3-nitrotyrosine and Respiratory-Distress-Syndrome

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

Peroxynitrite is involved in the pathogenesis of pulmonary diseases such as asthma, occupational pulmonary diseases and acute respiratory distress syndrome (ARDS) due to excessive production of nitric oxide or superoxide or both. Lornoxicam, a new oxicam derivative, is a potent anti-inflammatory agent. In this study, we evaluated the role of lornoxicam in a peroxynitrite-induced pulmonary and tracheal injury model by measuring myeloperoxidase (MPO) activity, malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) levels in lung tissue and bronco-alveolar lavage fluid. The study protocol was based on three experimental groups as treatment (T), control (C) and peroxynitrite (P). Each group was subdivided into three subgroups as 2nd, 24th and 48th hour groups. P and T groups were injected intratracheal peroxynitrite. The T group received intraperitoneal lornoxicam before and 24h after peroxynitrite installation. Tissue and serum MDA, MPO values and tissue 3-NT value of the treatment and control groups were found significantly lower than the peroxynitrite group at the 2nd, 24th and 48th hours (p<0.05). Similarly, values obtained from bronco-alveolar lavage fluid examination in the control and treatment groups were significantly less than those in the peroxynitrite group (p<0.01). Therefore, Lornoxicam has been found to be effective in attenuating peroxynitrite induced pulmonary and tracheal injury in rats.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchoalveolar Lavage Fluid; Female; Lung; Male; Malondialdehyde; Peroxidase; Peroxynitrous Acid; Piroxicam; Rats; Respiratory Distress Syndrome; Trachea; Tracheal Diseases; Tyrosine

Tyrosine nitration is a nitric oxide-derived post-translational modification of proteins. Elevated levels of specific plasma proteins modified by tyrosine nitration have been detected during acute and chronic inflammatory conditions, including acute lung injury (ALI). In the present study we examined whether circulating immunoglobulins against nitrated proteins are present in the plasma of subjects with clinically documented ALI. Affinity chromatography using covalently linked 3-nitrotyrosine was employed to identify plasma proteins that bind to this unusual amino acid. Western blotting and liquid chromatography-tandem mass spectrometry of in-gel digested protein bands revealed that the major proteins eluted from the affinity column were IgM and IgG. An enzyme-linked immunosorbent assay (ELISA) based on competition of horseradish peroxidase-derivatized 3-nitrotyrosine binding to plasma with unlabeled 3-nitrotyrosine was developed and validated. Using this ELISA, the levels of immunoglobulins that recognize 3-nitrotyrosine were significantly higher in the plasma of subjects with ALI compared with both normal control subjects and subjects with major trauma who did not develop ALI (0.36+/- 0.14 versus 0.03 +/- 0.05, and 0.25 +/- 0.15; P < 0.001 and P = 0.006, respectively). These data indicate that tyrosine-nitrated proteins induce the production of specific immunoglobulins during acute phase response and inflammation.

Topics: Adult; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulins; Ligands; Male; Reproducibility of Results; Respiratory Distress Syndrome; Risk Factors; Tyrosine; Wounds and Injuries

To clarify the role of macrophage class A scavenger receptors (SR-A, CD204) in oxidative lung injury, we examined lung tissue of SR-A deficient (SR-A(-/-)) and wild-type (SR-A(+/+)) mice in response to hyperoxic treatment. Protein levels of bronchoalveolar lavage fluid (BALF) and pulmonary oedema (wet : dry weight ratios) were higher in SR-A(-/-) mice than those in SR-A(+/+) mice. Cumulative survival was significantly decreased in SR-A(-/-) mice. However, there were no differences in BALF macrophage and neutrophil count between the two groups. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that messenger RNA (mRNA) levels of the inducible nitric oxide synthase (iNOS) were increased during hyperoxic injury, and this increase was more prominent in SR-A(-/-) mice. Expression levels of iNOS in alveolar macrophages after hyperoxia in vivo and in vitro were higher in SR-A(-/-) macrophages compared with SR-A(+/+) macrophages. Immunohistochemistry using anti-nitrotyrosine antibodies revealed distinctive oxidative stress in the injured lung in both groups, but it was more remarkable in the SR-A(-/-) mice. After hyperoxic treatment, pulmonary mRNA levels of tumour necrosis factor-alpha(TNF-alpha) were elevated more rapidly in SR-A(-/-) mice than in SR-A(+/+) mice. Together these results suggest that SR-A expression attenuates hyperoxia-induced lung injury by reducing macrophage activation.

Topics: Animals; Blotting, Western; Bronchoalveolar Lavage Fluid; Hyperoxia; Immunohistochemistry; Macrophage Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase Type II; Oxidative Stress; Respiratory Distress Syndrome; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scavenger Receptors, Class A; Tumor Necrosis Factor-alpha; Tyrosine

