Lamas S, Michel T, Brenner B M, Marsden P A
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Am J Physiol. 1991 Oct;261(4 Pt 1):C634-41. doi: 10.1152/ajpcell.1991.261.4.C634.
Nitric oxide (NO) release accounts for the biological activity of endothelium-derived relaxing factor. Given that tumor necrosis factor-alpha (TNF-alpha) has been implicated as an important mediator in septic shock, we explored whether TNF-alpha enhances L-arginine-dependent synthesis of NO and L-citrulline in endothelial cells. The release of NO was detected in a coincubation bioassay where measurement of guanosine 3',5'-cyclic monophosphate (cGMP) production in reporter monolayers, namely glomerular mesangial cells or fetal lung fibroblasts, reflected activation of soluble guanylate cyclase. Reporter monolayer cGMP content was greater in the presence of TNF-alpha-treated bovine aortic and renal artery endothelial cells than in the presence of vehicle-treated endothelial cells. TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were first evident at 8 h and maximal at 16-24 h. In addition, TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were abrogated by hemoglobin and methylene blue, blunted by N omega-nitro-L-arginine and augmented by superoxide dismutase and the calcium agonist bradykinin. These observations suggested that TNF-alpha enhanced release of NO. Furthermore, the formation of L-[14C]citrulline from L-[14C]arginine, as determined by quantitative cation-exchange chromatography and thin-layer chromatography, was enhanced by TNF-alpha in a time- and concentration-dependent manner. Thus it is evident that endothelial cells release NO for a prolonged period in response to TNF-alpha and transiently when stimulated with calcium agonists. The prolonged release of NO from TNF-alpha-stimulated endothelial cells may be implicated in the pathogenesis of septic shock.
一氧化氮（NO）的释放是内皮源性舒张因子生物活性的原因。鉴于肿瘤坏死因子-α（TNF-α）被认为是脓毒性休克中的一种重要介质，我们探讨了TNF-α是否会增强内皮细胞中L-精氨酸依赖性的NO和L-瓜氨酸合成。在共孵育生物测定中检测到NO的释放，其中在报告单层细胞（即肾小球系膜细胞或胎儿肺成纤维细胞）中测量鸟苷3',5'-环磷酸（cGMP）的产生反映了可溶性鸟苷酸环化酶的激活。在存在经TNF-α处理的牛主动脉和肾动脉内皮细胞的情况下，报告单层细胞的cGMP含量高于存在经载体处理的内皮细胞的情况。TNF-α刺激的内皮依赖性增加报告单层细胞cGMP含量在8小时时首次明显，在16 - 24小时时达到最大值。此外，TNF-α刺激的内皮依赖性增加报告单层细胞cGMP含量被血红蛋白和亚甲蓝消除，被Nω-硝基-L-精氨酸减弱，并被超氧化物歧化酶和钙激动剂缓激肽增强。这些观察结果表明TNF-α增强了NO的释放。此外，通过定量阳离子交换色谱法和薄层色谱法测定，TNF-α以时间和浓度依赖性方式增强了L-[14C]瓜氨酸从L-[14C]精氨酸的形成。因此，很明显内皮细胞在响应TNF-α时会长时间释放NO，而在用钙激动剂刺激时会短暂释放。TNF-α刺激的内皮细胞中NO的长时间释放可能与脓毒性休克的发病机制有关。
