3-nitrotyrosine and Lung-Diseases

Since the discovery of nitric oxide (NO), an intracellular signal transmitter, the role of NO has been investigated in various organs. In the respiratory system, NO derived from the constitutive type of NO synthase (cNOS, NOS1, NOS3) induces bronchodilation and pulmonary vasodilatation to maintain homeostasis. In contrast, the roles of excessive NO derived from the inducible type of NOS (iNOS, NOS2) in airway and lung inflammation in inflammatory lung diseases including bronchial asthma and chronic obstructive pulmonary disease (COPD) are controversial. In these inflammatory lung diseases, excessive nitrosative stress has also been observed. In asthma, some reports have shown that nitrosative stress causes airway inflammation, airway hyperresponsiveness, and airway remodeling, which are the features of asthma, whereas others have demonstrated the anti-inflammatory role of NO derived from NOS2. In the case of refractory asthma, more nitrosative stress has been reported to be observed in such airways compared with that in well-controlled asthmatics. In COPD, reactive nitrogen species (RNS), which are NO and NO-related molecules including nitrogen dioxide and peroxynitrite, cause lung inflammation, oxidative stress, activation of matrix metalloproteinase, and inactivation of antiprotease, which are involved in the pathophysiology of the disease. In the present paper, we review the physiological and pathophysiological effects of NO and NO-related molecules in the respiratory system and in inflammatory lung diseases.

Topics: Animals; Asthma; Humans; Inflammation; Lung; Lung Diseases; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; S-Nitrosothiols; Signal Transduction; Tyrosine

The formation of peroxynitrite and nitrotyrosine was examined in a variety of in vitro and in vivo animal models and its relation to cell or tissue damage was examined. polymorphonuclear leukocyte (PMN)-induced injury to cardiac myocytes endothelial cells, activated PMN produced peroxynitrite. Peroxynitrite appears to be responsible for the injury but it was not a major mediator of endothelial cell injury. In the experiment of ischemia-reperfusion injury of the rat brain nitrotyrosine was formed in the peri-infarct and core-of infarct regions. The degradation curve of nitrotyrosine revealed that its t(1/2) was about 2.2 hours. In the radiation-induced lung injury of rats, nitrotyrosine was also formed but it was not the sole mechanism for the injury. Levels of nitrotyrosine correlated with the severity of myocardial dysfunction in the canine model of cytokine-induced cardiac injury. Inhibition of NO generation abolished the formation of peroxynitrite and nitrotyrosine in all experiments. In conclusion; although nitrotyrosine is formed in a variety of pathological conditions where the generation of NO is increased, its presence does not always correlate with the severity of injury.

Topics: Animals; Brain Ischemia; Cell Survival; Cells, Cultured; Coculture Techniques; Disease; Dogs; Endothelium, Vascular; Lung Diseases; Male; Myocardium; Neutrophils; Radiation Injuries; Rats; Reperfusion Injury; Tyrosine

Topics: Animals; Free Radicals; Humans; Inflammation; Lung; Lung Diseases; Nitric Oxide; Tyrosine

The diffuse parenchymal lung diseases (DPLDs) are a group of clinicopathological entities which have recently undergone reclassification. The commonest type of idiopathic DPLD is interstitial pulmonary fibrosis (PF), which is histologically characterized by usual interstitial pneumonia (UIP), with inflammatory changes in the alveoli and subsequent collagen deposition. A similar type of inflammatory change can also be seen with connective tissue disorders. Many mediators are involved, but it is difficult to study these in a non-invasive manner in patients. The aim of the study detailed in this paper was to investigate inflammatory and oxidative stress biomarkers in PF and correlate these with lung function. 20 PF patients and 20 controls participated in the study. Exhaled breath condensate (EBC) was collected over 10 min using a refrigerated condenser, after fractional exhaled nitric oxide (FeNO) and carbon monoxide (eCO) measurement. EBC total nitrogen oxides (NOx), hydrogen peroxide (H(2)O(2)), 8-isoprostane (8-iso), 3-nitrotyrosine (3-NT), pH and total protein were measured. EBC biomarkers were significantly raised in PF compared with controls: EBC 3-NT (2.5 (0.7-8.9) versus 0.3 (0.1-1.1) ng ml(-1), p = 0.02); pH (7.6 ± 0.3 versus 7.4 ± 0.2, p = 0.004); 8-isoprostane (0.2 (0.1-0.4) versus 0.08 (0.04-0.2) ng ml(-1), p = 0.04) and total protein (24.7 ± 21.1 versus 10.7 ± 7.0 µg ml(-1), p = 0.008). FeNO and eCO were also increased (8.6 (7.1-10.4) versus 6.6 (5.6-7.8) ppb, p = 0.04, and 4.5 ± 1.7 versus 2.7 ± 0.7 ppm, p = 0.001, respectively), but no significant differences were found for NOx or H(2)O(2). In conclusion, inflammatory and oxidative stress biomarkers are raised in patients with PF compared with controls. EBC may be useful for detecting and monitoring lung inflammation in PF.

