Zhang David X, Zou Ai-Ping, Li Pin-Lan
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee 53226, USA.
Am J Physiol Heart Circ Physiol. 2003 Feb;284(2):H605-12. doi: 10.1152/ajpheart.00697.2002. Epub 2002 Nov 7.
We tested the hypothesis that ceramide induces endothelial dysfunction in small coronary arteries via NADPH oxidase-mediated superoxide and resulting peroxynitrite formation. With the use of dihydroethidium as a superoxide indicator, C(2)-ceramide was found to increase superoxide production in the endothelial cells of small coronary arteries, which was inhibited by the NADPH oxidase inhibitors N-vanillylnonanamide, apocynin, and diphenylene iodonium. NADPH oxidase expression was confirmed in endothelial cells, as indicated by the immunoblotting of its subunits gp91(phox) and p47(phox). C(2)-ceramide increased NADPH oxidase activity by 52%, which was blocked by NADPH oxidase inhibitors but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes. By Western blot analysis, ceramide-induced NADPH oxidase activation was found to be associated with the translocation of p47(phox) to the membrane. In isolated and pressurized small coronary arteries, N-vanillylnonanamide, apocynin, or uric acid, a peroxynitrite scavenger, largely restored the inhibitory effects of ceramide on bradykinin- and A-23187-induced vasorelaxation. With the use of nitrotyrosine as a marker, C(2)-ceramide was found to increase peroxynitrite in small coronary arteries, which could be blocked by uric acid. We conclude that NADPH oxidase-mediated superoxide production and subsequent peroxynitrite formation mediate ceramide-induced endothelial dysfunction in small coronary arteries.
神经酰胺通过NADPH氧化酶介导的超氧化物生成以及由此产生的过氧亚硝酸盐形成，诱导小冠状动脉内皮功能障碍。使用二氢乙锭作为超氧化物指示剂，发现C(2)-神经酰胺可增加小冠状动脉内皮细胞中的超氧化物生成，而NADPH氧化酶抑制剂N-香草基壬酰胺、白杨素和二亚苯基碘鎓可抑制这种生成。通过对其亚基gp91(phox)和p47(phox)进行免疫印迹分析，证实内皮细胞中有NADPH氧化酶表达。C(2)-神经酰胺使NADPH氧化酶活性增加了52%，这被NADPH氧化酶抑制剂所阻断，但未被一氧化氮合酶、黄嘌呤氧化酶和线粒体电子传递链酶的抑制剂所阻断。通过蛋白质印迹分析发现，神经酰胺诱导的NADPH氧化酶激活与p47(phox)向细胞膜的转位有关。在分离并加压的小冠状动脉中，N-香草基壬酰胺、白杨素或过氧亚硝酸盐清除剂尿酸，在很大程度上恢复了神经酰胺对缓激肽和A-23187诱导的血管舒张的抑制作用。使用硝基酪氨酸作为标志物，发现C(2)-神经酰胺可增加小冠状动脉中的过氧亚硝酸盐，而尿酸可阻断这种增加。我们得出结论，NADPH氧化酶介导的超氧化物生成以及随后的过氧亚硝酸盐形成介导了神经酰胺诱导的小冠状动脉内皮功能障碍。
