Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells
Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells
复制标题
DOI：
10.1111/j.1471-4159.2004.02577.x
复制
发表时间：
2004-09-01
影响因子：
4.7
通讯作者：
Kagan, VE
中科院分区：
文献类型：
--
作者：
Jiang, JF;Borisenko, GG;Kagan, VE
环氧合酶-2（考克斯-2）被认为通过产生自由基诱导神经元氧化应激。虽然氧自由基不直接参与考克斯-2-催化循环，但超氧阴离子自由基已被反复报道在考克斯-2相关的氧化应激中起关键作用。为了解决这一争议，我们的特点是生产自由基的PC 12细胞中，考克斯-2的表达是操纵基因或直接的蛋白质转染，并比较它们与那些产生的重组考克斯-2在无细胞系统。利用自旋捕获器α-（4-吡啶基-1-氧化物）-N-叔丁基硝酮、5，5-二甲基-1-吡咯啉-N-氧化物和4-（（9-吖啶羰基）氨基）-2，2，6，6-四甲基哌啶-1-氧自由基（Ac-Tempo），我们观察到花生四烯酸（AA）依赖的血红素重组考克斯-2产生碳中心自由基。未检测到氧自由基或硫代自由基。考克斯-2还催化AA依赖的抗坏血酸单电子共氧化为抗坏血酸自由基。接下来，我们使用了两种不同的细胞中考克斯-2表达方法，PCXII细胞表达异丙基-1-硫代-β-D-吡喃半乳糖苷诱导型考克斯-2，PC 12细胞使用蛋白质递送试剂Chariot转染考克斯-2。在这两种模型中，考克斯-2依赖AA诱导产生的碳中心自由基记录使用自旋陷阱和Ac-Tempo。在转染考克斯-2的细胞中，无论是自旋捕集器还是荧光探针二氢乙锭均未检测到氧自由基的形成。在抗坏血酸存在下，检测到AA诱导的考克斯-2依赖性抗坏血酸自由基。AA引起一个重要的和选择性的氧化的主要磷脂，磷脂酰丝氨酸（PS）。PS不是考克斯-2的直接底物，但在AA存在下被共氧化。自由基生成和PS氧化被抑制考克斯-2抑制剂，尼氟灭酸，尼美舒利，或NS-398。因此，考克斯-2产生的碳中心自由基，而不是氧自由基或硫代自由基是负责AA攻击的PC 12细胞过表达考克斯-2的氧化应激。
Cyclo-oxygenase-2 (COX-2) is believed to induce neuronal oxidative stress via production of radicals. While oxygen radicals are not directly involved in COX-2-catalytic cycle, superoxide anion radicals have been repeatedly reported to play a critical role in COX-2-associated oxidative stress. To resolve the controversy, we characterized production of free radicals in PC12 cells in which COX-2 expression was manipulated either genetically or by direct protein transfection and compared them with those generated by a recombinant COX-2 in a cell-free system. Using spin-traps alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide and 4-((9-acridinecarbonyl) amino)-2,2,6,6- tetramethylpiperidine-1-oxyl (Ac-Tempo), we observed arachidonic acid (AA)-dependent production of carbon-centered radicals by heme-reconstituted recombinant COX-2. No oxygen radicals or thiyl radicals have been detected. COX-2 also catalyzed AA-dependent one-electron co-oxidation of ascorbate to ascorbate radicals. Next, we used two different approaches of COX-2 expression in cells, PCXII cells which express isopropyl-1-thio-beta-D-galactopyranoside inducible COX-2, and PC12 cells transfected with COX-2 using a protein delivery reagent, Chariot. In both models, COX-2-dependent AA-induced generation of carbon-centered radicals was documented using spin-traps and Ac-Tempo. No oxygen radical formation was detected in COX-2-transfected cells by either spin-traps or fluorogenic probe, dihydroethidium. In the presence of ascorbate, AA-induced COX-2-dependent ascorbate radicals were detected. AA caused a significant and selective oxidation of one of the major phospholipids, phosphatidylserine (PS). PS was not a direct substrate for COX-2 but was co-oxidized in the presence of AA. The radical generation and PS oxidation were inhibited by COX-2 inhibitors, niflumic acid, nimesulide, or NS-398. Thus, COX-2 generated carbon-centered radicals but not oxygen radicals or thiyl radicals are responsible for oxidative stress in AA-challenged PC12 cells overexpressing COX-2.