Beltrán B, Orsi A, Clementi E, Moncada S
The Wolfson Institute for Biomedical Research, The Cruciform Building, University College London, Gower Street, London WCIE 6BT.
Br J Pharmacol. 2000 Mar;129(5):953-60. doi: 10.1038/sj.bjp.0703147.
The effect of prolonged exposure to nitric oxide on enzymes involved in cell metabolism was investigated in T lymphocyte-derived Jurkat and L929 fibroblast human cell lines using a constant concentration of nitric oxide (1.5 microM) released by the nitric oxide donor DETA-NO (0.5 mM). Nitric oxide inhibited immediately the respiration of the cells acting reversibly at complex IV. With time, the inhibition became progressively persistent, i.e. not reversed by trapping of nitric oxide with oxyhaemoglobin, and was preceded by a decrease in the concentration of the intracellular reduced glutathione. This persistent effect of nitric oxide on respiration was due to inhibition of complex I activity which could be reversed by addition of reduced glutathione or by cold light, suggesting that it was due to S-nitrosylation of thiols necessary for the activity of the enzyme. The activity of other enzymes also known to be susceptible to inhibition by S-nitrosylation, i.e. glyceraldehyde-3-phosphate dehydrogenase and glutathione reductase, was progressively decreased by exposure to nitric oxide with a similar time course to that observed for the inhibition of complex I. Furthermore, inhibition of these enzymes only occurred when the concentrations of reduced glutathione had previously fallen and could be prevented by increasing the intracellular concentrations of reduced glutathione. Our results suggest that S-nitrosylation of different enzymes by nitric oxide may occur only if the reducing potential of the cells is impaired.
使用一氧化氮供体DETA-NO（0.5 mM）释放的恒定浓度一氧化氮（1.5 microM），在T淋巴细胞衍生的Jurkat和L929成纤维细胞人细胞系中研究了长时间暴露于一氧化氮对参与细胞代谢的酶的影响。一氧化氮立即抑制细胞呼吸，在复合体IV处可逆地起作用。随着时间的推移，抑制作用逐渐持续，即不能通过与氧合血红蛋白捕获一氧化氮来逆转，并且在此之前细胞内还原型谷胱甘肽的浓度会降低。一氧化氮对呼吸的这种持续作用是由于复合体I活性的抑制，这可以通过添加还原型谷胱甘肽或冷光来逆转，表明这是由于该酶活性所需的硫醇的S-亚硝基化。已知其他也易受S-亚硝基化抑制的酶，即甘油醛-3-磷酸脱氢酶和谷胱甘肽还原酶，通过暴露于一氧化氮而逐渐降低，其时间进程与复合体I抑制所观察到的相似。此外，只有当还原型谷胱甘肽的浓度先前已经下降时，这些酶的抑制才会发生，并且可以通过增加细胞内还原型谷胱甘肽的浓度来预防。我们的结果表明，只有当细胞的还原电位受损时，一氧化氮才可能对不同的酶进行S-亚硝基化。