To monitor cascade of events following alveolar extravasation of blood due to exposure to shock wave (SW), we conducted spatiotemporal assessment of myeloperoxidase (MPO), heme oxygenase 1 (HO-1), Cu,Zn superoxide dismutase (SOD-1), transferrin (TRF), 3-nitrotyrosine (3NTyr), alveolar endothelial cadherin (VE-CDH), and the CD11b adhesion molecules on leukocytes using electron microscopy, electron paramagnetic resonance spectroscopy, immunofluorescence imaging, and immunoblotting. Accumulation of HO-1, MPO, 3NTyr, and SOD-1 in HIL at the first 12 h was associated with transmigration of inflammatory leucocytes (ILK) into hemorrhagic lesions (HLs). Biodegradation of extravasated hemoglobin (exvHb) and deposition of iron in alveoli occurred at 3-56 h post-exposure and was preceded by LKC degranulation and accumulation of MPO, HO-1, and SOD-1 in HLs. These alterations were accompanied by appearance of heme and non-heme iron complexes in HLs. A significant increase in TRF-bound [Fe(3+)] (i.e., 14.6 +/- 5.3 microM vs. 4.8 +/- 2.1 microM immediately after exposure) and non-TRF complexes of [Fe(3+)] (i.e., 4.5 +/- 1.8 microM vs. < 0.3 microM immediately after exposure) occurred at 24 h post-exposure. Transmigrations of ILK, nitroxidative stress, and iron deposition in endothelial and epithelial cells were accompanied by destruction of endothelial integrity at 3 h post-exposure, and alveolar capillary network and necrotic changes in the pulmonary epithelial cells at 24-56 h post-exposure.

Topics: Animals; Antigens, CD; Blast Injuries; Cadherins; Cell Movement; Endothelial Cells; Epithelial Cells; Heme Oxygenase-1; Hemoglobins; Hemorrhage; Inflammation; Iron; Leukocytes; Male; Oxidative Stress; Peroxidase; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Superoxide Dismutase; Superoxide Dismutase-1; Transferrin; Tyrosine

To examine the effects of combined burn and smoke inhalation injury on hypoxic pulmonary vasoconstriction, 3-nitrotyrosine formation, and respiratory function in adult sheep.. Prospective, placebo-controlled, randomized, single-blinded trial.. University research laboratory.. Twelve chronically instrumented ewes.. Following a baseline measurement, sheep were randomly allocated to either healthy controls (sham) or the injury group, subjected to a 40%, third-degree body surface area burn and 48 breaths of cotton smoke according to an established protocol (n = 6 each). Hypoxic pulmonary vasoconstriction was assessed as changes in pulmonary arterial blood flow (corrected for changes in cardiac index) in response to left lung hypoxic challenges performed at baseline and at 24 and 48 hrs postinjury.. Combined burn and smoke inhalation was associated with increased expression of inducible nitric oxide (NO) synthase, elevated NO2/NO3 (NOx) plasma levels (12 hrs, sham, 6.2 +/- 0.6; injury, 16 +/- 1.6 micromol.L; p < .01) and increased peroxynitrite formation, as indicated by augmented lung tissue 3-nitrotyrosine content (30 +/- 3 vs. 216 +/- 8 nM; p < .001). These biochemical changes occurred in parallel with pulmonary shunting, progressive decreases in Pao2/Fio2 ratio, and a loss of hypoxic pulmonary vasoconstriction (48 hrs, -90.5% vs. baseline; p < .001). Histopathology revealed pulmonary edema and airway obstruction as the morphologic correlates of the deterioration in gas exchange and the increases in airway pressures.. This study provides evidence for a severe impairment of hypoxic pulmonary vasoconstriction following combined burn and smoke inhalation injury. In addition to airway obstruction, the loss of hypoxic pulmonary vasoconstriction may help to explain why blood gases are within physiologic ranges for a certain time postinjury and then suddenly deteriorate.

Topics: Animals; Burns; Female; Hypoxia; Lung; Multiple Trauma; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Pulmonary Gas Exchange; Random Allocation; Respiratory Distress Syndrome; Sheep; Single-Blind Method; Smoke Inhalation Injury; Survival Analysis; Tyrosine; Vasoconstriction

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

To investigate the effects of inhaled nitric oxide (NO) on pulmonary inflammation, apoptosis, peroxidation and protein nitration in a rat model of acute lung injury (ALI) induced by meconium.. Twenty-four healthy male Sprague-Dawley rats were randomly devided into 3 groups (n=8): meconium-induced ALI with intratracheal instillation of 1 mL/kg saline (Mec/saline group), continuous inhalation of NO at 20 muL/L. (Mec/iNO), and the control group (control). Electromicroscopic examination was used to determine the extent of epithelial apoptosis. TUNEL was used to detect DNA fragmentation in pulmonary apoptotic cells, expressed as the apoptosis index (AI). Western blotting was used to detect pulmonary inducible NO synthase (iNOS) expression. RT-PCR was used to detect interleukin (IL)-1beta mRNA expression. Cell count in bronchoalveolar lavage (BAL), myeloperoxidase (MPO) activity, as well as malondialdehyde (MDA) and nitrotyrosine formation, the markers of toxic NO-superoxide pathway in rat lung parenchyma specimens, were also examined.. Expression of iNOS protein and IL-1beta mRNA were increased significantly in the Mec/saline group (both P<0.01) compared with the control group. BAL cell count, MPO activity, lung injury score, pulmonary AI, MDA level and nitrotyrosine formation were also increased significantly (all P<0.01). The meconium-induced iNOS protein and IL-1beta mRNA expression were inhibited significantly by NO inhalation when compared with the Mec/saline group (both P<0.01). BAL cell count, MPO activity and lung injury score were also decreased significantly (P<0.01 or P<0.05). However, there were no statistical differences in MDA level, nitrotyrosine formation or pulmonary AI between the Mec/saline and Mec/iNO groups. Electromicroscopic examination revealed a significant degree of epithelial apoptosis in both the Mec/saline and Mec/iNO groups.. Early continuous inhalation of NO 20 muL/L may protect the lungs from inflammatory injury, but does not decrease epithelial apoptosis or lung nitrotyrosine formation. Inhalation of NO alone is not associated with a detectable increase in oxidant stress.

