Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura of the rat.

To determine whether asbestos inhalation induces the formation of reactive nitrogen species, three groups of rats were exposed intermittently over 2 wk to either filtered room air (sham-exposed) or to chrysotile or crocidolite asbestos fibers. The rats were killed at 1 or 6 wk after exposure. At 1 wk, significantly greater numbers of alveolar and pleural macrophages from asbestos-exposed rats than from sham-exposed rats demonstrated inducible nitric oxide synthase protein immunoreactivity. Alveolar macrophages from asbestos-exposed rats also generated significantly greater nitrite formation than did macrophages from sham-exposed rats. Strong immunoreactivity for nitrotyrosine, a marker of peroxynitrite formation, was evident in lungs from chrysotile- and crocidolite-exposed rats at 1 and 6 wk. Staining was most evident at alveolar duct bifurcations and within bronchiolar epithelium, alveolar macrophages, and the visceral and parietal pleural mesothelium. Lungs from sham-exposed rats demonstrated minimal immunoreactivity for nitrotyrosine. Significantly greater quantities of nitrotyrosine were detected by ELISA in lung extracts from asbestos-exposed rats than from sham-exposed rats. These findings suggest that asbestos inhalation can induce inducible nitric oxide synthase activation and peroxynitrite formation in vivo, and provide evidence of a possible alternative mechanism of asbestos-induced injury to that thought to be induced by Fenton reactions.     

Peroxynitrite is an important mediator in thermal injury-induced lung damage

OBJECTIVE: Intestinal ischemia and reperfusion injury was known to cause postinjury multiple organ failure by neutrophil and unclear nonneutrophil factors. Peroxynitrite formed by the rapid reaction between superoxide and nitric oxide, is a toxic substance that contributes to tissue injury in a number of biological systems. In this study, the role of nitric oxide and neutrophils on lung damage after burn was investigated. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the rat. INTERVENTIONS: In experiment 1, specific pathogen-free Sprague-Dawley rats underwent 35% total body surface area burn. At 4, 8, 16, and 24 hrs after burn, intestinal mucosa and lung tissue were harvested for myeloperoxidase (MPO) assay, blood was collected for measurement of peroxynitrite-mediated oxidation of dihydrorhodamine 123, and pulmonary microvascular dysfunction was quantified by measuring the extravasation of Evans blue dye. In experiment 2, polymorphonuclear granulocyte antibody (0.12 mL/100 g administered intraperitoneally 16 hrs before burn), S-methylisothiourea (7.5 mg/kg, intraperitoneally, immediately after burn), a specific inducible nitric oxide synthase inhibitor, and sterile saline (15 mL/kg, intraperitoneally, immediately after burn) were given to different groups of thermally injured animals individually. The plasma dihydrorhodamine 123 oxidation level, intestinal and lung MPO activity, lung permeability, and lung histology were evaluated at 8 hrs after burn. The cellular localization of nitrotyrosine, a marker for peroxynitrite reactivity, was also examined by immunostaining. In experiment 3, 3-morpholinosydnonimine (10 mM, intraperitoneally), a peroxynitrite donor, was given to nonburned rats to examine the peroxynitrite effect on lung inducible nitric oxide synthase expression. MEASUREMENTS AND MAIN RESULTS: The level of MPO activity in intestine and lung, blood dihydrorhodamine 123 oxidation, and lung permeability were increased up to 2-fold, 2.5-fold, 2-fold, and 2-fold of normal, respectively, at 8 hrs after burn. S-methylisothiourea injection significantly decreased (p <.05) 31% of the lung MPO activity, 41% of the blood peroxynitrite level, 54% of the lung permeability, and the lung peroxynitrite production in burned rats. Polymorphonuclear granulocyte antibody pretreatment significantly decreased 60% of the intestinal MPO, 92% of the blood peroxynitrite level, and 56% the lung MPO activity in burned rats, but the lung permeability was only slightly decreased by polymorphonuclear granulocyte antibody pretreatment. Furthermore, 3-morpholinosydnonimine increased the lung inducible nitric oxide synthase messenger RNA levels. CONCLUSIONS: Thermal injury induces blood dihydrorhodamine 123 oxidation, intestinal and lung neutrophil deposition, lung nitrotyrosine production, and lung damage. Both specific inhibition of inducible nitric oxide synthase and polymorphonuclear granulocyte antibody pretreatment decrease blood dihydrorhodamine 123 oxidation and intestinal and lung neutrophil deposition, but only inducible nitric oxide synthase inhibition with S-methylisothiourea reduces lung peroxynitrite production and thermal injury-induced lung damage. Nitric oxide and the ensuing peroxynitrite production in lung play a more important role than neutrophil in contributing to thermal injury-induced lung damage.     

Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.

Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10(9) M-1s-1) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections were incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury.     

对一氧化碳中毒迟发性脑病相关因素的研究

目的 观察一氧化碳中毒（COP）后大鼠脑组织一氧化氮合酶（NOS）、外周血内皮细胞计数、血小板体积变化、血小板CD61表达、多形核白细胞粘附分子（CD11b/CD18)表达、海马神经细胞凋亡细胞百分比及线粒体膜电位的改变。方法 Wistar大鼠共16只随机分成正常对照组及一氧化碳中毒组各8只,制作COP动物模型,用流式细胞仪测定大鼠脑组织一氧化氮合酶（NOS）、外周血内皮细胞计数、血小板体积变化、血小板CD61表达、多形核白细胞粘附分子（CD11b/CD18)表达的动态变化,海马神经细胞凋亡细胞百分比及线粒体膜电位的改变,用分光光度法测定血浆组织型纤溶酶原激活剂（tPA)和抑制剂（PAI）。结果 COP后NOS水平降低,血小板活性增高,纤溶系统变化不明显。细胞粘附分子表达增加,线粒体膜电位降低,凋亡细胞数增加。结论 COP后血小板活必增高,细胞粘附分子表达增强,导致脑微小动物血栓形成趋势并引起白细胞浸润造成脑组织损伤,是COP迟发性脑病的重要发病因素之一。     

急性CO中毒大鼠脑组织NO NOS活性变化及纳洛酮的干预效应

目的　探讨急性一氧化碳 (CO)中毒大鼠脑组织中一氧化氮 (NO)和一氧化氮合成酶 (NOS)活性的变化及纳洛酮的治疗作用。方法　健康Wister大鼠 4 5只 ,随机分为 3组 :正常、CO染毒组 (中毒组 )、CO染毒后纳洛酮治疗组 (观察组 )。采用改良的Griess法和分光度法 ,分别测定大鼠大、小脑组织NO和NOS活性。结果　急性CO中毒后大鼠大、小脑组织中NO和NOS活性明显升高 ,与正常组比较P <0 0 1;应用纳洛酮治疗后 ,脑组织中NO和NOS活性明显降低 ,与中毒组比较P <0 0 1。结论　急性CO中毒后大鼠脑组织中NO和NOS活性增高 ,NO、NOS活性改变可能参与了急性CO中毒脑损伤的病理生理过程 ,纳洛酮治疗可降低急性CO中毒大鼠脑组织中NO和NOS活性。     

Elevated serum ubiquitin C-terminal hydrolase-L1 levels in patients with carbon monoxide poisoning

ObjectiveUbiquitin C-terminal hydrolase-L1 (UCH-L1) has been established as a reliable and potential biomarker of neuronal damage after acute neurologic insults, such as ischemic stroke, subarachnoid hemorrhage, and traumatic brain injury. However, the effect of serum UCH-L1 levels has not been investigated in carbon monoxide (CO)-poisoned patients. The aim of the present study was to evaluate whether serum UCH-L1 levels are a reliable marker of brain damage and the association of UCH-L1 with outcome.Design and methodsThis case–control study enrolled 46 CO-poisoned subjects and 30 controls. Using an enzyme-linked immunosorbent assay (ELISA) kit, we studied the temporal profile of serum UCH-L1 levels at 6, 12, 24 and 48 h after acute CO poisoning. Poisoning severity was assessed using the Glasgow Coma Scale (GCS) score. Long-term outcome was assessed using the Glasgow Outcome Scale (GOS) at 6 months after poisoning.ResultsCompared with controls, CO-poisoned patients had significantly elevated serum levels of UCH-L1 at each time point after poisoning. There were significantly higher levels of UCH-L1 in CO-poisoned patients with a lower GCS score as well as in those with a poor 6-month outcome dichotomized GOS.ConclusionsSerum levels of UCH-L1 appear to have potential clinical utility in providing valuable information about poisoning severity and outcome after CO poisoning.     

Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability.

