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Influence of tissue nitration on tissue damage with thermal injury
Abstract
A gas mediator, nitric oxide, is converted into peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent
oxidant, which injures various tissues and organs by nitration of tyrosine residue in protein and enhances the inflammatory
response in the prolonged phase. In this study, the authors investigated the relationship between peroxynitrite-mediated tissue
nitration and tissue damage with thermal injury using an experimental burn model. The content of nitrotyrosine in the burned
tissue significantly increased 1 to 6 h after injury. The nitrotyrosine content in the burned ear significantly decreased
with 100 mg/kg of LNAME administration. Vascular hyperpermeability was also significantly suppressed in the iNOS antibody
immunoneutralized mice 6 h after injury. There was a positive correlation between the severity of tissue damage, an indicator
of which is the increase in the weight of the burned ear along with the development of edema after injury, and the concentration
of nitrotyrosine in the wound tissues. Nitrotyrosine-like immune reactants were also diffusely detected in the burned region
and the surrounding areas. These results indicate that peroxynitrite is produced in the surrounding burned region and a reaction
of nitration of tissue tyrosine is involved with tissue damage at the burn wound. Therefore, to prevent the systemic vascular
hyperpermeability and tissue damage in a large area burn or severe burn patients, the administration of NOS inhibitors or
radical erasers may be easy to manage generally by inhibition of peroxynitrite formation.
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Reaction of nitric oxide (NO.) with superoxide radical generates peroxy-nitrite, which can decompose to products that nitrate aromatic amino acids. Such nitro-aromatics may be 'markers' of NO.-dependent oxidative damage. Blood serum and synovial fluid from patients with the inflammatory joint disease rheumatoid arthritis contain 3-nitrotyrosine. By contrast, body fluids from normal subjects and patients with osteoarthritis contain no detectable 3-nitrotyrosine; much lower levels were found in serum from patients in the early stages of rheumatoid arthritis. This is evidence that NO. plays a role in joint damage in rheumatoid arthritis.
Oxidant-mediated toxicity resulting from acute pulmonary inflammation has been demonstrated in acute lung injury. A potent biological oxidant, peroxynitrite, is formed by the near diffusion-limited reaction of nitric oxide with Superoxide. In addition to having hydroxyl radical-like oxidative reactivity, peroxynitrite is capable of nitrating phenolic rings, including protein-associated tyrosine residues. Nitric oxide does not directly nitrate tyrosine residues, therefore, demonstration of tissue nitrotyrosine residues infers the action of peroxynitrite or related nitrogen-centered oxidants. Lung tissue was obtained from formalin-fixed, paraffinembedded autopsy specimens, and specific polyclonal and monoclonal antibodies to nitrotyrosine were visualized by diaminobenzidene-peroxidase staining. Acute lung injury resulted in intense staining throughout the lung, including lung interstitium, alveolar epithelium, proteinaceous alveolar exudate, and inflammatory cells. In addition, staining of the vascular endothelium and subendothelial tissues was present in those patients with sepsis-induced acute lung injury. Antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. Reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. In control specimens with no overt pulmonary disease, there was only slight staining of the alveolar septum. These results demonstrate that nitrogen-derived oxidants are formed in human acute lung injury and suggest that peroxynitrite may be an important oxidant in inflammatory lung disease.
