Kumagai Y, Midorikawa K, Nakai Y, Yoshikawa T, Kushida K, Homma-Takeda S, Shimojo N
Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, Ibaraki, Japan.
Eur J Pharmacol. 1998 Nov 6;360(2-3):213-8. doi: 10.1016/s0014-2999(98)00666-9.
6-Anilino-5,8-quinolinedione (LY83583) has been widely used as an agent to reduce levels of nitric oxide (NO)-dependent cGMP in tissues. We report here that suppression of NO formation and production of superoxide during enzymatic reduction of LY83583 by neuronal NO synthase appeared to be potentially involved in the pharmacological action caused by LY83583. LY83583 suppressed neuronal NO synthase activity of 20,000 x g rat cerebellar supernatant preparation in a concentration-dependent manner (IC50 value = 12.9 microM). A kinetic study revealed that LY83583 is a competitive inhibitor with respect to NADPH, with a Ki value of 2.57 microM. With purified neuronal NO synthase it was found that LY83583 was a potent inhibitor of NO formation by the enzyme and served as efficient substrate for reduction with a specific activity of 173 nmol of NADPH oxidized per mg of protein per minute. The reductase activity was stimulated about 19.8-fold by addition of CaCl2/calmodulin, indicating that the presence of CaCl2/calmodulin is essential to express maximal activity of LY83583 reduction. Although LY83583 was a good substrate for both NADPH-cytochrome P450 reductase (P450 reductase) and DT-diaphorase, these flavin enzymes-catalyzed reductions of LY83583 were less than the neuronal NO synthase-mediated reduction in the presence of CaCl2/calmodulin. Enzymatic generation of superoxide during reduction of LY83583 by neuronal NO synthase, P450 reductase or DT-diaphorase was confirmed by electron spin resonance (ESR) experiments. Thus the present results indicate that a benzoquinone derivative LY83583 appears to interact with the P450 reductase domain on neuronal NO synthase, resulting in inhibition of NO formation and superoxide generation, which is involved in suppression of intracellular cGMP content.
6-苯胺基-5,8-喹啉二酮（LY83583）已被广泛用作降低组织中一氧化氮（NO）依赖性环鸟苷酸（cGMP）水平的药物。我们在此报告，神经元型一氧化氮合酶在酶促还原LY83583过程中对NO形成的抑制作用以及超氧化物的产生，似乎可能参与了LY83583引起的药理作用。LY83583以浓度依赖性方式抑制20,000 x g大鼠小脑上清液制剂中的神经元型一氧化氮合酶活性（IC50值 = 12.9 microM）。动力学研究表明，LY83583是NADPH的竞争性抑制剂，Ki值为2.57 microM。使用纯化的神经元型一氧化氮合酶发现，LY83583是该酶形成NO的有效抑制剂，并作为还原的有效底物，其比活性为每分钟每毫克蛋白质氧化173 nmol NADPH。通过添加CaCl2/钙调蛋白，还原酶活性被刺激约19.8倍，表明CaCl2/钙调蛋白的存在对于表达LY83583还原的最大活性至关重要。尽管LY83583是NADPH-细胞色素P450还原酶（P450还原酶）和DT-黄递酶的良好底物，但在存在CaCl2/钙调蛋白的情况下，这些黄素酶催化的LY83583还原作用小于神经元型一氧化氮合酶介导的还原作用。通过电子自旋共振（ESR）实验证实了神经元型一氧化氮合酶、P450还原酶或DT-黄递酶在还原LY83583过程中超氧化物的酶促生成。因此，目前的结果表明，苯醌衍生物LY83583似乎与神经元型一氧化氮合酶上的P450还原酶结构域相互作用，导致NO形成和超氧化物生成受到抑制，这与细胞内cGMP含量的抑制有关。
