Vo Phuong A, Lad Bhavini, Tomlinson James A P, Francis Stephanie, Ahluwalia Amrita
Clinical Pharmacology, William Harvey Research Institute, Barts and The London, Charterhouse Square, London, EC1M 6BQ, United Kingdom.
J Biol Chem. 2005 Feb 25;280(8):7236-43. doi: 10.1074/jbc.M411317200. Epub 2004 Dec 6.
Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.
诱导型一氧化氮合酶（iNOS）产生的一氧化氮（NO）是脓毒症诱导的低血压的原因，并且在随后的多器官功能衰竭中起主要作用。本研究旨在阐明内皮源性NO在脂多糖（LPS）诱导的分离大鼠主动脉环iNOS表达中的作用。暴露于LPS（1μg/ml，5小时）导致主动脉环中去氧肾上腺素预收缩张力的逆转（70.7±3.2%）。这种舒张与iNOS表达和核因子κB（NF-κB）激活有关。iNOS蛋白的阳性免疫反应定位于LPS处理过的主动脉环的中层和外膜层。去除内皮使主动脉环对LPS诱导的舒张产生抗性（8.9±4.5%）。对这些环进行蛋白质免疫印迹显示不存在iNOS表达。然而，用NO供体二乙胺- NONOate（0.1μM）处理去内皮的环，可使LPS诱导的舒张（61.6±6.6%）和iNOS表达恢复到与内皮完整的动脉相当的水平。使用格尔德霉素和根赤壳菌素（热休克蛋白90的抑制剂）阻断内皮型一氧化氮合酶（eNOS）的激活，在内皮完整的动脉中可抑制LPS诱导的舒张和LPS诱导的iNOS表达（分别为9.0±8.0%和2.0±6.2%）。此外，野生型小鼠经LPS处理（12.5mg/kg，静脉注射，15小时）导致血浆[NOx]测量值显著升高，而在eNOS基因敲除动物中降低了约50%。此外，在野生型组织中明显的LPS诱导的血管反应性变化和iNOS表达，在取自eNOS基因敲除动物的组织中被显著抑制。总之，这些数据表明eNOS衍生的NO部分通过激活NF-κB来调节LPS诱导的iNOS表达。本研究首次证明了血管eNOS在脓毒症中的促炎作用。