Objective: To determine whether burn-induced peroxynitrite production and expression of lung inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, CXCR2, macrophage inflammatory protein (MIP)-2, and neutrophil chemokine (KC) are mediated by the c-Jun NH2-terminal kinase (JNK). Design: Prospective, experimental study. Setting: Research laboratory at a university hospital. Subjects: Thermal injury models in the mice. Interventions: In experiment 1, specific pathogen-free C57/BL6 mice were subjected to 30% total body surface area third-degree burn over shaved back. At 0 hr, 2 hrs, 4 hrs, and 6 hrs after burn, lung tissues of those mice were harvested for JNK activity assay, AP-1 DNA-binding activity, and pJNK immunohistochemistry. In experiment 2, a specific JNK inhibitor, SP600125, was given (30 mg/kg intraperitoneally) to mice immediately postburn to suppress the JNK activity. At 8 hrs after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine (DHR) 123 oxidation. Lung tissues were harvested for myeloperoxidase (MPO) determination, ICAM-1, VCAM-1, CXCR2, KC, MIP-2, interleukin-1β, and interleukin-6 messenger RNA expression; iNOS immunohistochemical staining; and histologic studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. Measurements and Main Results: The JNK activity and AP-1 DNA-binding activity of lung tissue significantly increased to a peak at 2 hrs and 4 hrs, respectively, after thermal injury. Immunohistochemical study demonstrated that the increase of the pJNK was mostly from the bronchiole epithelial cells. This increase of MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased six-fold, nine-fold, and four-fold after burn. SP600125 administration obliterated the thermal injury-induced JNK activity, AP-1 DNA-binding activity, and iNOS expression in lung tissue. SP600125 treatment also significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 54%, 8%, and 47%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. Furthermore, SP600125 abolished thermal injury-induced ICAM-1, VCAM-1, CXCR2, MIP-2, and KC but not interleukin-1β and interleukin-6 messenger RNA levels of lung tissues. Conclusions: Thermal injury induces lung tissue JNK activation and AP-1 DNA-binding activity mainly from airway epithelial cells. Thermal injury-induced peroxynitrite production and lung iNOS, ICAM-1, and VCAM-1 expression are mediated by the JNK signaling. JNK inhibition decreases thermal injury-induced lung neutrophil infiltration and subsequently pulmonary hyperpermeability.
Background: Recent studies have suggested that adults who sustain burns of less than 15% total body surface area display elevated plasma nitrate levels, indicating increased production of nitric oxide. The present study was initiated to confirm whether plasma nitrate is elevated in minor burn injury and, if so, whether it heralds the onset of a systemic inflammatory response to that injury.
Methods: Plasma samples were taken from 98 control and 10 burns patients.
Results: The mean plasma nitrate level for nine burns patients with a mean total body surface area burnt of 7.65% (range, 4-15%) was 42.83 micro mol/L on day 1. This was not significantly different from that of a control population of 98 preoperative plastic surgery patients: 36.91 micro mol/L (p = 0.162). Eight of 10 burns patients showed a decrease in plasma nitrate to 27.47 micro mol/L by day 3 (p = 0.046). Elevated nitrate levels were seen in 2 of 10 burns patients. One had concurrent smoke-inhalation injury preceding multiple organ dysfunction, and one was treated with a cream containing cerium nitrate (Flammacerium, Duphar Laboratories, Southhampton, United Kingdom).
Conclusions: For patients who sustain minor burns, plasma levels of nitrate decrease from those of mean normal controls with time unless there is multiple organ dysfunction or the patient receives extraneous nitrate.
The relationship between histamine release and edema formation was studied after thermal injury of the rat paw. Injury of varying degrees of severity was produced by immersing the paw in hot water at 48, 53 and 56°C for 30 sec. Histamine was measured by a specific enzymatic methylation procedure. The amine disappeared from paw tissue and appeared in appreciable quantities in tissue fluid and blood serum after moderate (53°C) or severe injury (56°C). The time course of this release appeared to be related directly to edema formation. Both were delayed 30 min after moderate injury (53°C), but were immediate after severe injury (56°C). Once edema was fully developed no further release of histamine occurred. The edema subsided slowly after moderate injury (53°C) but after severe injury (56°C) the swelling persisted and extensive tissue necrosis developed with eventual loss of tissue. In animals depleted of tissue histamine by pretreatment with compound there was a marked reduction in edema and tissue damage. These results suggested that histamine release was closely related to the initial development of edema and may also influence the later stages of inflammation.
The production of both nitric oxide (NO) and superoxide increases in septic shock. The cogeneration of these molecules is known to yield peroxynitrite, which preferentially nitrates tyrosine residues of protein and nonprotein origins. We present evidence of peroxynitrite production in septic shock by measuring plasma nitrotyrosine. The nitrotyrosine was measured by an HPLC C-18 reverse-phase column and ultraviolet detector in chronic renal failure patients with or without septic shock, and in healthy volunteers. Plasma nitrite+nitrate (NOx) was also measured to evaluate NO production. Nitrotyrosine was selected as an index for production of peroxynitrite because the direct measurement of peroxynitrite in vivo is difficult. Patients with renal failure were selected in order to minimize nitrotyrosine excretion through the kidney. Plasma nitrotyrosine levels were not detectable in volunteers, 28.0 ± 12.3 μM (1.6 ± 1.1% of total tyrosine) in renal failure patients without septic shock, and 118.2 ± 22.0 μM (5.5 ± 1.2% of total tyrosine) in patients with septic shock. NOx levels were also higher in patients with septic shock than in patients without septic shock (173.9 ± 104.7 vs. 75.6 ± 19.1 μM). Although renal failure itself increases plasma concentrations of both molecules, the higher levels in patients with septic shock suggest that peroxynitrite is generated and the nitration of tyrosine residues is increased in this disease. Copyright © 1997 Elsevier Science Inc.