Topics: Biomarkers; Breath Tests; Carbon Monoxide; Exhalation; Female; Humans; Hydrogen Peroxide; Lung Diseases; Male; Nitric Oxide; Oxidative Stress; Pulmonary Fibrosis; Spirometry; Tyrosine

More than 100 million people in the United States live in areas that exceed current ozone air quality standards. In addition to its known pulmonary effects, environmental ozone exposures have been associated with increased hospital admissions related to cardiovascular events, but to date, no studies have elucidated the potential molecular mechanisms that may account for exposure-related vascular impacts. Because of the known pulmonary redox and immune biology stemming from ozone exposure, we hypothesized that ozone inhalation would initiate oxidant stress, mitochondrial damage, and dysfunction within the vasculature. Accordingly, these factors were quantified in mice consequent to a cyclic, intermittent pattern of ozone or filtered air control exposure. Ozone significantly modulated vascular tone regulation and increased oxidant stress and mitochondrial DNA damage (mtDNA), which was accompanied by significantly decreased vascular endothelial nitric oxide synthase protein and indices of nitric oxide production. To examine influences on atherosclerotic lesion formation, apoE-/- mice were exposed as above, and aortic plaques were quantified. Exposure resulted in significantly increased atherogenesis compared with filtered air controls. Vascular mitochondrial damage was additionally quantified in ozone- and filtered air-exposed infant macaque monkeys. These studies revealed that ozone increased vascular mtDNA damage in nonhuman primates in a fashion consistent with known atherosclerotic lesion susceptibility in humans. Consequently, inhaled ozone, in the absence of other environmental toxicants, promotes increased vascular dysfunction, oxidative stress, mitochondrial damage, and atherogenesis.

Topics: Air Pollutants; Animals; Aorta; Atherosclerosis; Blood Pressure; DNA Damage; DNA, Mitochondrial; Heart Rate; Lung Diseases; Macaca mulatta; Male; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Diseases; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type III; Nitrites; Oxidants; Oxidative Stress; Ozone; Superoxide Dismutase; Tyrosine

Asbestos induces generation of reactive oxygen and nitrogen species in laboratory studies. Several such species can be measured non-invasively in humans in exhaled breath condensate (EBC) but few have been evaluated. This study aimed to assess oxidative stress and lung inflammation in vivo.. Eighty six men were studied: sixty subjects with asbestos-related disorders (asbestosis: 18, diffuse pleural thickening (DPT): 16, pleural plaques (PPs): 26) and twenty six age- and gender-matched normal individuals.. Subjects with asbestosis had raised EBC markers of oxidative stress compared with normal controls [8-isoprostane (geometric mean (95% CI) 0.51 (0.17-1.51) vs 0.07 (0.04-0.13) ng/ml, p<0.01); hydrogen peroxide (13.68 (8.63-21.68) vs 5.89 (3.99-8.69) microM, p<0.05), as well as increased EBC total protein (17.27 (10.57-28.23) vs 7.62 (5.13-11.34) microg/ml, p<0.05), and fractional exhaled nitric oxide (mean+/-SD) (9.67+/-3.26 vs 7.57+/-1.89ppb; p<0.05). EBC pH was lower in subjects with asbestosis compared with subjects with DPT (7.26+/-0.31 vs 7.53+/-0.24; p<0.05). There were no significant differences in exhaled carbon monoxide, EBC total nitrogen oxides and 3-nitrotyrosine between any of the asbestos-related disorders, or between these and controls.. In asbestos-related disorders, markers of inflammation and oxidative stress are significantly elevated in subjects with asbestosis compared with healthy individuals but not in pleural diseases.

