Stuehr D J, Fasehun O A, Kwon N S, Gross S S, Gonzalez J A, Levi R, Nathan C F
Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York 10021.
FASEB J. 1991 Jan;5(1):98-103. doi: 10.1096/fasebj.5.1.1703974.
The cofactor requirements of macrophage nitric oxide (NO.) synthase suggest involvement of an NADPH-dependent flavoprotein. This prompted us to test the effect of the flavoprotein inhibitors diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodoniumdiphenyl (ID) on the NO. synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO. synthesis by mouse macrophages, their lysates, and partially purified macrophage NO. synthase. Inhibition of NO. synthase by these agents was potent (IC50's 50-150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP+, or 2'5'-ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5-diphosphoribose, protected NO. synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once NO. synthase was inhibited by DPI, neither NADPH nor FAD could restore its activity. DPI also inhibited acetylcholine-induced relaxation of norepinephrine-preconstricted rabbit aortic rings (IC50 300 nM). Inhibition of acetylcholine-induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine-induced vasoconstriction itself nor on vasorelaxation induced by the NO.-generating agent sodium nitroprusside. These results suggest that NO. synthesis in both macrophages and endothelial cells depends on an NADPH-utilizing flavoprotein. As a new class of NO. synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO(.).
巨噬细胞一氧化氮（NO.）合酶对辅助因子的需求表明有一个依赖NADPH的黄素蛋白参与其中。这促使我们测试黄素蛋白抑制剂二亚苯基碘鎓（DPI）、二 - 2 - 噻吩基碘鎓（DTI）和碘鎓二苯基（ID）对巨噬细胞和内皮细胞NO.合酶的作用。DPI、DTI和ID完全抑制了小鼠巨噬细胞、其裂解物以及部分纯化的巨噬细胞NO.合酶的NO.合成。这些试剂对NO.合酶的抑制作用很强（IC50为50 - 150 nM），是不可逆的，依赖于时间和温度，且与酶催化无关。DPI的抑制作用可被NADPH、NADP + 或2'5'-ADP阻断，但不能被NADH阻断。同样，FAD或FMN可保护NO.合酶免受DPI的抑制，而核黄素或腺苷5 - 二磷酸核糖则不能。NADPH和FAD都不与DPI反应。一旦NO.合酶被DPI抑制，NADPH和FAD都不能恢复其活性。DPI还抑制乙酰胆碱诱导的去甲肾上腺素预收缩兔主动脉环的舒张（IC50为300 nM）。在DPI被洗脱后，乙酰胆碱诱导的舒张抑制至少持续2小时。相反，DPI对去甲肾上腺素本身诱导的血管收缩以及由NO.生成剂硝普钠诱导的血管舒张均无影响。这些结果表明，巨噬细胞和内皮细胞中的NO.合成均依赖于一种利用NADPH的黄素蛋白。作为一类新型的NO.合酶抑制剂，DPI及其类似物可能在分析NO(.)的生理和病理生理作用方面很有用。
