Effects of Reactive Oxygen and Nitrogen Metabolites on RANTES- and IL-5-Induced Eosinophil Chemotactic Activity in Vitro

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

An Inhibitor of Inducible Nitric Oxide Synthase and Scavenger of Peroxynitrite Prevents Diabetes Development in NOD Mice

Peroxynitrite (ONOO(-)) is a highly reactive oxidant produced by the interaction of the free radicals superoxide (O*-2) and nitric oxide (NO(*)). In a previous study, we found that peroxynitrite is formed in islet beta-cells of nonobese diabetic (NOD) mice. Here, we report that guanidinoethyldisulphide (GED), a selective inhibitor of inducible nitric oxide synthase (iNOS) and scavenger of peroxynitrite prevents diabetes in NOD mice. GED treatment of female NOD mice, starting at age 5 weeks, delayed diabetes onset (from age 12 to 22 weeks) and significantly decreased diabetes incidence at 30 weeks (from 80% to 17%). GED did not prevent pancreatic islet infiltration by leukocytes; however, beta-cells that stained positive for nitrotyrosine (a marker of peroxynitrite) were significantly decreased in islets of GED-treated mice (1+/-1%) compared with vehicle-treated mice (30+/-9%). In addition, GED significantly inhibited nitric oxide and nitrotyrosine formation and decreased destruction of beta-cells in NOD mouse islets incubated in vitro with the combination of proinflammatory cytokines interleukin 1-beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). These findings indicate that both superoxide and nitric oxide radicals contribute to islet beta-cell destruction in autoimmune diabetes via peroxynitrite formation in the beta-cells.     

Peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells.

The proinflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma), are cytotoxic to pancreatic islet beta cells, possibly by inducing nitric oxide and/or oxygen radical production in the beta cells. Peroxynitrite, the reaction product of nitric oxide and the superoxide radical, is a strong oxidant and cytotoxic mediator; therefore, we hypothesized that peroxynitrite might be a mediator of cytokine-induced islet beta-cell destruction. To test this hypothesis we incubated islets isolated from human pancreata with the cytokine combination of IL-1beta, TNFalpha, and IFNgamma. We found that these cytokines induced significant increases in nitrotyrosine, a marker of peroxynitrite, in islet beta cells, and the increase in nitrotyrosine preceded islet-cell destruction. Peroxynitrite mimicked the effects of cytokines on nitrotyrosine formation and islet beta-cell destruction. L-N(G)-monomethyl arginine, an inhibitor of nitric oxide synthase, prevented cytokine-induced nitric oxide production but not hydrogen peroxide production, nitrotyrosine formation, or islet beta-cell destruction. In contrast, guanidinoethyldisulphide, an inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite, prevented cytokine-induced nitric oxide and hydrogen peroxide production, nitrotyrosine formation, and islet beta-cell destruction. These results suggest that cytokine-induced peroxynitrite formation is dependent upon increased generation of superoxide (measured as hydrogen peroxide) and that peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells.     

Expression of nitric oxide synthases and formation of nitrotyrosine and reactive oxygen species in inflammatory bowel disease