Hypotension during septic shock, which may reflect increased synthesis of the potent vasodilator nitric oxide (NO), is often refractory to vasoconstrictors. We describe the effects of NO synthase inhibition in two patients with life-threatening septic shock in whom conventional therapy had failed to restore blood pressure. NG-monomethyl-L-arginine (L-NMMA) caused dose-dependent increases in blood pressure and systemic vascular resistance in both patients, and a similar effect was observed in the second patient after treatment with NG-nitro-L-arginine methyl ester (L-NAME). These findings indicate that NO synthase induction contributes to the pathogenesis of septic shock, and that inhibition of NO synthase may represent a novel therapeutic option.
NO is a major messenger molecule which regulates immunity and vascular tone, serves as a neurotransmitter and participates in wound healing. It has also been known to be increased in vivo by thermal injury. Urinary nitrate excretion and tissue levels of NO synthase activity were measured after a non-lethal thermal injury. Urinary nitrate excretion decreased by 90% 24-48 h after injury, but dramatically increased by 10-fold thereafter for 30-40 days after injury and remained elevated until the wound healed. This response was inhibited by a competitive inhibitor of NO synthase. These rates of urinary nitrate excretion suggest that NO production is dramatically affected by injury in a pattern that is distinct from that observed after lipopolysaccharide administration. On the basis of these findings, we suggest that the hyperdynamic cardiovascular and hypermetabolic responses seen to continue weeks after thermal injury could be a result of the autocrine and paracrine effects of NO generated locally within the tissues in addition to that generated by inflammatory cells.
Oxidant-mediated toxicity resulting from acute pulmonary inflammation has been demonstrated in acute lung injury. A potent biological oxidant, peroxynitrite, is formed by the near diffusion-limited reaction of nitric oxide with superoxide. In addition to having hydroxyl radical-like oxidative reactivity, peroxynitrite is capable of nitrating phenolic rings, including protein-associated tyrosine residues. Nitric oxide does not directly nitrate tyrosine residues, therefore, demonstration of tissue nitrotyrosine residues infers the action of peroxynitrite or related nitrogen-centered oxidants. Lung tissue was obtained from formalin-fixed, paraffin-embedded autopsy specimens, and specific polyclonal and monoclonal antibodies to nitrotyrosine were visualized by diaminobenzidene-peroxidase staining. Acute lung injury resulted in intense staining throughout the lung, including lung interstitium, alveolar epithelium, proteinaceous alveolar exudate, and inflammatory cells. In addition, staining of the vascular endothelium and subendothelial tissues was present in those patients with sepsis-induced acute lung injury. Antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. Reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. In control specimens with no overt pulmonary disease, there was only slight staining of the alveolar septum. These results demonstrate that nitrogen-derived oxidants are formed in human acute lung injury and suggest that peroxynitrite may be an important oxidant in inflammatory lung disease.
The anti-inflammatory mechanism of iodine-enriched egg was investigated in mice by means of arachidonic acid-induced ear inflammation. The lipid fraction of iodine-enriched egg was capable of suppressing the increase in ear weight induced by arachidonic acid in a dose-dependent manner. The lipid fraction was further separated into neutral and polar lipid fractions. Of these two fractions, only the neutral lipid fraction was capable of suppressing LT-C4 production in arachidonic acid inflammation. Neither the neutral nor polar lipid fractions of ordinary egg, however, showed any anti-inflammatory effect. These results suggest that the anti-inflammatory activity of iodine-enriched egg is present in the neutral lipid fraction, and its mechanism is assumed to be inhibition of LT-C4 production.