Topics: Aged; Asbestos; Asbestosis; Biomarkers; Dinoprost; Forced Expiratory Volume; Humans; Lung Diseases; Male; Nitric Oxide; Oxidative Stress; Prognosis; Tyrosine

High-concentration oxygen therapy is used to treat tissue hypoxia, but hyperoxia causes lung injury. Overproduction of nitric oxide by nitric oxide synthase (NOS) is thought to promote hyperoxic lung injury. The present study was conducted to examine the role of inducible nitric oxide synthase (iNOS) in hyperoxic lung injury in mice.. Mice were exposed to >98% oxygen for 72 h, and ONO-1714 (0.05 mg/kg) (ONO) was subcutaneously administered to block iNOS. Hyperoxia significantly increased total cell count, protein concentration, and nitrites/nitrates in the bronchoalveolar lavage fluid and proinflammatory cytokines in the lung tissue. ONO significantly prevented the increases in all of these variables. ONO suppressed histologic evidence of lung injury. ONO markedly inhibited iNOS protein expression and nitrotyrosine production in lung homogenates. After exposure to hyperoxia, alveolar epithelial cells stained positively for 8-hydroxy-2'-deoxyguanosine, a proper marker of oxidative DNA damage by reactive oxygen species. ONO attenuated this finding.. NOS play important roles in the pathogenesis of hyperoxic lung injury. Selective iNOS inhibitors may be useful for the treatment of hyperoxic lung injury.

Topics: Amidines; Animals; Blotting, Western; Bronchoalveolar Lavage Fluid; Cell Differentiation; Cytokines; Drug Administration Schedule; Enzyme Inhibitors; Heterocyclic Compounds, 2-Ring; Hyperoxia; Immunohistochemistry; Injections, Subcutaneous; Lung; Lung Diseases; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Nitrogen Oxides; Tyrosine

Pulmonary manifestations of oxygen toxicity were studied and quantified in rats breathing >98% O(2) at 1, 1.5, 2, 2.5, and 3 ATA to test our hypothesis that different patterns of pulmonary injury would emerge, reflecting a role for central nervous system (CNS) excitation by hyperbaric oxygen. At 1.5 atmosphere absolute (ATA) and below, the well-recognized pattern of diffuse pulmonary damage developed slowly with an extensive inflammatory response and destruction of the alveolar-capillary barrier leading to edema, impaired gas exchange, respiratory failure, and death; the severity of these effects increased with time over the 56-h period of observation. At higher inspired O(2) pressures, 2-3 ATA, pulmonary injury was greatly accelerated but less inflammatory in character, and events in the brain were a prelude to a distinct lung pathology. The CNS-mediated component of this lung injury could be attenuated by selective inhibition of neuronal nitric oxide synthase (nNOS) or by unilateral transection of the vagus nerve. We propose that extrapulmonary, neurogenic events predominate in the pathogenesis of acute pulmonary oxygen toxicity in hyperbaric oxygenation, as nNOS activity drives lung injury by modulating the output of central autonomic pathways.