Topics: Administration, Inhalation; Animals; Apoptosis; Interleukin-1; Lung; Male; Malondialdehyde; Meconium; Nitric Oxide; Nitric Oxide Synthase Type II; Peroxidase; Random Allocation; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; RNA, Messenger; Tyrosine

Rosiglitazone, a potent agonist of peroxisome proliferator-activated receptor (PPAR)-gamma, exerts anti-inflammatory effects in vitro and in vivo. This study was designated to determine the effects of rosiglitazone on endotoxin-induced acute lung injury in rats.. Prospective, experimental study.. University research laboratory.. Thirty-six male Wistar rats.. All the animals were randomly assigned to one of six groups (n = 6 per group) and were given either lipopolysaccharide (6 mg/kg intravenously) or saline, pretreated with rosiglitazone (0.3 mg/kg intravenously) or vehicle (10% dimethyl sulphoxide) 30 mins before lipopolysaccharide. The selective PPAR-gamma antagonist GW9662 (0.3 mg/kg intravenously) or its vehicle (10% dimethyl sulphoxide) was given 20 mins before rosiglitazone.. Endotoxemia for 4 hrs induced evident lung histologic injury and edema, both of which were significantly attenuated by rosiglitazone pretreatment. The protective effects of rosiglitazone were correlated with the reduction by 71% of the increase of myeloperoxidase activity and the reduction by 84% of the increase of malondialdehyde in the lung tissue. The pulmonary hyperproduction of nitric oxide was reduced by 82% of the increase related to lipopolysaccharide challenge. Pretreatment with rosiglitazone also markedly suppressed lipopolysaccharide-induced expression of inducible nitric oxide synthase messenger RNA and protein in the lung, as demonstrated by reverse transcription-polymerase chain reaction or Western blot analysis. Immunohistochemical analysis revealed that rosiglitazone inhibited the formation of nitrotyrosine, a marker for peroxynitrite reactivity, in the lung tissue. In addition, the specific PPAR-gamma antagonist GW9662 antagonized the effects of rosiglitazone.. This study provides evidence, for the first time, that the PPAR-gamma agonist rosiglitazone significantly reduces endotoxin-induced acute lung injury in rats.

Topics: Anilides; Animals; Endotoxemia; Lipopolysaccharides; Lung; Male; Nitric Oxide; Nitric Oxide Synthase Type II; PPAR gamma; Rats; Rats, Wistar; Respiratory Distress Syndrome; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Tyrosine

Topics: Animals; Humans; Lung; Nitric Oxide; Nitric Oxide Synthase Type II; PPAR gamma; Rats; Respiratory Distress Syndrome; Rosiglitazone; Thiazolidinediones; Tyrosine

We have previously demonstrated the protective role of urinary trypsin inhibitor (UTI) against acute inflammatory lung injury induced by lipopolysaccharide (LPS) using UTI-deficient (-/-) mice and corresponding wild-type (WT) mice. The protection was mediated, at least partly, through inhibition of the enhanced local expression of proinflammatory cytokines, chemokines, and intercellular adhesion molecule-1. In the present study, we addressed whether UTI regulates oxidative stress generated by LPS challenge in the lung. UTI (-/-) and WT mice were treated intratracheally with vehicle or LPS (125 microg/kg). After LPS challenge in both genotypes of mice, the lung levels of mRNA for inducible nitric oxide synthase and hemo oxygenase-1 were elevated, but to a greater extent in UTI (-/-) mice than in WT mice. Immunohistochemistry showed that the formations of 8-hydroxy-2'-deoxyguanosine and nitrotyrosine in the lung were more intense in UTI (-/-) mice than in WT mice after LPS challenge. These results indicate that endogenous UTI is protective against acute lung injury induced by bacterial endotoxin, at least partly, via the antioxidative properties.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Deoxyguanosine; Gene Expression; Glycoproteins; Heme Oxygenase-1; Lipopolysaccharides; Lung; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Oxidative Stress; Respiratory Distress Syndrome; Tyrosine

Acute lung injury (ALI) is characterized by increased alveolar cytokines, inflammatory cell infiltration, oxidative stress, and alveolar cell apoptosis. Previous work suggested that xanthine oxidoreductase (XOR) may contribute to oxidative stress in ALI as a product of the vascular endothelial cell. We present evidence that cytokine induced lung inflammation and injury involves activation of XOR in the newly recruited mononuclear phagocytes (MNP). We found that XOR was increased predominantly in the MNP that increase rapidly in the lungs of rats that develop ALI following intratracheal cytokine insufflation. XOR was recovered from the MNP largely converted to its oxygen radical generating, reversible O-form, and alveolar MNP exhibited increased oxidative stress as evidenced by increased nitrotyrosine staining. Cytokine insufflation also increased alveolar cell apoptosis. A functional role for XOR in cytokine-induced inflammation was demonstrated when feeding rats two different XOR inhibitors, tungsten and allopurinol, decreased MNP XOR induction, nitrotyrosine staining, inflammatory cell infiltration, and alveolar cell apoptosis. Transfer of control or allopurinol treated MNP into rat lungs confirmed a specific role for MNP XOR in promoting lung inflammation. These data indicate that XOR can contribute to lung inflammation by its expression and conversion in a highly mobile inflammatory cell population.

