced in Group Sepsis. Pretreatment with ONO1714 dose-dependently attenuated sepsis-induced diaphragmatic contractile profiles and endurance capacity. CLP increased plasma nitrite/nitrate (NOx; stable NO metabolites), and diaphragm malondialdehyde (MDA; a product of lipid peroxidation), positive immunostaining for nitrotyrosine (peroxynitrite footprint), and iNOS activity. ONO1714 attenuated the increase. This beneficial effect of ONO1714 may be attributable, in part, to inhibition of peroxynitrite-induced lipid peroxidation in the diaphragm.. Sepsis impairs diaphragmatic contractility and endurance capacity, which may be involved in acute respiratory failure. Pretreatment with ONO1714, a new selective inducible nitric oxide synthase inhibitor, attenuated sepsis-induced diaphragmatic dysfunction in hamsters.

Topics: Amidines; Animals; Cricetinae; Diaphragm; Enzyme Inhibitors; Heterocyclic Compounds, 2-Ring; Immunohistochemistry; In Vitro Techniques; Malondialdehyde; Mesocricetus; Muscle Contraction; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Respiration Disorders; Sepsis; Tyrosine

Sepsis or peritonitis impairs diaphragmatic contractility and endurance capacity. Peroxynitrite, a powerful oxidant formed by superoxide and nitric oxide, has been implicated in the pathogenesis. Propofol scavenges this reactive molecule. The authors conducted the current study to evaluate whether propofol prevents diaphragmatic dysfunction induced by septic peritonitis.. Forty male Golden-Syrian hamsters (120-140 g) were randomly classified into five groups. Groups sham and sham-propofol 50 underwent sham laparotomy alone, whereas groups sepsis, sepsis-propofol 25, and sepsis-propofol 50 underwent cecal ligation with puncture. Groups sham and sepsis received infusion of intralipid, whereas groups sham-propofol 50, sepsis-propofol 25, and sepsis-propofol 50 received propofol at rates of 50, 25, and 50 mg.kg(-1).h(-1), respectively. Intralipid or propofol was subcutaneously infused from 3 h before surgery until 24 h after operation, when all hamsters were killed. Diaphragmatic contractility and fatigability were assessed in vitro using diaphragm muscle strips. Peroxynitrite formation in the diaphragm was assessed by nitrotyrosine immunostaining. Plasma nitrite-nitrate concentrations and diaphragmatic concentrations of malondialdehyde were determined. Using another set of animals, diaphragmatic inducible nitric oxide synthase activity was also measured.. Twitch, tetanic tensions, and tensions during fatigue trials were reduced in group sepsis compared with group sham. In group SEPSIS, diaphragm malondialdehyde and inducible nitric oxide synthase activity, and plasma nitrite-nitrate concentrations increased, and positive immunostaining for nitrotyrosine residues was found. Propofol attenuated these changes.. Pretreatment with propofol attenuated diaphragmatic dysfunction induced by septic peritonitis in hamsters assessed by contractile profiles and endurance capacity. This beneficial effect of propofol may be caused, in part, by inhibition of lipid peroxidation in the diaphragm caused by the powerful oxidant.

Topics: Animals; Cricetinae; Diaphragm; Dose-Response Relationship, Drug; Endotoxins; Immunohistochemistry; In Vitro Techniques; Male; Mesocricetus; Muscle Contraction; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peritonitis; Propofol; Sepsis; Tyrosine

The production of large amounts of nitric oxide (NO) by the inducible form of nitric oxide synthase (iNOS) and the subsequent production of peroxynitrite (OONO-) are believed to be major factors in the hemodynamic abnormalities of sepsis. This finding is based on data from rats and mice but has not been established in other species. Therefore, we examined the role of iNOS in lipopolysaccharide (LPS)-treated pigs, which have a hemodynamic pattern with sepsis that is more similar to humans than rats. Pigs were anesthetized, ventilated, and given LPS (n = 12), 20 microg/kg over 2 h, or saline (n = 7). They were killed after 2 (n = 8 LPS, 7 control) or 4 h (4 LPS). We measured cardiac output (CO), mean arterial (Part), and pulmonary and central venous pressures. We evaluated NO production by measuring expired NO, and plasma nitrate/nitrite concentration, NOS activity (in lung tissue), and iNOS protein by Western analysis, and immunohistochemistry (lung and liver), as well as iNOS mRNA by Northern analysis (liver and lung). We also measured nitrotyrosine as evidence of OONO- production by slot blot, Western analysis, and immunohistochemistry. By 2 h, Part fell and CO did not change so that systemic vascular resistance decreased from 21.5+/-2.9 to 12.7+/-3.1 mmHg x L(-1) x min (P < 0.05) and remained at 11.3+/-1.7 mmHg x L(-1) x min in the animals observed for 4 h. Plasma nitrate/nitrite, expired NO, and NOS activity did not change. We found no iNOS in tissues by Western analysis with 5 different antibodies but detected a small amount of iNOS by immunohistochemistry in inflammatory cells and small vessels. There was a small increase in iNOS mRNA in liver and lung. Despite the minimal increase in iNOS, nitrotyrosine was increased in small vessels and in inflammatory cells. In conclusion, caution should be used when extrapolating the septic response in rodents to other species, for the pattern of iNOS induction is very different.