Nitric oxide (NO) and reactive oxygen species (ROS) are important mediators in the pathogenesis of inflammatory bowel disease (IBD). NO in IBD can be either harmful or protective. NO can react with superoxide anions (O2.-), yielding the toxic oxidizing agent peroxynitrite (ONOO-). Peroxynitrite induces nitration of tyrosine residues (nitrotyrosine), leading to changes of protein structure and function. The aim of this study was to identify the cellular source of inducible nitric oxide synthase (iNOS) and to localize superoxide anion-producing cells in mucosal biopsies from patients with active IBD. Additional studies were performed to look at nitrotyrosine formation as a measure of peroxynitrite-mediated tissue damage. For this, antibodies against iNOS, endothelial NOS (eNOS), and nitrotyrosine were used. ROS-producing cells were detected cytochemically. Inflamed mucosa of patients with active IBD showed intense iNOS staining in the epithelial cells. iNOS could not be detected in non-inflamed mucosa of IBD patients and control subjects. eNOS was present in blood vessels, without any difference in the staining intensity between IBD patients and control subjects. ROS-producing cells were increased in the lamina propria of IBD patients; a fraction of these cells were CD15-positive. Nitrotyrosine formation was found on ROS-positive cells. These results show that iNOS is induced in epithelial cells from patients with active ulcerative colitis or Crohn's disease. Nitration of proteins was detected only on the ROS-producing cells at some distance from the iNOS-producing epithelial cells. These findings indicate that tissue damage during active inflammation in IBD patients is probably more related to ROS-producing cells than to NO. One may speculate that NO has a protective role when during active inflammation other mucosal defence systems are impaired.     

Effects of reactive oxygen and nitrogen metabolites on eotaxin-induced eosinophil chemotactic activity in vitro

Peroxynitrite, an oxidant generated by the interaction between superoxide and nitric oxide (NO), has been implicated in the etiology of numerous disease processes. Several studies have shown that peroxynitrite-induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the eosinophil chemotactic responses of eotaxin incubated with and without peroxynitrite were evaluated. Peroxynitrite attenuated eotaxin-induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05). The inhibitory effects were not significant on ECA induced by leukotriene B(4) or complement-activated serum incubated with peroxynitrite. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate (an NO donor) or a combination of xanthine and xanthine oxidase to generate superoxide did not show an inhibitory effect on ECA induced by eotaxin. In contrast, 3-morpholinosydnonimine, a peroxynitrite generator, caused a concentration-dependent inhibition of ECA by eotaxin. Consistent with its capacity to reduce ECA, peroxynitrite treatment reduced eotaxin binding to eosinophils. Nitrotyrosine was detected in the eotaxin incubated with peroxynitrite. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of eotaxin binding to eosinophils and a reduction in ECA. These data demonstrate that peroxynitrite modulates the eosinophil migration by eotaxin, and suggest that oxidants may play an important role in regulation of eotaxin-induced eosinophil chemotaxis.     

B-type natriuretic peptide in reversible myocardial ischaemia

BACKGROUND: Coronary heart disease is associated with increased B-type natriuretic peptides (BNPs), and, although controversial, may cause exaggerated exercise-induced BNP secretion. We investigated BNP in relation to reversible myocardial ischaemia. Materials and methods: Serum N-terminal proBNP (NT-proBNP) was measured before and after an exercise electrocardiogram test (ETT) in 14 patients with and 45 patients without exercise-induced myocardial ischaemia. Statistical analysis was carried out on logarithmically transformed data. Results, however, are pre-transformed data. RESULTS: NT-proBNP increased with exercise both in ETT-positive patients (mean (SD) 71.4 (41.2) v 76.8 (44.0) ng/l; p<0.001) and ETT-negative patients (54.0 (61.2) v 60.1 (69.0) ng/l; p<0.001). Pre-exercise and post-exercise NT-proBNP were higher (p<0.05) in ETT-positive than in ETT-negative patients. Incremental NT-proBNP was similar in ETT-positive (4.7 (4.2) ng/l) and ETT-negative (6.2 (8.6) ng/l) patients. CONCLUSION: Serum NT-proBNP concentrations are higher in patients with exercise-induced myocardial ischaemia than in those without. Exercise-induced electrocardiographic myocardial ischaemia, however, is not associated with exaggerated BNP secretion.     