Vulnerable areas of atherosclerotic plaques often contain lipid-laden macrophages and display matrix metalloproteinase activity. We hypothesized that reactive oxygen species released by macrophage-derived foam cells could trigger activation of latent proforms of metalloproteinases in the vascular interstitium. We showed that in vivo generated macrophage foam cells produce superoxide, nitric oxide, and hydrogen peroxide after isolation from hypercholesterolemic rabbits. Effects of these reactive oxygens and that of peroxynitrite, likely to result from simultaneous production of nitric oxide and superoxide, were tested in vitro using metalloproteinases secreted by cultured human vascular smooth muscle cells. Enzymes in culture media or affinity-purified (pro-MMP-2 and MMP-9) were examined by SDS-PAGE zymography, Western blotting, and enzymatic assays. Under the conditions used, incubation with xanthine/xanthine oxidase increased the amount of active gelatinases, while nitric oxide donors had no noticeable effect. Incubation with peroxynitrite resulted in nitration of MMP-2 and endowed it with collagenolytic activity. Hydrogen peroxide treatment showed a catalase-reversible biphasic effect (gelatinase activation at concentrations of 4 microM, inhibition at > or = 10-50 microM). Thus, reactive oxygen species can modulate matrix degradation in areas of high oxidant stress and could therefore contribute to instability of atherosclerotic plaques.     

Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase.

Nitric oxide provokes vasodilation and inhibits platelet aggregation. We examined the effect of nitric oxide on superoxide anion production by three sources: activated intact neutrophils, xanthine oxidase/hypoxanthine, and the NADPH oxidase. Nitric oxide significantly inhibited the generation of superoxide anion by neutrophils exposed to either FMLP (10(-7)M) or PMA (150 ng/ml) (IC50 = 30 microM). To determine whether the effect of nitric oxide on the respiratory burst was due to simple scavenging of O2+, kinetic studies that compared effects on neutrophils and the cell-free xanthine oxidase system were performed. Nitric oxide inhibited O2+ produced by xanthine oxidase only when added simultaneously with substrate, consistent with the short half-life of NO in oxygenated solution. In contrast, the addition of nitric oxide to neutrophils 20 min before FMLP resulted in the inhibition of O2+ production, which suggests formation of a stable intermediate. The effect of nitric oxide on the cell-free NADPH oxidase superoxide-generating system was also examined: The addition of NO before arachidonate activation (t = -6 min) significantly inhibited superoxide anion production. Nitric oxide did not inhibit O2+ when added at NADPH initiation (t = 0). Treatment of the membrane but not cytosolic component of the oxidase was sufficient to inhibit O2+ generation. The data suggest that nitric oxide inhibits neutrophil O2+ production via direct effects on membrane components of the NADPH oxidase. This action must occur before the assembly of the activated complex.     

Presence of nitrotyrosine with minimal inducible nitric oxide synthase induction in lipopolysaccharide-treated pigs

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

The potential value of the protein S-100B level as a criterion for hyperbaric oxygen treatment and prognostic marker in carbon monoxide poisoned patients

Carbon monoxide (CO) poisoning resulting in diffuse tissue hypoxia. Cerebral hypoxia is a major cause of morbidity and mortality after CO poisoning. There are some clinical criteria that could help a physician to make a decision concerning the application of hyperbaric oxygenation therapy. However, it would be convenient to discover an objective biochemical serum marker that could help in the grade evaluation of CO poisoning and indication of therapy in CO-poisoned patients. We present two case reports where the established criteria for the CO poisoning were not optimum for the decision regarding therapy. It seems that the S-100B protein could be used as a biochemical marker of CO induced brain injury. S-100B values could perhaps help us to select patients for hyperbaric oxygen therapy and to predict the short and long term outcome.     

急性一氧化碳中毒大鼠迟发性神经元损伤与记忆功能改变

目的：观察急性一氧化碳中毒（CO）中毒大鼠脑内迟发神经元损伤和记忆功能的改变，探讨CO中毒导致的迟发性神经损伤和记忆功能改变两者之间的关系。方法：SD大鼠暴露在空气或CO（3451ppm）60min，暴露后1、3、5、7d处死大鼠，大脑经处理制成石蜡切片，行HE染色以观察脑内病理损伤程度，通过被动回辟跳台实验评估CO中毒对大鼠记忆保持巩固能力的影响。结果：大鼠海马CA1区发生迟发性神经元损伤。锥体细胞从CO暴露后第3天开始显著减少，被动回避跳台实验中，CO中毒大鼠从染毒后第5天开始跳台潜伏期明显缩短，记忆力部分丧失，出现迟发性健忘症（delayed amnesia,DA)。结论：急性CO中毒导致迟发性神经元损伤，迟发性神经元损伤引起DA。     

