Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012
Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012
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DOI：
10.1016/j.freeradbiomed.2003.10.012
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发表时间：
2004-01-01
影响因子：
7.4
通讯作者：
Munzel, T
中科院分区：
文献类型：
--
作者：
Daiber, A;August, M;Munzel, T
本文研究了化学发光染料8-氨基-5-氯-7-苯基吡啶并[3，4-d]哒嗪-1，4-（2 H，3 H）二酮钠盐（L-012）对超氧阴离子的检测能力。在人全血或分离的白细胞中，鲁米诺类似物L-012检测超氧化物的灵敏度高于鲁米诺、光泽精、腔肠素和荧光染料二氢乙啶。在对照（新西兰白色）和高脂血症（Watanabe遗传性高脂血症）家兔的分离白细胞和主动脉环中，L-012增强的化学发光成功检测了基础条件下和蛋白激酶C直接激活剂佛波醇12，13-二丁酸酯（PDBu）刺激下超氧化物形成的差异。PDBu的作用被胶毒毒素和蛋白激酶C抑制剂如白屈菜红碱消除，确定NAD（P）H氧化酶为重要的超氧化物源。使用电子顺磁共振和自旋陷阱5-二乙氧基磷酰基-5-甲基-1-吡咯啉-N-氧化物的实验显示，与光泽精相反，L-012不经历氧化还原循环。这些发现表明，L-012增强的化学发光是检测全血、炎症细胞和血管组织中超氧化物的灵敏可靠的探针。(C)2003年爱思唯尔公司All rights reserved.
In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt(L-012)to detect superoxide indifferent biological systems. In human wholeblood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hypertipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue. (C) 2003 Elsevier Inc. All rights reserved.