1. Peroxynitrite, a cytotoxic oxidant formed from the reaction of nitric oxide (NO) and superoxide is a mediator of cellular injury in ischaemia/reperfusion injury, shock and inflammation. Here we investigated whether L-buthionine-(S,R)-sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, alters endothelial and vascular smooth muscle injury in response to peroxynitrite in vitro and during endotoxic shock in vivo. 2. In human umbilical vein endothelial cells and in rat aortic smooth muscle cells, BSO (1 mM, for 24 h) enhanced, whereas glutathione (3 mM) or glutathione ethyl ester (3 mM) attenuated the peroxynitrite (100-1000 microM)-induced suppression of mitochondrial respiration (measured by the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan), formation of nitrotyrosine (detected by Western blotting), protein oxidation (measured by detection of 2,4 dinitrophenylhydrazine-reactive carbonyls), and DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) (measured by the incorporation of radiolabelled NAD+ into nuclear proteins and by the alkaline unwinding assay, respectively). Glutathione ethyl ester treatment reduced the BSO-induced enhancement of peroxynitrite-induced cytotoxicity. 3. In rat isolated thoracic aortic rings, BSO treatment (in vivo, at 1 g kg(-1) intraperitoneally (i.p.) for 24 h) enhanced, whereas pretreatment with glutathione (in vitro, 3 mM) attenuated the peroxynitrite-induced reduction of the contractions to noradrenaline, and the peroxynitrite-induced impairment of the endothelium-dependent relaxations to acetylcholine. 4. In BSO-pretreated rats, treatment with bacterial lipopolysaccharide (LPS, 15 mg kg(-1), i.p., for 6 h) caused a more pronounced vascular hyporeactivity and endothelial dysfunction ex vivo. BSO pretreatment also increased the degree of nitrotyrosine staining (detected by imunohistochemistry) in the aorta after LPS treatment. 5. In conclusion, our results demonstrate that L-buthionine-(S,R)-sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase enhances peroxynitrite- and endotoxic shock-induced vascular failure. Based on these findings, we suggest that endogenous glutathione plays an important protective role against peroxynitrite- and LPS-induced vascular injury.

中文翻译：

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γ-谷氨酰胺半胱氨酸合成酶抑制剂L-丁硫氨酸-（S，R）-磺胺嘧啶对过氧亚硝酸盐和内毒素性休克引起的血管衰竭的作用。

1.过氧亚硝酸盐，一种由一氧化氮（NO）和超氧化物反应形成的细胞毒性氧化剂，是缺血/再灌注损伤，休克和炎症中细胞损伤的介质。在这里，我们研究了γ-谷氨酰半胱氨酸合成酶的抑制剂L-丁硫氨酸-（S，R）-硫代亚胺（BSO）是否在体外和体内内毒素休克期间改变了对过氧亚硝酸盐的内皮和血管平滑肌损伤。2.在人脐静脉内皮细胞和大鼠主动脉平滑肌细胞中，BSO（1 mM，持续24 h）增强，而谷胱甘肽（3 mM）或谷胱甘肽乙酯（3 mM）减弱过氧亚硝酸盐（100-1000 microM）诱导的线粒体呼吸抑制（通过将3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）转化为甲maz来测量），硝基酪氨酸的形成（通过蛋白质印迹法检测），蛋白质氧化（通过检测2,4二硝基苯基肼反应性羰基化合物测量），DNA单链断裂和核酶聚（ADP-核糖）合成酶（PARS）的活化（通过将放射性标记的NAD +掺入到核蛋白中，并分别通过碱性解旋分析）。谷胱甘肽乙酯处理减少了BSO诱导的过氧亚硝酸盐诱导的细胞毒性增强。3.在大鼠离体的胸主动脉环中，BSO处理（体内，腹膜内（ip）1 g kg（-1）处24 h）增强，而谷胱甘肽预处理（体外3 mM）减弱了过氧亚硝酸盐诱导的还原收缩至去甲肾上腺素，以及过氧亚硝酸盐诱导的内皮依赖性舒张损伤乙酰胆碱。4。在用BSO预处理的大鼠中，细菌脂多糖（LPS，15 mg kg（-1），腹腔注射，持续6 h）治疗导致更明显的血管反应性降低和离体内皮功能障碍。BPS预处理还增加了LPS治疗后主动脉中硝基酪氨酸染色的程度（通过免疫组织化学检测）。5.总之，我们的结果表明，γ-谷氨酰半胱氨酸合成酶的抑制剂L-丁硫氨酸-（S，R）-亚砜胺可增强过氧亚硝酸盐和内毒素性休克引起的血管衰竭。基于这些发现，我们建议内源性谷胱甘肽对过氧亚硝酸盐和LPS诱导的血管损伤起着重要的保护作用。持续6 h）引起更明显的血管反应性低和离体内皮功能障碍。BPS预处理还增加了LPS治疗后主动脉中硝基酪氨酸染色的程度（通过免疫组织化学检测）。5.总之，我们的结果表明，γ-谷氨酰半胱氨酸合成酶的抑制剂L-丁硫氨酸-（S，R）-亚砜胺可增强过氧亚硝酸盐和内毒素性休克引起的血管衰竭。基于这些发现，我们建议内源性谷胱甘肽对过氧亚硝酸盐和LPS诱导的血管损伤起重要的保护作用。持续6 h）引起更明显的血管反应性低和离体内皮功能障碍。BSO预处理还增加了LPS治疗后主动脉中硝基酪氨酸染色的程度（通过免疫组织化学检测）。5.总之，我们的结果表明，γ-谷氨酰半胱氨酸合成酶的抑制剂L-丁硫氨酸-（S，R）-亚砜胺可增强过氧亚硝酸盐和内毒素性休克引起的血管衰竭。基于这些发现，我们建议内源性谷胱甘肽对过氧亚硝酸盐和LPS诱导的血管损伤起着重要的保护作用。γ-谷氨酰半胱氨酸合成酶抑制剂可增强过氧亚硝酸盐和内毒素休克引起的血管衰竭。基于这些发现，我们建议内源性谷胱甘肽对过氧亚硝酸盐和LPS诱导的血管损伤起着重要的保护作用。γ-谷氨酰半胱氨酸合成酶抑制剂可增强过氧亚硝酸盐和内毒素休克引起的血管衰竭。基于这些发现，我们建议内源性谷胱甘肽对过氧亚硝酸盐和LPS诱导的血管损伤起着重要的保护作用。