Preliminary report on nitric oxide-mediated oxidative damage in adolescent varicocele

BACKGROUND: The current study was designed to characterize the process of nitric oxide (NO) and peroxynitrite generation through the determination of nitrotyrosine concentration in the dilated veins of varicoceles in adolescents. METHODS: Ten adolescents with a median age of 13 years (range 12-17) affected by a left idiopathic varicocele (grade II and III) were studied. Whole blood samples were withdrawn from a peripheral vein at time of induction of anaesthesia, and from a dilated spermatic vein before ligation. Peripheral blood samples from five adolescents undergoing minor surgical procedures were used as controls. The nitrotyrosine concentration was evaluated by a sandwich enzyme-linked immunosorbent assay (ELISA), using a monoclonal anti-nitrotyrosine antibody and Western blot analysis. RESULTS: Plasma nitrotyrosine concentrations were significantly greater in the spermatic vein when compared with the peripheral vein (P = 0.031). Nitrotyrosine in plasma of controls did not show any significant difference in comparison with peripheral samples from varicocele patients. Western blot analysis confirmed the above data. CONCLUSIONS: In adolescents with a varicocele, there is an increase in nitrotyrosine concentration within the spermatic vein that can cause protein nitration and cytotoxicity via its reaction with various molecular targets. This could have repercussions on both sperm and testis function. We conclude that an oxidative stress status is present and should be considered as an indication for varicocele treatment in the adolescent.     

Peroxynitrite formation in radiation-induced salivary gland dysfunction in mice

Xerostomia frequently arises in patients with head and neck malignancies that are treated by radiation. However, the mechanisms responsible for the destruction of the salivary gland remain unknown. We previously established a xerostomia model of mice and identified the pathway through which nitric oxide (NO) affects the pathogenesis of radiation-induced salivary gland dysfunction. Although the toxicity of NO alone is modest, NO with superoxide anion (O2(*-)) rapidly forms peroxynitrite (ONOO), a more powerful toxic oxidant. In this study, we used the experimental model to examine: 1) when NO and O2(*-) production is maximum in the salivary gland after irradiation;2) whether peroxynitrite, as assessed by nitrotyrosine production, is responsible for salivary gland dysfunction; and 3) the effect of the iNOS selective inhibitor, aminoguanidine (AG), on nitrotyrosine formation. The increases in production of NO and O2(*-) in the salivary gland peaked on day 7 after irradiation. Nitrotyrosine detected immunohistochemically was significantly reduced by AG in the salivary gland. On the basis of these results, we concluded that NO together with O2(*-) forms the more reactive ONOO, which might be an important pathogenic factor in radiation-induced salivary gland dysfunction.     

Asymmetric dimethylarginine and nitric oxide levels as signs of endothelial dysfunction in Behcet's disease

Behcet's disease (BD) has been known for many years, yet the etiology of the systemic vasculitis remains unknown. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase. ADMA is involved in endothelial dysfunction in various vascular diseases and its level in BD is unclear. This study was performed to evaluate the relationship between ADMA and NO levels in plasma of patients with BD. There were 3 groups of 30 subjects: (a) controls, (b) BD patients with mucocutaneous involvement, and (c) BD patients with vascular involvement. Plasma NO levels were assayed by spectrophotometry and plasma ADMA levels were assayed by an ELISA test. Plasma ADMA levels were higher in both groups of BD patients than in the controls; the ADMA levels were higher in the BD patients with vascular involvement than in the mucocutaneous group. Plasma NO levels were lower in both groups of BD patients than in controls; plasma NO levels were lower in the BD patients with vascular involvement than in mucocutaneous group. In the combined groups of 60 BD patients, there was significant inverse correlation between the plasma concentrations of ADMA and NO (r = -0.570, p <0.001). Plasma lipid profiles did not differ significantly between the BD patients and the controls. These results are evidence for increased plasma ADMA levels and decreased plasma NO levels as risk factors for cardiovascular events in BD patients. Inhibition of NO synthesis by ADMA may contribute to vascular involvement in BD.     

Possible mechanism of bronchoprotection by SIN-1 in anaesthetized guinea pigs: roles of nitric oxide and peroxynitrite.

