Rochelle L G, Kruszyna H, Kruszyna R, Barchowsky A, Wilcox D E, Smith R P
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835.
Toxicol Appl Pharmacol. 1994 Sep;128(1):123-8. doi: 10.1006/taap.1994.1189.
Sodium nitroprusside (Na2[(CN)5FeNO], SNP), which is stable, diamagnetic, and not detectable by electron paramagnetic resonance (EPR) spectroscopy, can be activated by one-electron reduction. The initial product, which retains the five cyanides and is here called penta, has a distinctive EPR signal. Penta spontaneously dissociates the trans-cyanide ligand resulting in a second paramagnetic species called tetra, which has a different and distinctive EPR signal. Tetra is able to transfer its NO ligand to a suitable acceptor, and all four equatorial cyanides subsequently dissociate. However, excess free cyanide shifts the tetra-penta equilibrium in the direction of penta and prevents NO release. This study was an attempt to extend the above results on SNP reduction, which were obtained in a model hemoglobin system, to intact porcine cells by characterizing all EPR-detectable intermediates. When porcine aortic endothelial or smooth muscle cells in culture were incubated under anaerobic conditions with SNP, an EPR spectrum was obtained, which could be resolved into the signal for penta and a signal previously described as a nonheme iron-nitrosyl-sulfur complex, Fe-NOSR. Tetra was not detected. This FeNOSR has some differences in its stability and location from that described by others in activated macrophages. When incubations were carried out under air, penta could not be detected, but a somewhat diminished signal for FeNOSR was still detectable. When incubations were carried out in the presence of excess free cyanide, conditions under which reduced SNP does not nitrosylate hemoglobin, the penta signal became stronger and the FeNOSR signal, though decreased, was still observed. Depletion (95%) of intracellular reduced glutathione in endothelial cells had no effect on the FeNOSR signal strength. We conclude that SNP is activated in porcine endothelial cells by a one-electron reduction to penta, which apparently dissociates its trans-cyanide to form tetra which then goes on to form FeNOSR upon reaction with a membrane-bound thiol. Glutathione is not involved in any of these reactions.
硝普钠（Na2[(CN)5FeNO]，SNP）稳定、抗磁性且无法通过电子顺磁共振（EPR）光谱检测到，可通过单电子还原被激活。最初的产物保留了五个氰化物，在此称为五氰化物，具有独特的EPR信号。五氰化物会自发解离反式氰化物配体，产生第二种顺磁性物质，称为四氰化物，其具有不同且独特的EPR信号。四氰化物能够将其NO配体转移至合适的受体，随后所有四个赤道面氰化物都会解离。然而，过量的游离氰化物会使四氰化物 - 五氰化物平衡向五氰化物方向移动，并阻止NO释放。本研究试图通过表征所有EPR可检测的中间体，将上述在模型血红蛋白系统中获得的关于SNP还原的结果扩展至完整的猪细胞。当在厌氧条件下用SNP孵育培养的猪主动脉内皮细胞或平滑肌细胞时，获得了一个EPR光谱，该光谱可分解为五氰化物的信号和先前描述为非血红素铁 - 亚硝酰 - 硫复合物（Fe - NOSR）的信号。未检测到四氰化物。这种FeNOSR在稳定性和位置上与其他文献中描述的活化巨噬细胞中的有所不同。当在空气中进行孵育时，无法检测到五氰化物，但仍可检测到强度有所减弱的FeNOSR信号。当在过量游离氰化物存在的条件下进行孵育时，即还原型SNP不会使血红蛋白亚硝化的条件下，五氰化物信号变强，FeNOSR信号虽有所降低但仍可观察到。内皮细胞中细胞内还原型谷胱甘肽减少95%对FeNOSR信号强度没有影响。我们得出结论，SNP在猪内皮细胞中通过单电子还原被激活为五氰化物，五氰化物显然会解离其反式氰化物以形成四氰化物，然后四氰化物与膜结合的硫醇反应形成FeNOSR。谷胱甘肽不参与这些反应中的任何一个。