Endothelin is a well known vasoconstrictive peptides produced by endothelial cells and has been reported to regulate the systemic circulation. The authors investigated changes in endothelin in plasma and the surface of wounds induced with thermal injury using an experimental ear burn model in mice. At 0, 15, 30, 60, 120 and 180 minutes after thermal injury the plasma endothelin-like immunoreactant levels were 1.50 +/- 0.21, 1.86 +/- 0.36, 2.81 +/- 0.55, 2.62 +/- 0.27, 1.54 +/- 0.14 and 1.25 +/- 0.19 fmol/ml (N = 8), respectively. Endothelin-like immunoreactant levels in the plasma increased gradually until 30 minutes after the thermal injury. Endothelin-like immunoreactant content in the ear before thermal injury and at 60 minutes after injury were 7.04 +/- 0.64 and 8.61 +/- 1.24 fmol/ear (N = 8), respectively. The change in endothelin-like immunoreactant after thermal injury originated from endothelin 1,2; that is, the endothelin-1,2 content of the burned ear increased significantly 15 and 60 minutes after thermal injury to 12.52 +/- 0.68 and 11.58 +/- 1.04 fmol/ear, respectively, compared with 1.78 +/- 0.91 fmol/ear (N = 8) obtained before injury. These results suggested that endothelin 1,2 existed in the region of the wound caused by thermal injury.
Human burn injury is associated with an inflammatory response and related hyperdynamic cardiovascular profile. Increased production of nitric oxide (NO), a potent endogenous vasodilator, has been reported in patients with inflammatory states, including sepsis, but not after trauma other than burns. We studied whether plasma levels of the stable byproducts of NO, nitrite (NO2-) and nitrate (NO3-), are increased in burn patients.
Prospective controlled study.
In consecutive patients admitted to the intensive care unit of the burn center at the Queen Astrid Military Hospital in Brussels, plasma was drawn daily from day 1 to day 5 postadmission for determination of NO2-/NO3- levels (Griess' reaction). In a control group of nonseptic inpatients from the department of neurology in Erasme University Hospital who were matched for nutrition (30 to 40 kcal/kg/day of a standard enteral solution), plasma was drawn once for NO2-/NO3- determination.
The burn group included 16 patients (age 35 +/- 18 years, total burn surface area (TBSA) 37 +/- 19%) and the control group included six patients (age 64 +/- 18 years). For each comparison between the groups, NO2-/NO3- plasma levels were higher in those patients with burns than in the control group. In the burn group, there was no correlation between NO2-/NO3- plasma levels and TBSA, age, TBSA x age, blood pressure or time. However, in a subgroup of five burned patients who became septic during the study period, NO2-/NO3- plasma levels were slightly higher than in the non-infected patients (177 +/- 131 vs. 83 +/- 48 micromoles/L, NS).
Human burn injury is associated with an increase in NO production. In this small-size study, NO production was not proportional to burn area, and seemed to be further enhanced in septic patients.
Tumor necrosis factor-alpha (TNF alpha) is a central mediator in the pathogenesis of sepsis. It also interferes with the hemostatic system and exerts and a net procoagulant effect. Since TNF alpha may contribute to thrombotic complications in sepsis patients, we determined markers of thrombin activation, parameters of the fibrinolytic system (D-dimer, tissue plasminogen activator antigen (tPA) urinary type plasminogen activator antigen (uPA), plasminogen activator inhibitor antigen (PAI-1) and von Willebrand factor antigen (vWF) in 30 patients with sepsis or septic shock. All patients were treated with standard therapy, but 14 patients were treated additionally with an anti-TNF alpha monoclonal antibody (MAK 195F); 16 patients served as historical controls. No significant effect of the antibody on the parameters of the hemostatic system could be determined. Our data speak against a modulation of coagulation or the fibrinolytic system by the monoclonal anti-TNF alpha antibody MAK 195F in this cohort of sepsis patients.
The kinetics and role of nitric oxide (NO) on vascular permeability were studied using an ear thermal injury model. Vascular permeability was suppressed for 3 hours after a thermal injury by the preventive administration of nitric oxide synthase (NOS) inhibitors. The NO content in the injured region was significantly increased compared with the intact region. The plasma NO content was significantly increased in a biphasic pattern at 1 and 6 hours postinjury. NOS inhibitors administered as therapeutic treatment suppressed vascular permeability 1 and 6 hours postburn. Thus, NOS inhibitors may be effective in burn treatment.