BACKGROUND: S-morpholinosydnonimine (SIN-1) is thought to generate peroxynitrite. Recent reports suggested that peroxynitrite possessed a potent vascular relaxant activity via guanylate cyclase activation. However, no previous studies have examined the relaxant effect of peroxynitrite on airway smooth muscle. OBJECTIVE: To determine the mechanism of bronchoprotection by SIN-1, considering in particular the involvement of nitric oxide (NO) and peroxynitrite. METHODS: Peroxynitrite formation was assayed by monitoring the oxidizing activity of dihydrorhodamine 123, and NO was measured polarographically as a redox current in vitro. We examined the effect of SIN-1 delivered to the airway by ultrasonic nebulization against bronchoconstriction induced by acetylcholine in anaesthetized guinea pigs. RESULTS: SIN-1 produced peroxynitrite in a time- and concentration-dependent manner, but did not produce NO in vitro. However, when mixed with glutathione (GSH) and bronchoalveolar lavage fluid (BALF), peroxynitrite formation by SIN-1 was inhibited and SIN-1 induced the release of NO. SNAP (S-nitroso-N-acetyl-penicillamine) and SIN-1 each inhibited acetylcholine-induced bronchoconstriction in a dose-dependent manner in vivo. Though GSH alone did not have any effect on baseline airway resistance and acetylcholine-induced bronchoconstriction, pretreatment with GSH significantly enhanced SNAP- and SIN-1-induced bronchoprotection. In addition, pretreatment with carboxy-PTIO, a NO scavenger, completely inhibited bronchoprotective effect of SNAP on acetylcholine-induced bronchoconstriction, but partially inhibited SIN-1-induced bronchoprotection. CONCLUSION: These findings demonstrated that SIN-1 is a potent peroxynitrite-releasing compound and caused significant bronchoprotection against acetylcholine. The mechanism of bronchoprotection by SIN-1 appears to be mediated by peroxynitrite but also at least in part through NO regeneration, which may involve GSH and airway thiols as a consequence of exposure to peroxynitrite.     

Peroxynitrite-induced thymocyte apoptosis: the role of caspases and poly (ADP-ribose) synthetase (PARS) activation.

The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross-linking of T-cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno-histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10 microM) of peroxynitrite led to apoptosis, whereas higher concentrations (50 microM) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP-ribose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3-aminobenzamide (3-AB), or thymocytes from PARS-deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatment, while apoptosis induced by non-oxidative stimuli such as dexamethasone or anti-FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite-induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energy-conserving step, enabling the cell to complete the process of apoptosis.     

Central nitric oxide inhibition modifies metabolic adjustments induced by exercise in rats

The influence of the central nervous system on metabolic function is of interest in situations deviating from basal states, such as during exercise. Our previous study in rats demonstrated that central nitric oxide (NO) blockade increases metabolic rate, reducing mechanical efficiency during exercise. To assess the role of brain nitric oxide in the plasma glucose, lactate and free fatty acids (FFAs) concentrations of rats submitted to an incremental exercise protocol on a treadmill until fatigue, 1.43 micromol (2 microl) of N(omega)-nitro-l-arginine methyl ester (L-NAME, n=6), a NO synthase inhibitor, or 2 microl of 0.15M NaCl (SAL, n=6) was injected into the lateral cerebral ventricle (icv) of male Wistar rats immediately before exercise (starting at 10 m/min, with increments of 1m/min every 3 min until fatigue, 10% inclination). Blood samples were collected through a chronic jugular catheter at rest and during exercise until fatigue. During exercise, the L-NAME-treated animals had the following metabolic response compared to controls: (1) an increased hyperglycemic response during the first 60% of time to fatigue; (2) higher plasma lactate levels; and (3) a significant transitory increase in plasma free fatty acids during the dynamic phase of exercise that returned to basal levels earlier than controls during the steady state phase of exercise. In addition L-NAME-treated rats fatigued earlier than controls. The data indicate that the inhibition of the brain nitrergic system induced by icv L-NAME treatment disrupted the accuracy of the neural mechanism that regulates plasma glucose and free fatty acids mobilization during exercise in rats.     