Topics: Allopurinol; Animals; Apoptosis; Cell Differentiation; Cytokines; Enzyme Induction; Enzyme Inhibitors; Interferon-gamma; Interleukin-1; Lung; Male; Phagocytes; Pneumonia; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Tungsten; Tyrosine; Xanthine Oxidase

Nuclear factor-kappaB (NF-kappaB) is a transcription factor which plays a pivotal role in the induction of genes involved in the response to injury and inflammation. Calpain I inhibitor is a potent antioxidant which is an effective inhibitor of NF-kappaB. This study examined whether the postulate that calpain I inhibitor attenuates experimental acute pancreatitis.. In a murine model we measured NF-kappaB activation, expression of intercellular adhesion molecule (ICAM) 1, nitrotyrosine, inducible nitric oxide synthase (iNOS), nuclear enzyme poly(ADP-ribose) synthetase (PARS), myeloperoxidase, malondialdehyde, amylase and lipase and determined histological evidence of lung and pancreas injury in four groups: control (saline only), cerulein, calpain I inhibitor plus cerulein and calpain I inhibitor plus saline.. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterised by oedema, neutrophil infiltration, tissue haemorrhage and necrosis and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine, iNOS and PARS in the pancreas and lung of cerulein-treated mice. In contrast, pre-treatment with calpain I inhibitor markedly reduced: the degree of pancreas and lung injury; upregulation/expression of ICAM-1; staining for iNOS, nitrotyrosine and PARS; and lipid peroxidation. Additionally, calpain I inhibitor treatment significantly prevented the activation of NF-kappaB as suggested by the inhibition of IkappaB-alpha; degradation in the pancreas tissues after cerulein administration.. Taken together, our results clearly demonstrate that prevention of the activation of NF-kappaB by calpain I inhibitor ameliorates experimental murine acute pancreatitis.

Topics: Acute Disease; Analysis of Variance; Animals; Blotting, Western; Calpain; Ceruletide; Disease Models, Animal; Immunohistochemistry; Intercellular Adhesion Molecule-1; Lipid Peroxidation; Male; Mice; NF-kappa B; Nitric Oxide Synthase; Pancreatitis; Poly(ADP-ribose) Polymerases; Random Allocation; Respiratory Distress Syndrome; Tyrosine

Ischemia-reperfusion (IR) is characterized by microvascular disfunction and this involves both direct effected organ and remote organ by systemic inflammatory response. These remote effects of IR are most frequently observed in the lung and cardiovascular system. In this study we aim to determine lung damage which induced IR, and endothelial and microvascular disfunction using nitrosative markers. Previous studies suggest that caffeic acid phenethyl ester (CAPE) has some antioxidant effects. Therefore, we also investigated whether it has a role associated with nitric oxide during IR condition. Twenty-two adult male Wistar rats were divided into three groups: control (n= 7), IR (n= 7), and CAPE + IR (n= 8). 8 h IR period was performed on right hindlimb in the IR and the CAPE with IR group. In the CAPE with IR group, animals received CAPE 10 microM 1 h before the reperfusion. At the end of the reperfusion period, blood, bronchoalveolar lavage (BAL) and lung tissue were obtained, and were used for biochemical and histopathological examination. There was a significantly elevation in serum nitrate, BAL MPO, and leukocyte infiltration in the lung in the IR group compared to the CAPE + IR group. But, serum nitrite and lung 3-NT levels were not different between these groups. While nitrate (p< 0.0001), MPO (p< 0.0001) and leukocyte infiltration (chi2= 27.163, p= 0.0001), reduce by using CAPE before reperfusion, tissue 3-NT levels did not change. In conclusion, peripheral IR leads to systemic inflammatory responses and endothelial disfunction-induced NO production, and these harmful effects may reduced by CAPE.

Topics: Animals; Bronchoalveolar Lavage Fluid; Caffeic Acids; Capillary Permeability; Cytokines; Hindlimb; Lung; Male; Malondialdehyde; Neutrophil Infiltration; NF-kappa B; Phenylethyl Alcohol; Rats; Rats, Wistar; Reperfusion Injury; Respiratory Distress Syndrome; Tourniquets; Tyrosine

Ischemia-reperfusion (I/R) of remote organs is a common cause of lung injury. We observed that lung injury after partial hepatic I/R in mice coincides with the appearance of 3-nitrotyrosine (NT) in the lung tissue, a marker of peroxynitrite involvement and oxidant stress. Peroxynitrite can cause mitochondrial dysfunction by inactivation of manganese superoxide dismutase (MnSOD), the major antioxidant enzyme in mitochondria. Our aims were to examine whether pulmonary MnSOD is a target of nitration following hepatic I/R and whether nitrated MnSOD (N-MnSOD) correlates with acute lung injury.. Five 20-25-g male C57BL/6 mice underwent laparotomy, and atraumatic occlusion of the portal and arterial blood supply to the upper three lobes of the liver for 90 min. This warm ischemic period was followed by 4 h of reperfusion, and the animals were then euthanized. Lung injury was assessed by LDH and protein levels in bronchoalveolar lavage (BAL) fluid. Pulmonary MnSOD activity in pulmonary homogenates was measured by the cytochrome c reduction method. The presence of N-MnSOD was determined by immunoprecipitation (IP) and Western Blot analysis. Controls (N = 5) underwent sham operation.. Elevated plasma transaminases confirmed hepatic injury. Lung injury was demonstrated by elevation in BAL protein and LDH levels (495.7 (48.4) versus 644.9 (37.3) [p < 0.05] and 56.5 (11.8) versus 345.2 (80) [p < 0.01], respectively). Immunoprecipitation and Western blot demonstrated N-MnSOD in the lung tissue of I/R animals but not controls. MnSOD activity decreased following I/R (8.1 (0.7) versus 10.8 (0.3) [p < 0.05]).. Pulmonary MnSOD is both nitrated and inactivated following hepatic I/R and is associated with acute lung injury. These findings suggest that MnSOD incapacitance may contribute to I/R-induced lung injury and provide a therapeutic target in attenuating multisystem injury following hepatic I/R.