急性一氧化碳中毒大鼠记忆功能改变与迟发性神经元损伤的相关性

背景:急性CO中毒大鼠可能发生迟发性健忘症,与人急性CO中毒导致的迟发性神经综合征相似,所以实验拟通过对急性CO中毒大鼠的研究来探讨迟发性神经综合征的发病机制。目的:观察急性CO中毒大鼠脑内迟发性神经元损伤和记忆功能的改变,并分析两者之间的关系。设计:随机对照动物实验。单位:解放军第四军医大学唐都医院急诊科;西安高新医院检验中心;解放军北京解放军空军总医院;解放军第四军医大学航空航天医学系高压氧治疗中心。材料:实验于2005-07/11在解放军第四军医大学航空航天医学系航空病理学和分子生物学实验室进行。取健康雄性Sprague-Dawley(SD)大鼠50只随机分为对照组和染毒组各25只。方法:将染毒组清醒大鼠放入自制染毒罐中,然后向罐内注入体积分数为0.999的CO气体。大鼠在罐内静式吸入CO与空气的混合气体,CO平均体积分数为3.451×10-3,60min后出罐。对照组不干预。主要观察指标:①记忆能力:染毒前和暴露后1,3,5,7d进行大鼠跳台实验,以跳台潜伏期为评价记忆保持巩固能力的指标,跳台潜伏期越短,记忆能力越差。②脑组织病理改变:暴露后1,3,5,7d跳台实验后,两组各处死6只大鼠,取脑,行苏木精-伊红染色以观察脑内病理损伤程度和海马CA1区锥体细胞数。③应用SPSS 10.0软件分析海马CA1区锥体细胞数与组大鼠跳台潜伏期间的关系。结果:48只大鼠进入结果分析。①跳台潜伏期:CO暴露后1,3d,两组相比没有差别,但在CO暴露后第5和7天,染毒组明显短于对照组(P<0.05,0.01)。②海马CA1区锥体细胞数:CO暴露后1d,染毒组与正常对照相比没有明显改变,但是在CO暴露后3,5和7d即明显减少,CO暴露后7d,可以观察到15%的锥体细胞发生死亡。③海马CA1区锥体细胞数减少与染毒组大鼠跳台潜伏期缩短之间有明显的相关性(r=0.270,P<0.01)。具体的主要数据,研究的主要发现(给出统计学显著性检验值)结论:主要结论及其潜在的应用价值。急性CO中毒导致迟发性神经元损伤,迟发性神经元损伤引起迟发性健忘症。     

Role of nitric oxide and peroxynitrite in the cytokine-induced sustained myocardial dysfunction in dogs in vivo.

Studies in vitro suggested that inflammatory cytokines could cause myocardial dysfunction. However, the detailed mechanism for the cytokine-induced myocardial dysfunction in vivo remains to be examined. We thus examined this point in our new canine model in vivo, in which microspheres with and without IL-1beta were injected into the left main coronary artery. Left ventricular ejection fraction (LVEF) was evaluated by echocardiography for 1 wk. Immediately after the microsphere injection, LVEF decreased to approximately 30% in both groups. While LVEF rapidly normalized in 2 d in the control group, it was markedly impaired in the IL-1beta group even at day 7. Pretreatment with dexamethasone or with aminoguanidine, an inhibitor of inducible nitric oxide synthase, prevented the IL-1beta-induced myocardial dysfunction. Nitrotyrosine concentration, an in vivo marker of the peroxynitrite production by nitric oxide and superoxide anion, was significantly higher in the myocardium of the IL-1beta group than in that of the control group or the group cotreated with dexamethasone or aminoguanidine. There was an inverse linear relationship between myocardial nitrotyrosine concentrations and LVEF. These results indicate that IL-1beta induces sustained myocardial dysfunction in vivo and that nitric oxide produced by inducible nitric oxide synthase and the resultant formation of peroxynitrite are substantially involved in the pathogenesis of the cytokine-induced sustained myocardial dysfunction in vivo.     

