Ishii M, Shimizu S, Yamamoto T, Momose K, Kuroiwa Y
Department of Clinical Pharmacy and Pharmacology, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo, Japan.
Life Sci. 1997;61(7):739-47. doi: 10.1016/s0024-3205(97)00538-9.
The purpose of this study was to examine whether nitric oxide (NO) synthase dysfunction accompanied with decrease in tetrahydrobiopterin (BH4) content increases H2O2-induced endothelial cell death. Endothelial cell death was measured by the release of intracellular lactate dehydrogenase (LDH). Intracellular BH4 content was changed by pretreatment with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, or pretreatment with sepiapterin, a substrate for the salvage pathway of BH4 synthesis, and the intracellular content was measured by high performance liquid chromatography equipped with a fluorescence detector. Moreover, production of superoxide was detected by a chemiluminescence technique using MCLA, a Cypridina luciferin analogue, for the superoxide-sensitive probe. Pretreatment with DAHP (10 mM) for 24 h decreased intracellular BH4 content to 14% and increased H2O2-induced cell death. The toxic effect of DAHP was reduced by co-pretreatment with sepiapterin (100 microM) or treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mM), an inhibitor of NO synthase, but not by N(G)-methyl-L-arginine (L-NMA, 1 mM), the other inhibitor of NO synthase. Moreover, production of superoxide in endothelial cells induced by Ca2+-ionophore ionomycin (1 microM) increased by the pretreatment with DAHP, and the increase in superoxide production was blocked by L-NAME (1 mM) but not L-NMA (1 mM). Co-pretreatment with sepiapterin decreased the production of superoxide. These findings suggested that dysfunction of NO synthase with a decrease in BH4 content in endothelial cells produced superoxide instead of NO and increased the oxidative stress-induced endothelial cell death.
本研究的目的是检验一氧化氮（NO）合酶功能障碍伴随四氢生物蝶呤（BH4）含量降低是否会增加过氧化氢（H2O2）诱导的内皮细胞死亡。通过细胞内乳酸脱氢酶（LDH）的释放来测定内皮细胞死亡。用2,4 - 二氨基 - 6 - 羟基嘧啶（DAHP，一种GTP环水解酶I的抑制剂）预处理或用蝶呤（BH4合成补救途径的底物）预处理来改变细胞内BH4含量，并通过配备荧光检测器的高效液相色谱法测量细胞内含量。此外，使用MCLA（一种海萤荧光素类似物，作为超氧化物敏感探针）的化学发光技术检测超氧化物的产生。用10 mM DAHP预处理24小时可使细胞内BH4含量降至14%，并增加H2O2诱导的细胞死亡。与蝶呤（100 microM）共同预处理或用NO合酶抑制剂N(G)-硝基 - L - 精氨酸甲酯（L - NAME，1 mM）处理可降低DAHP的毒性作用，但用另一种NO合酶抑制剂N(G)-甲基 - L - 精氨酸（L - NMA，1 mM）处理则无效。此外，用DAHP预处理可增加由钙离子载体离子霉素（1 microM）诱导的内皮细胞中超氧化物的产生，而超氧化物产生的增加可被L - NAME（1 mM）阻断，但不能被L - NMA（1 mM）阻断。与蝶呤共同预处理可减少超氧化物的产生。这些发现表明，内皮细胞中NO合酶功能障碍伴BH4含量降低会产生超氧化物而非NO，并增加氧化应激诱导的内皮细胞死亡。