The nose contributes the large amount of nitric oxide (NO) to exhaled air. NO is a mediator of vasodilation and yields peroxynitrite (ONOO-) by reacting with superoxide (O2-). ONOO attacks tyrosine residues to form nitrotyrosine.
The aim of this study was to examine the pathophysiological role of NO in nasal mucosa in patients with perennial nasal allergy.
We measured nitrite and nitrate (NO2-/NO3-) and 3',5'-guanosine monophosphate (cyclic GMP) in nasal lavage fluid, and also measured haemoglobin concentration in nasal mucosa as an indicator of blood volume in the patients and healthy volunteers. The deleterious role of NO was also investigated by measuring nitrotyrosine in nasal mucosa.
The NO2-/NO3- concentration in the nasal lavage fluid was 39.5+/-2.8 microM in healthy volunteers (n=40), 42.4+/-3.0 microM in patients with mild allergy (mild group, n=32), and 88.7+/-6.6 microM in patients with severe allergy (severe group, n=61). In the patients whose symptoms were improved with treatment, NO2-/NO3- levels decreased to 45.7+/-10.4 microM. The concentration of cyclic GMP in nasal lavage fluid was higher in the severe group than in the healthy volunteers. The mucosal haemoglobin index was 88+/-4 in the healthy volunteers, 67+/-4 in the mild group, and 53+/-2 in the severe group. The formation of nitrotyrosine was expressed 0.58+/-10% to total tyrosine in the severe group (n=11), but was not found in non-allergy patients (n=9).
The production of NO was increased in patients with perennial nasal allergy, but the blood flow in the nasal mucosa of patients was reduced. Nitrotyrosine formation suggests that there is a process of ONOO(-)-induced damage in mucosa of patients with the perennial nasal allergy and this damage may limit the dilatation of blood vessels, despite the presence of excessive NO.
Nitric oxide (NO) is an important mediator in numerous physiological and pathophysiological events. After thermal injury an increase in plasma and urinary levels has been observed. The real importance of this fact is unknown. The stable NO derivatives (NO2-/NO3-) plasma concentrations were determined in 27 burned patients admitted to the Burn Unit at Santa Maria Hospital in Lisbon at first, third, fifth, seventh, ninth and 15th days and the values were compared with healthy controls (n=9). A significant increase (P<0.05) in burn patient determinations upon admission was found. The patients with inhalation injury revealed greater values compared to the other patients with statistical significance at 5th day (P<0.05). The patients who died showed a NO increase (0.397+/-0.138 vs. 0.267+/-0.017, P> 0.1, day 1) with significance at day 5 (0.615+/-0.223 vs. 0.154+/-0.048, P<0.05). The determinations in patients with sepsis were higher than in the other patients (P<0.01) at day 3. No relation with total burned surface area (TBSA) was found. For the first time, considering burned patients, a significant increase of NO was found in patients who died, in patients with inhalation injury and in patients in sepsis. The possible role of NO in burn injury is discussed. The authors suggest the possible role of NO determination as an indicator of sepsis. The role of NO synthesis inhibitors is discussed. Further studies are needed to clarify these questions.
Failure of GI tract mucosa to act as a barrier against bacterial translocation (BT) has been proposed as a potential source of sepsis and subsequent multiple organ failure post thermal injury. Nitric oxide (NO) is an inorganic radical produced by NO synthase (NOS) from L-arginine. Gut mucosal constitutive NOS (cNOS) provides protection for itself. In contrast to cNOS, inducible NOS (iNOS) releases far greater amounts of NO, promotes oxidative reactions and is responsible for tissue injury. Peroxynitrite formed by the rapid reaction between superoxide and NO, is a toxic substance that contributes to tissue injury in a number of biological systems. This study was designed to investigate the effect of iNOS specific inhibitor S-methylisothiourea (SMT) on the postburn intestinal mucosal barrier function and the possible mechanism of SMT's action. Female SPF Sprague Dawley rats underwent 35% total body surface area (TBSA) or sham burn. Either SMT or the same volume of saline was given (5 mg/kg, i.p. q 12 h) for 2 days to assess the effect of iNOS inhibition. On postburn day 2, the intestinal mucosal cNOS and iNOS activity were assayed by using Griess' reagent, the mesenteric lymph node (MLN), spleen and liver were collected and cultured for BT assay and the cellular localization of nitrotyrosine, a marker for peroxynitrite activity, was examined by immunostaining. After thermal injury in rats, administration of SMT for 2 days decreased the intestinal mucosal iNOS activity/ tNOS activity ratio and the BT incidence. Nitrotyrosine immunostaining of the intestinal mucosa showed a decrease in the SMT-treated group. These findings suggest that SMT, a specific inhibitor for iNOS improves the barrier function after burn by suppression of the intestinal mucosal iNOS activity. The decrease in NO production resulted in decreased formation of peroxynitrite and subsequently decreased damage of mucosal tissue.