Sesame oil attenuates multiple organ failure and increases survival rate during endotoxemia in rats

OBJECTIVE: To investigate the effects and the possible mechanism of sesame oil on multiple organ failure induced by lipopolysaccharide in rats. DESIGN: Laboratory in vivo study of the effects of sesame oil on serum aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, total bilirubin, blood urea nitrogen, creatinine, lipid peroxide, and nitric oxide concentrations. To assess the effect of sesame oil on xanthine oxidase, serum uric acid was measured. Furthermore, lipid peroxide concentrations in liver and kidney were determined. SETTING: University laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Blood testing. MEASUREMENT AND MAIN RESULTS: Serum aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, total bilirubin, blood urea nitrogen, creatinine, and uric acid concentrations were determined. Lipid peroxide was analyzed by using a commercial kit. Nitric oxide production was estimated by Griess reaction. Sesame oil ameliorated hepatic and renal damage in a dose-dependent manner and increased animal survival in lipopolysaccharide-treated rats. Sesame oil decreased lipid peroxide concentration in serum but not in liver and kidney. Serum nitrite production was unaffected by sesame oil ingestion. Furthermore, the activity of xanthine oxidase was reduced by sesame oil in lipopolysaccharide-challenged rats. CONCLUSION: Sesame oil ameliorated multiple organ failure and mortality via its inhibition of xanthine oxidase in lipopolysaccharide-dosed rats. Xanthine oxidase may play a critical role in sesame oil-associated organ protection during endotoxemia in rats.     

高压氧对急性一氧化碳中毒大鼠迟发型脑损伤影响的组织病理学特点

背景:临床上有3%～30%的急性一氧化碳中毒患者会发生一氧化碳中毒后迟发性脑病,出现以痴呆、精神症状和锥体外系症状为主的神经系统症状。目前,其发病机制还不清楚。目的:探讨一氧化碳中毒迟发性脑损伤的病理损伤机制,及高压氧对迟发性脑损伤的影响。设计:随机对照动物实验。单位:解放军第四军医大学航空航天医学系航空卫生教研室。材料:实验于2004-03在解放军第四军医大学航空航天医学系航空病理学和分子生物学实验室进行。取清洁级健康雄性Sprague-Dawley大鼠80只,单纯随机分为3组,正常对照组10只,模型组35只,高压氧组35只,后2组又分为染毒后6h、1,3,5,7,14,21d7个时间点,每个时间点5只。方法:①模型组:将大鼠放入染毒罐中熏吸入一氧化碳与空气的混合气体60min,一氧化碳的体积分数保持在2500×10-6,制备急性CO中毒动物模型。②高压氧组:同模型组造模,染毒后3h开始行高压氧治疗,压力0.2MPa,氧的体积分数保持在0.90以上熏整个过程共115min,染毒后前3d2次/d,之后1次/d,每周休息1d。③正常对照组不干预。主要观察指标:①采用组织病理学、免疫组织化学等方法检测大鼠染毒后各时间点大鼠脑组织病理改变的特点。②通过细胞超微结构观察和原位末端转移酶标记(TUNEL)等方法进行细胞凋亡的检测,观察急性各组大鼠脑神经元凋亡的发生情况。结果:造模后大鼠死亡率约为10%。①模型组大鼠脑内发生广泛的病理损伤,脑皮质、海马、纹状体和小脑等部位神经元出现变性坏死,其中大脑皮质、海马等部位损伤较重。苏木精-伊红、TUNEL染色和电镜观察表明大鼠海马神经元发生凋亡,凋亡神经元从染毒后第3天开始显著增加,第7天达到高峰穴P<0.01雪,以后逐渐减少。②高压氧组:与模型组相比,脑内神经元变性坏死明显减轻,各时间点大鼠海马区损伤均轻于模型组;凋亡神经元数目减少,尤以中毒后5和7d明显(P<0.01)。高压氧促进模型大鼠海马区Bcl-2蛋白表达熏尤以CO暴露后3,5d明显(P<0.01)。结论:①急性一氧化碳中毒大鼠出现广泛的迟发性神经元损伤,表现为迟发的神经元坏死和凋亡。②高压氧治疗可以有效减少变性坏死神经元熏促进凋亡抑制基因bcl-2表达熏从而抑制神经元坏死和凋亡。     

Time course of nitric oxide,peroxynitrite, and antioxidants in the endotoxemic heart