Topics: Animals; Biomarkers; Biopsy, Needle; Disease Models, Animal; Immunohistochemistry; Ischemia; Liver; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Reperfusion; Reperfusion Injury; Respiratory Distress Syndrome; Sensitivity and Specificity; Severity of Illness Index; Superoxide Dismutase; Tyrosine

Peroxynitrite is a potent cytotoxic free radical produced by the reaction of nitric oxide with the superoxide ion produced in conditions of oxidative stress. The purpose of the study was to examine the role of this reactive nitrogen species in lung ischemia-reperfusion injury.. Left lungs of male Long-Evans rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received FP-15 (a water-soluble iron containing metalloporphyrin that acts as a peroxynitrite decomposition catalyst). Injury was quantitated in terms of tissue neutrophil accumulation (myeloperoxidase content) and vascular permeability ((125)I bovine serum albumin [BSA] extravasation) and bronchoalveolar lavage cytokine, transcriptional factor and leukocyte content. Separate tissue samples were processed for immunohistology and nuclear protein analysis.. Lung vascular permeability was reduced in treated animals by 61% compared with control animals (p < 0.005). The protective effects of enhanced peroxynitrite decomposition correlated with a 72% reduction in tissue myeloperoxidase content (p < 0.001) and marked reductions in brochoalveolar lavage leukocyte accumulation. This correlated positively with the diminished expression of pro-inflammatory chemokines and nuclear transcription factors.. The deleterious effects of lung ischemia-reperfusion injury are in part mediated by the formation of peroxynitrite, as enhanced decomposition of this species is protective in this model. The development of potent water-soluble decomposition catalysts represents a potentially useful therapeutic tool in the prevention of lung ischemia-reperfusion injury after lung transplantation.

Topics: Animals; Bronchoalveolar Lavage Fluid; Capillary Permeability; Chemokines; Disease Models, Animal; Immunohistochemistry; Inflammation Mediators; Macrophages, Alveolar; Male; Models, Cardiovascular; NF-kappa B; Peroxidase; Peroxynitrous Acid; Rats; Rats, Long-Evans; Reactive Nitrogen Species; Reperfusion Injury; Respiratory Distress Syndrome; Severity of Illness Index; Statistics as Topic; Transcription Factor AP-1; Transcriptional Activation; Tyrosine

To investigate the effects of treatment with propofol administration at different time point in acute lung injury of endotoxin-induced shock rats.. 76 male wistar rats were randomly assigned to five groups: A) control group; B) endotoxemic group, receiving intravenous lipopolysaccharide (LPS) 8 mg.kg-1; C) pretreatment group, treated identically to endotoxemic group with the additional administration of propofol (5 mg.kg-1 bolus, followed by infusion at 10 mg.kg-1.h-1) of 1 hr prior to the injection of LPS; D) simultaneously treatment group, treated identically to endotoxemic group with the additional administration of propofol simultaneously with the injection of LPS; E) post-treatment group, which was treated identically to endotoxemic group except for administration of propofol 1 hr after the injection of LPS. PaO2, pH, MAP and survival rate were recorded and plasma NO, TNF-alpha were measured during 5-hr after the injection of LPS. After the rats were killed, lung tissue was sampled to measured expression of inducible nitric oxide synthase (iNOS), nitrotyrosine (NT), myeloperoxidase (MPO) activity, malondialdehyde (MDA), wet-to-dry lung weight ratio (W/D), and pulmonary permeability index (PPI).. Compared with the endotoxemic group, both the pretreatment and simultaneously treatment groups, significantly improved PaO2, pH, MAP and 5th hour survival rate of rats, and attenuated endotoxin-induced increased iNOSmRNA, NT expression, MPO activity and MDA level in lung tissue, and decreased pulmonary microvascular permeability, TNF-alpha, NO in plasma. But these beneficial efficacies were blunted in the post-treatment group.. These findings showed that propofol administration may provide protective effects on acute lung injury in endotoxin-induced shock.

Topics: Analysis of Variance; Anesthetics, Intravenous; Animals; Blotting, Western; Hemodynamics; Lipopolysaccharides; Lung; Male; Malondialdehyde; Nitric Oxide; Organ Size; Peroxidase; Propofol; Random Allocation; Rats; Rats, Wistar; Respiratory Distress Syndrome; Reverse Transcriptase Polymerase Chain Reaction; Survival Rate; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine

In the present study, we evaluated the effect of Celecoxib, a selective COX-2 inhibitor, in an acute model of lung injury induced by carrageenan administration in the rats. Injection of carrageenan into the pleural cavity of rats elicited an acute inflammatory response characterized by: fluid accumulation in the pleural cavity which contained a large number of polymorphonuclear neutrophils (PMNs) as well as an infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of prostaglandin E(2) (PGE(2)), tumor necrosis factor alpha (TNFalpha), and interleukin-1beta. All parameters of inflammation were attenuated by Celecoxib. Furthermore, carrageenan induced an upregulation of the adhesion molecules ICAM-1 and P-selectin, as well as nitrotyrosine and poly(ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine and PARS was reduced by Celecoxib. These results clearly confirmed that COX-2 plays a critical role in the development of the inflammatory response by altering key components of the inflammatory cascade. Therefore, selective inhibitor of COX-2 such as Celecoxib, offers a therapeutic approach for the management of various inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Celecoxib; Cytokines; Disease Models, Animal; Erythrocytes; Intercellular Adhesion Molecule-1; Male; P-Selectin; Pleurisy; Poly(ADP-ribose) Polymerases; Pyrazoles; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Sulfonamides; Tyrosine

Topics: Humans; Lung; Nitric Oxide; Prognosis; Respiratory Distress Syndrome; Tyrosine

Nitric oxide (NO) end-products (nitrate and nitrite) are present in bronchoalveolar lavage (BAL) fluid of patients with inflammatory lung diseases. Reactive oxygen-nitrogen intermediates damage macromolecules by oxidation or nitration of critical residues in proteins. The goal of this study was to measure NO end-products (nitrate+ nitrite), in BAL fluid before and after the onset of acute respiratory distress syndrome (ARDS) and to determine if these products are associated with expression of inducible nitric oxide synthase enzyme (iNOS) in BAL cells and nitration of BAL proteins. We performed bronchoalveolar lavage (BAL) in patients at risk for ARDS (n = 19), or with ARDS (n = 41) on Days 1, 3, 7, 14, and 21 after onset, and measured total nitrite (after reducing nitrate to nitrite) and protein-associated nitrotyrosine concentration in each BAL fluid sample. Cytospin preparations of BAL cells were analyzed by immunocytochemistry for iNOS and nitrotyrosine. Nitrate+nitrite were detected in BAL fluid from patients at risk for ARDS, and for as long as 21 d after the onset of ARDS. Nitrotyrosine was detectable in all BAL fluid samples for as long as 14 d after the onset of ARDS (range, 38.8 to 278.5 pmol/mg of protein), but not in BAL of normal volunteers. Alveolar macrophages of patients with ARDS were positive for iNOS and nitrotyrosine, and remained positive for as long as 14 d after onset of ARDS. The BAL nitrate+nitrite did not predict the onset of ARDS, but the concentration was significantly higher on Days 3 and 7 of ARDS in patients who died. Thus, NO end products accumulate in the lungs before and after onset of ARDS; iNOS is expressed at high levels in AM during ARDS; and nitration of intracellular and extracellular proteins occurs in the lungs in ARDS. The data support the concept that NO-dependent pathways are important in the lungs of patients before and after the onset of ARDS.

Topics: Adult; Bronchoalveolar Lavage Fluid; Female; Follow-Up Studies; Humans; Intensive Care Units; Macrophages, Alveolar; Male; Middle Aged; Nitric Oxide; Positive-Pressure Respiration; Reference Values; Respiratory Distress Syndrome; Tyrosine

Alveolar type II cells (type II cells) play a crucial role in the progression and repair of lung inflammation and injury. We investigated whether inducible nitric oxide synthase (iNOS) was expressed and nuclear factor-kappaB (NF-kappaB) was activated in type II cells in lung injury. After injecting lipopolysaccharide (LPS) or saline in the rat, the lungs were excised and type II cells were isolated. iNOS and its mRNA were expressed both in lung tissue and isolated type II cells in response to LPS. The lungs from saline-treated rats showed only minimal expression of iNOS. Electrophoretic mobility shift assay revealed that expression of NF-kappaB in the nuclear extracts was augmented by LPS, and p5O/NFkappaB was expressed in type II cells in LPS-treated rats. Intraperitoneal dexamethasone almost completely inhibited the iNOS expression and attenuated the activation of NF-kappaB in the LPS-treated lung. These findings suggest that type II cells can be a source of NO production in lung injury,and that the effects of corticosteroids may be in part through inhibition of both iNOS expression and NF-kappaB activation.

Topics: Animals; Dexamethasone; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Enzymologic; Glucocorticoids; Immunohistochemistry; Lipopolysaccharides; Male; NF-kappa B; NF-kappa B p50 Subunit; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; RNA, Messenger; Specific Pathogen-Free Organisms; Tyrosine

The present study identifies proteins modified by nitration in the plasma of patients with ongoing acute respiratory distress syndrome (ARDS). The proteins modified by nitration in ARDS were revealed by microsequencing and specific antibody detection to be ceruloplasmin, transferrin, alpha(1)-protease inhibitor, alpha(1)-antichymotrypsin, and beta-chain fibrinogen. Exposure to nitrating agents did not deter the chymotrypsin-inhibiting activity of alpha(1)-antichymotrypsin. However, the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of alpha(1)-protease inhibitor were reduced to 50.3 +/- 1.6 and 60.3 +/- 5.3% of control after exposure to the nitrating agent. In contrast, the rate of interaction of fibrinogen with thrombin was increased to 193.4 +/- 8.5% of the control value after exposure of fibrinogen to nitration. Ferroxidase activity of ceruloplasmin and elastase-inhibiting activity of the alpha(1)-protease inhibitor in the ARDS patients were significantly reduced (by 81 and 44%, respectively), whereas alpha(1)-antichymotrypsin activity was not significantly altered. Posttranslational modifications of plasma proteins mediated by nitrating agents may offer a biochemical explanation for the reported diminished ferroxidase activity, elevated levels of elastase, and fibrin deposits detected in patients with ongoing ARDS.