Recent studies on smoke inhalation injury have been focused on nitric oxide (NO) as an essential factor of progressive lung injury. We studied the effects of inducible nitric oxide synthase (iNOS) inhibition on inhalation injury in sheep. Sheep (n = 14) were prepared surgically for chronic study. After recovery period, the sheep received 48 breaths of cotton smoke. The animals were then randomised into two groups: MEG group [30 mg/kg mercaptoethylguanidine (MEG), selective inhibitor of iNOS and peroxynitrite scavenger, was given 1 h after injury and then 8 h for 41 h, n = 7] and control group (0.9% NaCl, n = 7). All animals were ventilated mechanically, and airway blood flow was measured using colored microspheres. In the control group, following significant increase in airway blood flow, deterioration in the PaO2/FiO2 ratio was observed. Whereas in the MEG group, it was not observed. In addition, the MEG group did not show significant increase in pulmonary vascular resistance and intrapulmonary shunt fraction. Lung wet/dry ratios, a marker of pulmonary edema, were significantly lower in the MEG group. At 48 h after injury, lung tissue-conjugated dienes, an index of lung oxidative tissue injury, were significantly lower in the MEG group than in the control group. Our data suggest that 1) iNOS-NO produced in the airway circulation plays a major role on the significant increase in airway blood flow, which may contribute to the spread of injury from injured airway to the lung parenchyma; 2) iNOS-NO induced in the pulmonary circulation contributes to the loss of hypoxic pulmonary vasoconstriction; and 3) iNOS-NO plays an important role on the lung oxidative tissue injury.
The role of nitric oxide and related synthase in thermal injury was investigated by using models of experimental burn to evaluate severity from the aspect of vascular permeability. Thermal injuries were produced in the murine right ear by pinching with a pair of preheated tweezers. Immediately thereafter, Evans blue dye was intravenously administered, and the mice injured with burns were sacrificed at various times. The burned ears were collected and hydrolyzed, and the level of extracted dye was measured as an indicator of inflammation. Vascular hyperpermeability was suppressed by the administration of nitric oxide synthase inhibitors. LNAME not only suppressed vascular hyperpermeability in thermal injuries in a dose-dependent manner but was also effective with either prophylactic or therapeutic administration. Although aminoguanidine also suppressed the inflammatory response, it had no effect on the early inflammatory phase. Nitric oxide synthase is well known to have two types of isozymes. Aminoguanidine, an inhibitor specific to inducible nitric oxide synthase, suppressed the late phase 6 h after injury, suggesting that inducible nitric oxide synthase is involved in inflammatory responses of thermal injuries. These results also demonstrated that inducible nitric oxide synthase-like protein stained the burned region immunohistochemically. Therefore, both types of enzymes mediating nitric oxide affect inflammatory responses, i.e., vascular hyperpermeability, and their regulation may lead to the development of new therapy for thermal injuries.
A gas mediator, nitric oxide is converted to peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of the tyrosine residues of proteins, and it enhances the late response of inflammation. The determination of nitrated tyrosine, nitrotyrosine, which is a stable final metabolite of peroxynitrite, provides an important indicator of tissue disorders caused by peroxynitrite. This paper reports a competitive solid-phase immunoassay for measuring nitrotyrosine in various biological specimens. In this study, peroxidase-conjugated nitrotyrosine was prepared by reaction of nitrotyrosine with 1,4-benzoquinone treatment, and then it was allowed to compete with nitrotyrosine on an anti-nitrotyrosine antibody-coated 96-well multiplate. No amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at the concentrations higher than 1000 times the concentration of nitrotyrosine. The limit of detection of free nitrotyrosine was approximately 500 pg/mL (2 nM) at a competition ratio (B/B(o)%) of 80%. The newly developed enzyme immunoassay (EIA) method was used for assay of nitrotyrosine in biological specimens, with the following results: (i) Lipopolysaccharide (LPS) activation of RAW264.7 cells induced a significant increase in nitrotyrosine production compared to that with nonactivated cells. N(omega)-nitro-L-arginine methyl ester decreased nitrotyrosine production with either LPS-activated or nonactivated RAW cells. There is a relationship between nitrotyrosine production and nitrite ion. (ii) The nitrotyrosine level detected in the plasma specimens from healthy volunteers was 35.21 +/- 4.87 ng/mL (135.4 +/- 18.7 nM). (iii) The concentration of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients was 41.40 +/- 20.96 ng/mL (159.02 +/- 80.6 nM). Thus, the EIA method combines sensitivity and specificity with the ability to process a large number of specimens to quantify nitrotyrosine produced with in vivo and in vitro sources.
