Mayer B, Brunner F, Schmidt K
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria.
Biochem Pharmacol. 1993 Jan 26;45(2):367-74. doi: 10.1016/0006-2952(93)90072-5.
Methylene blue appears to inhibit nitric oxide-stimulated soluble guanylyl cyclase and has been widely used for inhibition of cGMP-mediated processes. We report here that endothelium-dependent relaxation of isolated blood vessels and NO synthase-dependent cGMP formation in cultured endothelial cells were both markedly more sensitive to inhibition by methylene blue than effects induced by direct activation of soluble guanylyl cyclase. These discrepancies were also observed when superoxide dismutase (SOD) was present to protect NO from inactivation by superoxide anion. Subsequent experiments showed that formation of L-citrulline by purified NO synthase was completely inhibited by 30 microM methylene blue (IC50 = 5.3 and 9.2 microM in the absence and presence of SOD, respectively), whereas guanylyl cyclase stimulated by S-nitrosoglutathione was far less sensitive to the drug (50% inhibition at approximately 60 microM, and maximal inhibition of 72% at 1 mM methylene blue). Experimental evidence indicated that oxidation of NADPH, tetrahydrobiopterin or reduced flavins does not account for the inhibitory effects of methylene blue. Our data suggest that methylene blue acts as a direct inhibitor of NO synthase and is a much less specific and potent inhibitor of guanylyl cyclase than hitherto assumed.
亚甲蓝似乎能抑制一氧化氮刺激的可溶性鸟苷酸环化酶，并且已被广泛用于抑制环磷酸鸟苷（cGMP）介导的过程。我们在此报告，离体血管的内皮依赖性舒张以及培养的内皮细胞中一氧化氮合酶依赖性cGMP的形成，对亚甲蓝抑制作用的敏感性均明显高于可溶性鸟苷酸环化酶直接激活所诱导的效应。当存在超氧化物歧化酶（SOD）以保护一氧化氮不被超氧阴离子灭活时，也观察到了这些差异。随后的实验表明，纯化的一氧化氮合酶形成L-瓜氨酸的过程被30微摩尔亚甲蓝完全抑制（在不存在和存在SOD的情况下，半数抑制浓度分别为5.3和9.2微摩尔），而由S-亚硝基谷胱甘肽刺激的鸟苷酸环化酶对该药物的敏感性要低得多（在约60微摩尔时抑制50%，在1毫摩尔亚甲蓝时最大抑制率为72%）。实验证据表明，烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、四氢生物蝶呤或还原型黄素的氧化并不能解释亚甲蓝的抑制作用。我们的数据表明，亚甲蓝作为一氧化氮合酶的直接抑制剂，其作为鸟苷酸环化酶抑制剂的特异性和效力比迄今所认为的要低得多。