Topics: Animals; Blood Gas Analysis; Blotting, Western; Disease Models, Animal; Hemodynamics; Lipopolysaccharides; Liver; Lung; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Sepsis; Swine; Tyrosine

Despite recent investigations, the mechanisms responsible for intestinal epithelial injury during endotoxemia remain unclear. The present study tests the hypothesis that epithelial necrosis and/or apoptosis correlate with nitric oxide (NO) dysregulation in a nonischemic model of sepsis-induced ileal injury. To test this hypothesis, a well-established in situ, autoperfused, feline ileal preparation was employed. After endotoxin (lipopolysaccharide [LPS], 3 mg/ kg, intravenously; n = 9) or vehicle (control; n = 5) treatment, ileal segments were obtained at baseline, 2 and 4 h for simultaneous evaluations of cellular and mitochondrial ultrastructure, immunoprevalence of inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (a stable biomarker of peroxynitrite), and histochemical evidence of apoptosis. Epithelial necrosis was prominent by 2 h post-LPS, despite unaltered global ileal tissue oxygen content, blood volume, and blood flow. Significant evidence of apoptosis and increases in the immunoprevalence of iNOS and 3-nitrotyrosine were not evident until 4 h post-LPS. These results suggest that the early ileal mucosal necrosis may be due to LPS-induced activation of inflammatory pathways and/or microcirculatory disturbances, whereas NO dysregulation may participate in later events, including protein nitration and epithelial apoptosis.

Topics: Animals; Apoptosis; Cats; Ileum; Immunohistochemistry; Intestinal Mucosa; Lipopolysaccharides; Male; Mitochondria; Multiple Organ Failure; Necrosis; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxygen Consumption; Sepsis; Tyrosine

Topics: Alprostadil; Animals; Arginine; Burns; Enzyme Inhibitors; Humans; Ischemia; Luminescent Measurements; Models, Biological; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Oxidative Stress; Reperfusion Injury; Sepsis; Shock; Superoxides; Tyrosine

Recently, it was proposed that zymosan, a nonbacterial agent, causes cellular injury by inducing the production of peroxynitrite and consequent poly-(ADP-ribose) synthetase (PARS activation). Here we investigated whether in vivo N-acetylcysteine treatment inhibits cellular injury in macrophages collected from rats subjected to zymosan-induced shock. Macrophages harvested from the peritoneal cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, and by nitrotyrosine. Furthermore, zymosan-induced shock caused a suppression of macrophage mitochondrial respiration, DNA strand breakage, and reduction of cellular levels of NAD+. In vivo treatment with N-acetylcysteine (40, 20, and 10 mg/kg, intraperitoneally, 1 and 6 h after zymosan) significantly reduced in a dose-dependent manner peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitochondrial respiration, and the loss of cellular levels of NAD+. Our study supports the view that the antioxidant and anti-inflammatory effect of N-acetylcysteine is also correlated with the inhibition of peroxynitrite production. In conclusion, N-acetylcysteine may be a novel pharmacological approach to prevent cell injury in inflammation.

Topics: Acetylcysteine; Animals; Cell Membrane Permeability; Disease Models, Animal; DNA Damage; Dose-Response Relationship, Drug; Energy Metabolism; Free Radical Scavengers; Macrophages, Peritoneal; Male; NAD; Nitrates; Nitric Oxide; Rats; Rats, Sprague-Dawley; Sepsis; Shock; Tyrosine; Zymosan

Production of nitric oxide via the cytokine-mediated activation of myocardial inducible nitric oxide synthase decreases myocardial contractility. Whether myocardial dysfunction is mediated directly by nitric oxide or indirectly through the formation of secondary reaction products, such as peroxynitrite, has not been established. Peroxynitrite, but not nitric oxide, reacts with the phenolic ring of tyrosine to form the stable product 3-nitro-L-tyrosine. Demonstration of tissue nitrotyrosine residues, therefore, infers the presence of peroxynitrite or related nitrogen-centered oxidants.. Retrospective analysis of human autopsy specimens.. University pathology and basic science laboratories.. Formalin-fixed, paraffin-embedded myocardial tissue samples were obtained from 11 patients with a diagnosis of sepsis, seven patients with a diagnosis of viral myocarditis, and five control patients without clinical or pathologic cardiac disease.. None.. Specific antibodies to nitrotyrosine were utilized to detect nitrotyrosine residues in human autopsy specimens. Cardiac tissue obtained from patients with myocarditis or sepsis demonstrated intense nitrotyrosine immunoreactivity in the endocardium, myocardium, and coronary vascular endothelium and smooth muscle. In contrast, connective tissue elements were without appreciable immunohistochemical staining. Nitrotyrosine antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin, but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. In situ reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. Densitometric analysis of nitrotyrosine immunoreactivity demonstrated significantly higher values for specimens from myocarditis and sepsis patients when compared with control tissue specimens.. These results demonstrate the formation of peroxynitrite within the myocardium during inflammatory disease states, suggesting a role for peroxynitrite in inflammation-associated myocardial dysfunction.

Topics: Adolescent; Autopsy; Child; Child, Preschool; Densitometry; Female; Humans; Infant; Infant, Newborn; Male; Myocarditis; Nitrates; Nitrosation; Retrospective Studies; Sepsis; Tyrosine