Topics: Animals; Behavior, Animal; Blood Gas Analysis; Body Fluids; Bronchoalveolar Lavage Fluid; Hyperoxia; L-Lactate Dehydrogenase; Lung; Lung Diseases; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Oxygen; Pneumonia; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Survival Analysis; Tyrosine; Vagotomy

Asbestosis is a chronic form of interstitial lung disease characterized by inflammation and fibrosis that results from the inhalation of asbestos fibers. Although the pathogenesis of asbestosis is poorly understood, reactive oxygen species may mediate the progression of this disease. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) can protect the lung against a variety of insults; however, its role in asbestosis is unknown. To determine if EC-SOD plays a direct role in protecting the lung from asbestos-induced injury, intratracheal injections of crocidolite were given to wild-type and ec-sod-null mice. Bronchoalveolar lavage fluid (BALF) from asbestos-treated ec-sod-null mice at 24 h, 14 days, or 28 days posttreatment showed increased inflammation and total BALF protein content compared to that of wild-type mice. In addition, lungs from ec-sod-null mice showed increased hydroxyproline content compared to those of wild-type mice, indicating a greater fibrotic response. Finally, lungs from ec-sod-null mice showed greater oxidative damage, as assessed by nitrotyrosine content compared to those of their wild-type counterparts. These results indicate that depletion of EC-SOD from the lung increases oxidative stress and injury in response to asbestos.

Topics: Animals; Asbestos, Crocidolite; Bronchoalveolar Lavage Fluid; Hydroxyproline; Inflammation; Lung; Lung Diseases; Lung Injury; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidative Stress; Superoxide Dismutase; Tyrosine

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

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

Abnormal growth and development of lymphatic pulmonary structures leads to severe hypoxia in congenital pulmonary lymphangiectasis (CPL). This case study aims to determine the cellular source and topographical distribution of the nitric oxide synthases in CPL. It studies the post mortem tissue of a term newborn with the clinical course and histological findings of CPL and three controls without pulmonary pathology. It was found that endothelial cells of pulmonary arteries and lymphatic structures stained significantly more for endothelial nitric oxide synthase protein in the CPL patient compared to the controls. The authors conclude that synthesis of endothelial nitric oxide synthase is upregulated in vascular and lymphatic endothelial cells in congenital pulmonary lymphangiectasis.

Topics: Adult; Endothelium, Lymphatic; Endothelium, Vascular; Female; Fetal Hypoxia; Humans; Immunoenzyme Techniques; Infant; Infant, Newborn; Lung; Lung Diseases; Lymphangiectasis; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Pregnancy; Reference Values; Tyrosine

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

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

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

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

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human alpha/sickle beta-globin (halphabetaS) transgene (SCD mice) or are heterozygous for the normal murine beta-globin gene and express the halphabetaS transgene (mbeta+/-, halphabetaS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing *NO generation and predisposing the lung to vaso-occlusion.

Topics: Acute Disease; Anemia, Sickle Cell; Animals; Disease Models, Animal; Hemoglobin, Sickle; HSP90 Heat-Shock Proteins; Humans; Hypoxia; Lung Diseases; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Tyrosine

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

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

gamma-Glutamyl transferase (GGT) is critical to glutathione homeostasis by providing substrates for glutathione synthesis. We hypothesized that loss of GGT would cause oxidant stress in the lung. We compared the lungs of GGT(enu1) mice, a genetic model of GGT deficiency, with normal mice in normoxia to study this hypothesis. We found GGT promoter 3 (P3) alone expressed in normal lung but GGT P3 plus P1, an oxidant-inducible GGT promoter, in GGT(enu1) lung. Glutathione content was barely decreased in GGT(enu1) lung homogenate and elevated nearly twofold in epithelial lining fluid, but the fraction of oxidized glutathione was increased three- and fourfold, respectively. Glutathione content in GGT(enu1) alveolar macrophages was decreased nearly sixfold, and the oxidized glutathione fraction was increased sevenfold. Immunohistochemical studies showed glutathione deficiency together with an intense signal for 3-nitrotyrosine in nonciliated bronchiolar epithelial (Clara) cells and expression of heme oxygenase-1 in the vasculature only in GGT(enu1) lung. When GGT(enu1) mice were exposed to hyperoxia, survival was decreased by 25% from control because of accelerated formation of vascular pulmonary edema, widespread oxidant stress in the epithelium, diffuse depletion of glutathione, and severe bronchiolar cellular injury. These data indicate a critical role for GGT in lung glutathione homeostasis and antioxidant defense in normoxia and hyperoxia.