OBJECTIVES: To determine the time course for myocardial production of nitric oxide, peroxynitrite, and glutathione, to determine the activities of the myocardial antioxidant enzymes glutathione peroxidase, superoxide dismutase, and glutathione reductase throughout endotoxemia and into recovery, and to correlate the levels of these variables to left ventricular contractility in endotoxemia. DESIGN: Rats were treated with lipopolysaccharide. Endotoxemic hearts were examined at baseline, 4, 16, 24, and 48 hrs after lipopolysaccharide. Saline time-control groups were treated identically. SETTING: A pulmonary research laboratory of a university teaching hospital. MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide administration resulted in decreased contractility at 16 hrs as assessed by the isolated papillary muscle technique. Contractility recovered by 24 hrs. Myocardial glutathione content initially increased, but it was decreased from baseline by 16 hrs, as was glutathione peroxidase activity. Both superoxide dismutase and glutathione reductase activities were increased early (4 hrs) and remained elevated throughout the course of the experiment. Myocardial nitric oxide content (assessed by the chemiluminescence technique) was increased by 4 hrs and was markedly elevated by 16 hrs. Nitric oxide levels remained elevated despite recovery of contractility at 24 hrs. Similarly, peroxynitrite (assessed by measurement of 3-nitrotyrosine by high-pressure liquid chromatography) was elevated at 16 hrs and remained elevated despite normalization of contractility at 24 and 48 hrs. CONCLUSIONS: Myocardial dysfunction in endotoxemia correlates mainly with decreased glutathione content and glutathione peroxidase activity rather than nitric oxide or peroxynitrite formation. These data indicate that lipopolysaccharide-induced myocardial dysfunction is not solely caused by elevated myocardial nitric oxide levels but rather caused by the sum of complex interactions between various oxygen- and nitrogen-derived radicals.     

Evidence for the involvement of nitric oxide in 3,4-methylenedioxymethamphetamine-induced serotonin depletion in the rat brain

Production of reactive oxygen and/or nitrogen species has been thought to contribute to the long-term depletion of brain dopamine and serotonin (5-HT) produced by amphetamine derivatives, i.e., methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA). In the present study, the effects of nitric-oxide synthase (NOS) inhibitors were examined on the long-term depletion of striatal dopamine and/or 5-HT produced by the local perfusion of malonate and MDMA or the systemic administration of MDMA. The effect of MDMA on nitric oxide formation and nitrotyrosine concentration also was determined. Perfusion with MDMA and malonate resulted in a 34% reduction of 5-HT and 49% reduction of dopamine concentrations in the striatum. The systemic administration of NOS inhibitors, N(omega)-nitro-l-arginine methyl ester hydrochloride and S-methyl-l-thiocitrulline (S-MTC), and the peroxynitrite decomposition catalyst Fe(III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride attenuated the MDMA- and malonate-induced depletion of striatal dopamine and 5-HT. S-MTC also attenuated the depletion of 5-HT in the striatum produced by the systemic administration of MDMA without attenuating MDMA-induced hyperthermia. Additionally, the systemic administration of MDMA significantly increased the formation of nitric oxide and the nitrotyrosine concentration in the striatum. These results support the conclusion that MDMA produces reactive nitrogen species in the rat that contribute to the neurotoxicity of this amphetamine analog.     

Oxidative inactivation of nitric oxide and endothelial dysfunction in stroke-prone spontaneous hypertensive rats

This study tested the hypothesis that increased nitric oxide (NO) inactivation and concurrent peroxynitrite formation is responsible for endothelial dysfunction in the spontaneously hypertensive stroke-prone rat (SHRSP). In SHRSP, the aortic vasorelaxation to acetylcholine (ACh) was decreased (p < 0.05), but NO production was unchanged. Nitrotyrosine staining, a footprint of peroxynitrite (ONOO(-)) formation, was detected. Exposure of SHRSP to a high-salt, high-fat diet (SFD) further exacerbated hypertension and accelerated end-organ disease. A severe endothelial dysfunction [maximal ACh relaxation: 49.8 +/- 2.1 versus 94.5 +/- 1.8% in Wistar-Kyoto rats (WKY), p < 0.01], increased basal NO production (482 +/- 17 versus 356 +/- 21 nM, p < 0.01), decreased ACh-stimulated NO production (57 +/- 6 versus 112 +/- 6 nM, p < 0.01), extensive inducible NO synthase and nitrotyrosine staining, elevated nitrotyrosine content (21-fold increase over WKY), and a high percentage of cells with DNA damage were observed in the aortic tissues from these animals. Treatment of SHRSP on SFD with carvedilol restored ACh-induced vasorelaxation and NO production, inhibited nitrotyrosine formation, reduced vascular cell DNA damage, and reduced end-organ injury. These data demonstrate that endothelial dysfunction was caused by increased NO inactivation alone (SHRSP) or in combination with decreased NO production from endothelial NO synthase (SHRSP on SFD). Antioxidant treatment with carvedilol exerted significant vascular protective effects, attenuated end-organ damage, and decreased mortality under these conditions.