Differences in the metabolic and hormonal response to exercise between racing cyclists and untrained individuals.

1. Six well-trained cyclists and six untrained subjects were studied during and immediately after four successive 7 min periods of exercise at 30, 45, 60 and 75% of their maximal work capacity. 2. Venous blood samples were taken at rest, at the end of each exercise period and 5 min following the end of exercise, for estimation of metabolites in blood and plasma insulin, growth hormone, cortisol and catecholamines. 3. The results showed significant differences in the mobilization and utilization of muscle fuels between the athletically fit cyclists and the untrained group. In the cyclists, glucose, glycerol and free fatty acid concentrations were higher, but lactate, pyruvate and alanine were lower than in the untrained subjects during exercise. 4. Plasma catecholamines rose in both groups during exercise but the rise was significantly less in the racing cyclists. Plasma insulin was depressed to a greater extent in the untrained subjects during exercise and plasma glucagon rose to a greater extent during strenuous exercise and remained elevated after the end of exercise in the untrained group. Plasma human growth hormone rose to a greater extent during exercise and remained elevated after the end of exercise in the untrained group. Plasma cortisol fell at low and moderate exercise rates in both groups, but to a smaller extent in the cyclists. Cortisol values rose at higher workloads and were significantly higher in the cyclists at the end of exercise. 5. It is concluded that there are significant differences in the metabolic and hormonal responses to exercise between athletically trained and untrained individuals, even when the physically fit subjects work at the same percentage of their maximal capacity as the unfit subjects.     

A computational model for free radicals transport in the microcirculation

Nitric oxide (NO), superoxide (O(2)(-)), and peroxynitrite (ONOO(-)) interactions in pathophysiologic conditions such as cardiovascular disease, hypertension, and diabetes have been studied extensively in vivo and in vitro. A reduction in bioavailability of NO is a common event that is known as the endothelial dysfunction in these conditions. Despite intense investigation of NO biotransport and O(2)(-) and ONOO(-) biochemical interactions in vasculature, we have very little quantitative knowledge of distributions and concentrations of NO, O(2)(-), and ONOO(-) under normal physiologic and pathophysiologic conditions. Based on fundamental principles of mass balance, vessel geometry, and reaction kinetics, we developed a mathematical model of these free radicals transport in and around an arteriole during oxidative stress. We investigated the role of O(2)(-) and ONOO(-) in inactivating vasoactive NO. The model predictions include (a) NO interactions with oxygen, O(2)(-), and ONOO(-) have relatively little effect on the NO level in the vascular smooth muscle under physiologic conditions; (b) superoxide diffuses only a few microns from its source, whereas peroxynitrite diffuses over a larger distance; and (c) reduced superoxide dismutase levels significantly increase O(2)(-) and peroxynitrite concentrations and decrease NO concentration. Model results indicate that the reduced NO bioavailability and enhanced peroxynitrite formation may vary depending on the location of oxidative stress in the microcirculation, which occurs at diverse vascular cell locations in diabetes, aging, and cardiovascular diseases. The results will have significant implications for our understanding of these free radical interactions in physiologic and pathophysiologic conditions resulting from endothelial dysfunction.     

吸烟对血管内皮依赖性舒张功能的影响

目的 观察吸烟对和因管内皮依赖性舒张功能的影响,探讨吸烟对心血管危害的机制。     

Biochemical characterization of reactive nitrogen species by eosinophil peroxidase in tyrosine nitration