Topics: Acute Disease; Adult; alpha 1-Antichymotrypsin; Blood Proteins; Blotting, Western; Carbon Dioxide; Ceruloplasmin; Enzyme Activation; Fibrinogen; Humans; In Vitro Techniques; Nitrates; Nitric Oxide; Oxidative Stress; Precipitin Tests; Respiratory Distress Syndrome; Superoxides; Tyrosine

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

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

To assess the contribution of poly (adenosine 5'-diphosphate ribose) synthetase (PARS) to the development of bacterial lipopolysaccharide (LPS)-induced acute lung injury and vascular failure in pigs.. Four groups of anesthetized, paralyzed, and mechanically ventilated domestic white pigs. Group 1 served as control, whereas Escherichia coli LPS (20 microg/kg/h) was continuously infused in group 2. Group 3 received 20 mg/kg injection of 3-aminobenzamide (a selective inhibitor of PARS activity) 15 minutes before LPS infusion. Only 3-aminobenzamide and not LPS was injected in group 4. All animals were examined for 180 minutes. Systemic and pulmonary hemodynamics and lung mechanics were measured during the experimental period. Lung wet/dry ratio, bronchoalveolar lavage (BAL) protein levels and cell counts and lung nitrotyrosine (footprint of peroxynitrite) immunostaining were also measured in a few animals.. LPS infusion evoked a progressive decline in systemic arterial pressure, a small increase in cardiac output, and biphasic elevation of pulmonary arterial pressure. Lung compliance declined progressively, whereas lung and total respiratory resistance rose significantly after LPS infusion. Prominent nitrotyrosine immunostaining was detected around small airways and pulmonary endothelium of LPS-infused animals. No significant changes in lung wet/dry ratio and BAL protein levels and cell counts were produced by LPS infusion. Pretreatment with 3-aminobenzamide did not alter the systemic and pulmonary hemodynamic responses to LPS infusion but eliminated the rise in pulmonary and total respiratory resistance.. We concluded that PARS activation plays an important role in the changes of lung mechanics associated with LPS-induced acute lung injury but had no role in vascular failure.

Topics: Animals; Benzamides; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Enzyme Inhibitors; Escherichia coli Infections; Female; Hemodynamics; Immunohistochemistry; Lipopolysaccharides; Male; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proteins; Pulmonary Circulation; Respiratory Distress Syndrome; Respiratory Mechanics; Shock, Septic; Swine; Tyrosine

Nitrated tyrosine, implicated in protein dysfunction, is increased in various tissues in association with diverse pathological processes. Angiotensin converting enzyme (ACE) is a luminal vascular endothelial enzyme whose dysfunction is an early sign of endothelial injury. ACE contains a tyrosine critical for its enzymatic activity. Others have shown that nitrite exacerbates the ACE dysfunction of cultured endothelial cells in contact with activated polymorphonuclear neutrophils (PMN). We hypothesized that exogenous nitrite would enhance endothelial ACE dysfunction associated with PMN activation in the isolated lung. Rats received lipopolysaccharide (LPS) 2 h prior to isolated lung perfusion with Ficoll containing buffer. Either formyl-Met-Leu-Phe (fMLP, 10(-7) M) or phorbol myristate acetate (PMA, 10(-7) M) was used to activate PMN in lungs treated or not treated with 300-microM nitrite. A first pass indicator dilution method and first order reaction kinetics were used to determine ACE activity, while lung Ficoll content served as an index of vascular permeability. Both fMLP and PMA decreased endothelial ACE activity and increased pulmonary artery pressure, edema and vascular permeability. Exogenous nitrate did not potentiate the decrease in ACE activity, the lung injury or nitrotyrosine immunoreactivity of lung homogenates. In contrast to observations in cultured endothelial cells, our findings in the whole lung are compatible with the speculation of others that the rat lung has an unidentified factor, which minimizes accumulation of nitrated proteins.

Topics: Animals; Blood Pressure; Capillary Permeability; Carcinogens; Dimethyl Sulfoxide; Endothelium, Vascular; Free Radical Scavengers; Lipopolysaccharides; Lung; Male; Models, Animal; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitrites; Peptidyl-Dipeptidase A; Rats; Rats, Wistar; Respiratory Distress Syndrome; Tetradecanoylphorbol Acetate; Tyrosine

Topics: Animals; Lung; Oxygen; Rats; Respiratory Distress Syndrome; Sodium-Potassium-Exchanging ATPase; Tyrosine

Inhaled nitric oxide (.NO) is used to improve gas exchange and reduce pulmonary vascular resistance (PVR) in patients with the acute respiratory distress syndrome (ARDS). Although controlled studies have shown no survival benefit, some investigators have suggested that inhaled.NO may have antiinflammatory properties under these circumstances. In contrast, others have speculated that.NO given by inhalation could be cytotoxic, as it combines with superoxide at near diffusion-limited rates to produce the highly reactive oxidant peroxynitrite (ONOO(-)). We therefore quantified levels of 3-nitrotyrosine, a marker for ONOO(-) formation, in bronchoalveolar lavage fluid (BAL) from patients with ARDS receiving inhaled.NO, and from patients with comparable lung injury who were not so treated. We also measured levels of 3-chlorotyrosine as an index of neutrophil activation to assess indirectly the effects of inhaled.NO on lung inflammation. Patients receiving .NO had increased levels of 3-nitrotyrosine (6.76 +/- 2.79 versus 0.4 +/- 0.15 nmol/mg of protein, p < 0.05) and 3-chlorotyrosine (7.97 +/- 2.74 versus 1. 53 +/- 1.09 nmol/mg of protein, p < 0.05) in BAL protein compared with controls. In patients with ARDS, inhaled.NO increases the formation of 3-nitrotyrosine and is accompanied by an increase in levels of 3-chlorotyrosine (a marker of neutrophil activation). The possible long-term consequences of these observations remain to be evaluated.