Topics: Animals; Antibody Specificity; Female; gamma-Glutamyltransferase; Glutathione; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hyperoxia; Lung; Lung Diseases; Male; Membrane Proteins; Mice; Mice, Mutant Strains; Oxidative Stress; Oxygen; RNA, Messenger; Survival Rate; Tyrosine

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

Topics: Acute Disease; Animals; Bronchoalveolar Lavage Fluid; Enzyme Induction; L-Lactate Dehydrogenase; Lung; Lung Diseases; Male; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Proteins; Radiation Injuries, Experimental; Rats; Rats, Wistar; Tyrosine

We reported that systemic keratinocyte growth factor (KGF) given before bone marrow transplantation (BMT) prevents allogeneic T cell-dependent lung inflammation assessed on Day 7 post-BMT, but the antiinflammatory effects of KGF were impaired in mice injected with both T cells and conditioning regimen of cyclophosphamide (Cy). Intratracheal KGF is known to stimulate the expression of surfactant protein A (SP-A), an oxidant-sensitive T cell immunomodulator produced by alveolar type II cells. We hypothesized that systemic KGF up-regulates SP-A after allogeneic BMT, and the addition of Cy may interfere with the ability of KGF to enhance SP-A production. The subcutaneous administration of recombinant human KGF (5 mg/kg on Days -6, -5, and -4 pre-BMT) increased SP-A protein and mRNA in allogeneic T cell-recipient irradiated mice measured on Day 7 post-BMT. In contrast, the same KGF treatment in irradiated mice given T cells and Cy failed to up-regulate SP-A mRNA and protein expression. In mixed lymphocyte reaction experiments designed to simulate the in vivo model, the addition of human SP-A (5-50 microg) to alloactivated T cells suppressed the production of interleukin-2 in a dose-dependent fashion. We conclude that the systemic pre-BMT injection of KGF in recipients of allogeneic T cells up-regulates SP-A, which may contribute to the early antiinflammatory effects of KGF. The protective KGF-mediated SP-A production is abolished in mice given alloreactive T cells plus Cy.

Topics: Animals; Bone Marrow Transplantation; Bronchoalveolar Lavage Fluid; Cyclophosphamide; Female; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Growth Substances; Immunosuppressive Agents; Interleukin-2; Keratinocytes; Lung Diseases; Lymphocyte Culture Test, Mixed; Lymphocyte Depletion; Mice; Mice, Inbred Strains; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; T-Lymphocytes; Transplantation Conditioning; Tyrosine; Up-Regulation

Acute chest syndrome (ACS) is the most common form of acute pulmonary disease associated with sickle cell disease. To investigate the possibility that alterations in endothelial cell (EC) production and metabolism of nitric oxide (NO) products might be contributory, we measured NO products from cultured pulmonary EC exposed to red blood cells and/or plasma from sickle cell patients during crisis. Exposure to plasma from patients with ACS caused a 5- to 10-fold increase in S-nitrosothiol (RSNO) and a 7- to 14-fold increase in total nitrogen oxide (NO(x)) production by both pulmonary arterial and microvascular EC. Increases occurred within 2 h of exposure to plasma in a concentration-dependent manner and were associated with increases in endothelial nitric oxide synthase (eNOS) protein and eNOS enzymatic activity, but not with changes in nitric oxide synthase (NOS) III or NOS II transcripts, inducible NOS (iNOS) protein nor iNOS enzymatic activity. RSNO and NO(x) increased whether plasma was obtained from patients with ACS or other forms of vasoocclusive crisis. Furthermore, an oxidative state occurred and oxidative metabolites of NO, particularly peroxynitrite, were produced. These findings suggest that altered NO production and metabolism to damaging oxidative molecules contribute to the pathogenesis of ACS.

Topics: Animals; Blood Physiological Phenomena; Cattle; Cells, Cultured; Endothelium, Vascular; Glutathione; Hemoglobin SC Disease; Humans; Lung Diseases; Mercaptoethanol; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Nitroso Compounds; RNA, Messenger; S-Nitrosothiols; Sulfhydryl Compounds; Tyrosine