Nunes Carla, Almeida Leonor, Laranjinha João
Laboratory of Biochemistry, Faculty of Pharmacy and Center for Neurosciences and Cell Biology, University of Coimbra, Couraça dos Apóstolos, 51, r/c, 3000 Coimbra, Portugal.
Neurochem Int. 2005 Aug;47(3):173-82. doi: 10.1016/j.neuint.2005.03.005.
The inhibition of mitochondrial respiration by nitric oxide (.NO) at cytochrome c oxidase level has been established as a physiological regulatory mechanism of mitochondrial function. Given, on the one hand, the potential involvement of .NO and dopamine metabolism in mitochondrial dysfunction associated with neurodegeneration and, on the other hand, the reported interaction of .NO with dihydroxyphenylacetic acid (DOPAC), a major mitochondrial-associated dopamine metabolite, we examined the combined effects of .NO and DOPAC on the respiratory chain of isolated rat brain mitochondria. Whereas dopamine or DOPAC induced no measurable effects on the mitochondrial respiration rate, a mixture of .NO with DOPAC inhibited the rate in a way stronger than that exerted by .NO. This effect was noticed with actively respiring (state 3) and resting (state 4) mitochondria. At variance with DOPAC, dopamine failed to potentiate .NO inhibitory effects. The inhibition was dependent on the concentration of both compounds, .NO and DOPAC, and exhibited characteristics similar to those exerted by .NO, namely: it was reversible and dependent on the concentration of oxygen. Analysis of respiratory enzymatic activities demonstrated a selective inhibition at the level of cytochrome c oxidase (complex IV). Insights into the chemical mechanisms underlying the inhibitory effect were inferred from experiments using metmyoglobin (a ligand for .NO and derived species, such as nitroxyl anion) and ferrocyanide (a reductant of .NO, producing nitroxyl anion). Whereas metmyoglobin decreased the inhibition, ferrocyanide potentiated the inhibition. Moreover, a mixture of ferrocyanide with .NO reproduced the effects exerted by the mixture of .NO with DOPAC. The results are consistent with the notion of a reaction of .NO with DOPAC producing a nitric oxide-derived compound(s), which inhibit O2 uptake at the cytochrome oxidase level. Although the mechanism in question remains to be clearly elucidated it is suggested that the .NO/DOPAC-dependent inhibition of cytochrome oxidase may involve nitroxyl anion. The significance of these observations for mitochondrial dysfunction inherent in Parkinson's disease is discussed.
一氧化氮（·NO）在细胞色素c氧化酶水平对线粒体呼吸的抑制作用已被确立为线粒体功能的一种生理调节机制。一方面，鉴于·NO和多巴胺代谢可能参与与神经退行性变相关的线粒体功能障碍，另一方面，据报道·NO与二羟基苯乙酸（DOPAC，一种主要的线粒体相关多巴胺代谢产物）存在相互作用，我们研究了·NO和DOPAC对分离的大鼠脑线粒体呼吸链的联合作用。多巴胺或DOPAC对线粒体呼吸速率没有可测量的影响，而·NO与DOPAC的混合物对呼吸速率的抑制作用比·NO单独作用时更强。在活跃呼吸（状态3）和静止（状态4）的线粒体中均观察到这种效应。与DOPAC不同，多巴胺未能增强·NO的抑制作用。这种抑制作用取决于·NO和DOPAC这两种化合物的浓度，并且表现出与·NO单独作用时相似的特征，即：它是可逆的，并且依赖于氧气浓度。呼吸酶活性分析表明，在细胞色素c氧化酶（复合体IV）水平存在选择性抑制。通过使用高铁肌红蛋白（·NO及其衍生物种如硝酰阴离子的配体）和亚铁氰化物（·NO的还原剂，产生硝酰阴离子）的实验推断出了抑制作用背后的化学机制。高铁肌红蛋白可降低抑制作用，而亚铁氰化物则增强抑制作用。此外，亚铁氰化物与·NO的混合物重现了·NO与DOPAC混合物所产生的效应。这些结果与·NO与DOPAC反应生成一种一氧化氮衍生化合物的观点一致，该化合物在细胞色素氧化酶水平抑制氧气摄取。尽管相关机制仍有待明确阐明，但提示·NO/DOPAC依赖的细胞色素氧化酶抑制作用可能涉及硝酰阴离子。本文讨论了这些观察结果对帕金森病内在线粒体功能障碍的意义。