Topics: Adult; Aged; Bronchoalveolar Lavage Fluid; Bronchodilator Agents; Cell Count; Chromatography, High Pressure Liquid; Female; Humans; Male; Middle Aged; Neutrophil Activation; Nitric Oxide; Respiratory Distress Syndrome; Tyrosine

To assess the degree, source, and patterns of oxidative damage to bronchoalveolar lavage proteins as a modification of amino acid residues in patients with acute respiratory distress syndrome (ARDS).. Prospective, controlled study.. Adult intensive care unit of a postgraduate teaching hospital.. Twenty-eight patients with established ARDS were studied and compared with six ventilated patients without ARDS and 11 normal healthy controls.. Supportive techniques appropriate to ARDS.. Evidence of oxidative modification of bronchoalveolar lavage fluid protein, indicative of the production of specific reactive oxidizing species, was sought using a high-performance liquid chromatography technique. Bronchoalveolar lavage fluid samples from patients with ARDS, ventilated intensive care controls, and normal healthy controls were analyzed. Concentrations of orthotyrosine were significantly higher in the ARDS group than in either control group (7.98 + 3.78 nmol/mg for ARDS, 0.67 + 0.67 for ventilated controls, and 0.71 + 0.22 for healthy controls; p < .05). Chlorotyrosine concentrations were also significantly increased in the ARDS group over either control group (4.82 + 1.07 nmol/mg for ARDS, 1.55 + 1.34 for ventilated controls, and 0.33 + 0.12 for healthy controls; p < .05). Nitrotyrosine concentrations were similarly significantly increased in the ARDS groups compared with each control group (2.21 + 0.65 nmol/mg for ARDS, 0.29 + 0.29 for ventilated controls, and 0.06 + 0.03 for healthy controls; p < .05). Chlorotyrosine and nitrotyrosine concentrations showed significant correlations with myeloperoxidase concentrations in bronchoalveolar lavage fluid, measured using an enzyme-linked immunosorbent assay in patients with ARDS. These findings suggest a possible relationship between inflammatory cell activation, oxidant formation, and damage to proteins in the lungs of these patients. Overall, our data strongly suggest heightened concentrations of oxidative stress in the lungs of patients with ARDS that lead to significantly increased oxidative protein damage.

Topics: Adolescent; Adult; Aged; Biomarkers; Bronchoalveolar Lavage Fluid; Case-Control Studies; Chromatography, High Pressure Liquid; Female; Humans; Hydroxyl Radical; Hydroxylation; Hypochlorous Acid; Linear Models; Male; Middle Aged; Neutrophil Activation; Neutrophils; Nitrates; Oxidants; Oxidative Stress; Proteins; Respiration, Artificial; Respiratory Distress Syndrome; Statistics, Nonparametric; Tyrosine

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

Topics: Animals; Dogs; Enzyme Inhibitors; Guanidines; Hemodynamics; Isothiuronium; Lipopolysaccharides; Lung; Neutrophils; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pulmonary Gas Exchange; Respiratory Distress Syndrome; Tyrosine

The role of nitric oxide (NO) in lung injury remains unclear. Both beneficial and detrimental roles have been proposed. In this study, we used mutant mice lacking the inducible nitric oxide synthase (iNOS) to assess the role of this isoform in sepsis-associated lung injury. Wild-type and iNOS knockout mice were injected with either saline or Escherichia coli endotoxin (LPS) 25 mg/kg and killed 6, 12, and 24 h later. Lung injury was evaluated by measuring lactate dehydrogenase activity in the bronchoalveolar lavage, pulmonary wet/dry ratio, and immunostaining for nitrotyrosine formation. In the wild-type mice, LPS injection elicited more than a 3-fold rise in lactate dehydrogenase activity, a significant rise in lung wet/dry ratio and extensive nitrotyrosine staining in large airway and alveolar epithelium, macrophages, and pulmonary vascular cells. This was accompanied by induction of iNOS protein and increased lung nitric oxide synthase activity. By comparison, LPS injection in iNOS knockout mice elicited no iNOS induction and no significant changes in lung NOS activity, lactate dehydrogenase activity, lung wet/dry ratio, or pulmonary nitrotyrosine staining. These results indicate that mice deficient in iNOS gene are more resistant to LPS-induced acute lung injury than are wild-type mice.

Topics: Animals; Blood Vessels; Bronchi; Bronchoalveolar Lavage Fluid; Coloring Agents; Disease Models, Animal; Endotoxins; Epithelium; Escherichia coli; Female; Follow-Up Studies; Isoenzymes; L-Lactate Dehydrogenase; Lipopolysaccharides; Lung; Macrophages, Alveolar; Male; Mice; Mice, Knockout; Mutation; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organ Size; Pulmonary Alveoli; Respiratory Distress Syndrome; Tyrosine; Vasodilator Agents