It is well known that eosinophils are involved in tyrosine nitration. In this study, we evaluated tyrosine nitration by rat eosinophils isolated from peritoneal fluid and constituent eosinophils in the stomach. Rat peritoneal eosinophils activated with 1 microM phorbol myristate acetate (PMA) and 50 microM NO2- showed immunostaining for nitrotyrosine only in smaller cells, despite the fact that eosinophils are capable of producing superoxide (O2*-) Free tyrosine nitrating capacity after incubation with PMA and NO2- was 4-fold higher in eosinophils than in neutrophils. Catalase and alpha- and gamma-tocopherol inhibited free tyrosine nitration by reactive nitrogen species from eosinophils but not that by peroxynitrite. Superoxide dismutase augmented free tyrosine nitration by activated eosinophils and peroxynitrite. The concentration of nitric oxide released from eosinophils was relatively low (0.32 microM/10(6) cells/h) and did not contribute to the formation of nitrotyrosine. On the other hand, most constituent eosinophils constituent in the rat stomach stimulated by PMA and NO2- showed tyrosine nitration capacity. These results suggest that intact cells other than apoptotic-like eosinophils eluted in the intraperitoneal cavity could not generate reactive species responsible for nitration by a peroxidase-dependent mechanism. In contrast, normal eosinophils in the stomach were capable of nitration, suggesting that the characteristics of eosinophils in gastric mucosa are different from those eluted in the peritoneal cavity.     

Peroxynitrite Exposure of Cells Cocultured with Macrophages

Peroxynitrite is a potent oxidant that is formed endogenously from NO and O2-. One approach for studying its toxicity involves the coculture of activated macrophages (producers of NO and O2-) with "target cells" (which make O2- but not NO). Because peroxynitrite concentrations in such experiments are too small and localized to measure, reaction-diffusion models were developed for situations ranging from isolated cells to many cells randomly distributed on a plate. In these models the macrophage population created a constant NO concentration in and near all cells, whereas fluxes of O2- into the media from individual macrophages led to localized extracellular formation of peroxynitrite. Target cells were found to have no effect on the peroxynitrite concentrations in other cells, whereas the macrophage effects were additive. The average concentration inside randomly distributed target cells increased with increasing macrophage number density, as entry of extracellular peroxynitrite grew in importance relative to intracellular formation. For high cell densities, large peroxynitrite membrane permeabilities, and low rates of intracellular synthesis, the surrounding macrophages were calculated to double or triple the peroxynitrite concentration in an average target cell. It was estimated that a macrophage enveloping a target cell could cause as much as a 10-fold increase in the concentration inside that target cell.     

Type 2 nitric oxide synthase and protein nitration in chronic lung infection

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.     

Local peroxynitrite formation contributes to early control of Cryptosporidium parvum infection

In intestinal inflammation, mucosal injury is often exacerbated by the reaction of NO with neutrophil-derived superoxide to form the potent oxidant peroxynitrite. Peroxynitrite also has antimicrobial properties that aid in the killing mechanism of macrophages and neutrophils. Cryptosporidium parvum parasitizes intestinal epithelium, resulting in loss of epithelial cells and mucosal inflammation. Synthesis of NO is significantly increased and arises from the induced expression of inducible nitric oxide synthase (iNOS) by the infected epithelium. Inhibition of iNOS results in intensified epithelial parasitism and oocyst excretion. We hypothesized that formation of peroxynitrite is restricted to sites of iNOS expression by the epithelium and contributes to host defense in C. parvum infection. Accordingly, the location and biological effects of peroxynitrite formation were examined in neonatal piglets infected with C. parvum. Infected piglets were treated daily with a selective iNOS inhibitor [L-N6-(1-iminoethyl)-lysine] or one of two peroxynitrite scavengers [5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron(III) or uric acid] or received vehicle. At peak infection, peroxynitrite formation was restricted to sites of iNOS expression by parasitized epithelium and lamina propria of the apical villi. Peroxynitrite formation was dependent on iNOS activity and was inhibited by treatment with peroxynitrite scavengers. Scavengers increased the number of intracellular parasites and the number of infected epithelial cells present per villus and significantly exacerbated oocyst excretion. Recovery from infection was not delayed by ongoing treatment with scavenger. The present results are the first to demonstrate an in vivo role for peroxynitrite formation in acute mucosal defense against a noninvasive intestinal epithelial pathogen.