DOPAMINE-INDUCED APOPTOSIS OF PC12 CELLS

多巴胺（DA）的细胞代谢产生H2 O2，其在铁的存在下进一步还原为羟基自由基。DA衍生的羟基自由基造成的细胞损伤被认为是导致帕金森病的原因。以前，我们提出，含有活性半胱氨酸残基的蛋白质的氧化DA衍生的过氧化氢可能是主要负责DA诱导的神经细胞凋亡，并在帕金森病的发病机制中发挥重要作用。在这里，我们研究了蛋白质氧化的羟基自由基和PC 12细胞凋亡之间的相关性。羟基自由基引起的蛋白质最常见的修饰之一是将羰基引入侧链。含羰基的蛋白质可以被二硝基苯肼特异性修饰，并通过二硝基苯残基抗体的免疫印迹分析检测。DA诱导的细胞凋亡的进展，测量细胞活力以及CPP 32，caspase家族的成员，裂解，与蛋白质羰基形成进行了比较。暴露于3 mM DA的PC 12细胞导致时间依赖性细胞死亡，在6 h时达到最大效应的一半。用3 mM DA处理也引起前体CPP 32的切割，这是不明显的前3小时，但几乎完成后6小时与DA处理。细胞死亡和CPP 32的激活被部分阻止NAC的存在。然而，通过免疫印迹分析检测到的羰基形成在DA治疗的6小时期间保持在背景水平，并且不受NAC的影响。看来，在PC 12细胞中游离铁的细胞内浓度是非常低的，没有铁的H2 O2的羟基自由基的细胞凋亡的转化没有进行到一个显着的程度。在支持这一前提，蛋白质羰基形成时，细胞内的铁水平增加处理PC 12细胞与铁和8-羟基喹啉的膜渗透复合物。这些结果表明，羟基自由基造成的损害是最小的，当PC 12细胞单独用DA处理，不太可能是细胞凋亡的直接原因。- 多巴胺，细胞凋亡，羟自由基，PC 12细胞，铁

Cellular metabolism of dopamine (DA) generates H2O2, which is further reduced to hydroxyl radicals in the presence of iron. Cellular damage inflicted by DA-derived hydroxyl radicals is thought to contribute to Parkinsons disease. Previously we proposed that the oxidation of proteins that contain reactive Cys residues by DA-derived H2O2 might be largely responsible for DA-induced apoptosis in neuronal cells and play an important role in the pathogenesis of Parkinsons disease. Here, we investigated the correlation between protein oxidation by hydroxyl radicals and apoptosis of PC12 cells. One of the most common modification of proteins caused by hydroxyl radicals is the introduction of carbonyl groups into the side chains. Carbonyl group- containing proteins can be specifically modified by dinitrophenylhydrazine and detected by immunoblot analysis with antibodies to dinitrophenyl residues. The progress of DA-induced apoptosis, measured by cell viability as well as the cleavage of CPP32, a member of the caspase family, was compared with protein carbonyl formation. Exposure of PC12 cells to 3 mM DA resulted in time-dependent cell death with half maximal effect at 6 h. Treatment with 3 mM DA also caused the cleavage of precursor CPP32, which was not obvious for the first 3 h but nearly completed 6 h after treatment with DA. Both the cell death and CPP32 activation were partially prevented by the presence of NAC. However, carbonyl group formation detected by immunoblot analysis remained at background levels over the 6 h period of DA treatment and was not affected by NAC. It appears that the intracellular concentration of free iron was very low in PC12 cells and without iron the conversion of H2O2 to hydroxyl radicals apoptosis did not proceed to a significant extent. In support of this premise, protein carbonyl formation occurred when the intracellular iron level was increased by treating PC12 cellss with a membrane permeable complex of iron and 8-hydroxyquinoline. These results indicate that damage caused by hydroxyl radicals is minimal when PC12 cells were treated with DA alone and is not likely to be the immediate cause of apoptosis. - dopamine, apoptosis, hydroxyl radicals, PC 12 cells, iron