The purpose of this study was to evaluate effects of early wound excision on changes in NO and endothelin-1 (ET-1) level in the plasma after extensive burn injury. The effects on vascular permeability and hepatic blood flow (HBF) were also assessed. Male Wistar rats were used for this study. A 30% total body surface area (TBSA) third-degree burn was made on the back. Then animals were divided into four groups. Burn group (n = 13), burn alone; infusion group (n = 13), burn injury and fluid resuscitation; early excision group (n = 13), burn injury, total wound excision at 30 min after the injury followed with immediate allogenic skin graft and fluid resuscitation; and the sham group (n = 15). The sham group and the early excision group did not show significant changes in the NO and ET-1 level in plasma during experimental period, while the burn group and the infusion group showed significant increase in the NO and ET-1. The early excision group also did not show hypovolemia, and the significant decrease in the HBF. These data suggest that the increased NO and ET-1 in plasma following thermal injury were originated from burned tissue and the removal of these injured tissue has beneficial effect on the vascular permeability and the changes in HBF.
To determine whether burn-induced peroxynitrite production and expression of lung inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, CXCR2, macrophage inflammatory protein (MIP)-2, and neutrophil chemokine (KC) are mediated by the c-Jun NH2-terminal kinase (JNK).
Prospective, experimental study.
Research laboratory at a university hospital.
Thermal injury models in the mice.
In experiment 1, specific pathogen-free C57/BL6 mice were subjected to 30% total body surface area third-degree burn over shaved back. At 0 hr, 2 hrs, 4 hrs, and 6 hrs after burn, lung tissues of those mice were harvested for JNK activity assay, AP-1 DNA-binding activity, and pJNK immunohistochemistry. In experiment 2, a specific JNK inhibitor, SP600125, was given (30 mg/kg intraperitoneally) to mice immediately postburn to suppress the JNK activity. At 8 hrs after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine (DHR) 123 oxidation. Lung tissues were harvested for myeloperoxidase (MPO) determination, ICAM-1, VCAM-1, CXCR2, KC, MIP-2, interleukin-1beta, and interleukin-6 messenger RNA expression; iNOS immunohistochemical staining; and histologic studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye.
The JNK activity and AP-1 DNA-binding activity of lung tissue significantly increased to a peak at 2 hrs and 4 hrs, respectively, after thermal injury. Immunohistochemical study demonstrated that the increase of the pJNK was mostly from the bronchiole epithelial cells. This increase of MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased six-fold, nine-fold, and four-fold after burn. SP600125 administration obliterated the thermal injury-induced JNK activity, AP-1 DNA-binding activity, and iNOS expression in lung tissue. SP600125 treatment also significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 54%, 8%, and 47%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. Furthermore, SP600125 abolished thermal injury-induced ICAM-1, VCAM-1, CXCR2, MIP-2, and KC but not interleukin-1beta and interleukin-6 messenger RNA levels of lung tissues.
Thermal injury induces lung tissue JNK activation and AP-1 DNA-binding activity mainly from airway epithelial cells. Thermal injury-induced peroxynitrite production and lung iNOS, ICAM-1, and VCAM-1 expression are mediated by the JNK signaling. JNK inhibition decreases thermal injury-induced lung neutrophil infiltration and subsequently pulmonary hyperpermeability.
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Sugao, Miyamae, Kawasaki 216-0015, Japan e-mail: h2inoue@marianna-u.ac.jp H. Inoue : M. Tomioka : M. Shimokawa : N. Kumagai Department of Plastic and Reconstructive Surgery, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki 216-0015, Japan References
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