Partial purification of a pramipexole-induced trophic activity directed at dopamine neurons in ventral mesencephalic cultures
Partial purification of a pramipexole-induced trophic activity directed at dopamine neurons in ventral mesencephalic cultures
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DOI：
10.1016/s0006-8993(98)00088-2
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发表时间：
1998-04
期刊：
影响因子：
2.9
通讯作者：
Z. Ling;C. Tong;P. Carvey
中科院分区：
文献类型：
--
作者：
Z. Ling;C. Tong;P. Carvey
我们之前证明，在普拉克索（PPX）和其他具有多巴胺（DA） d3特性的药物存在的情况下，暴露于腹侧中脑（VM）培养物的培养基可以增加受体VM培养物中DA神经元的生长和存活。这种营养活性是热不稳定的，在顶叶皮层培养物或用DA神经元毒素MPP+预处理的培养物中不存在。为了开始确定导致这种营养效应的蛋白质，我们比较了正常VM培养物的条件培养基，用PPX孵育的VM培养物，以及用MPP+预处理和PPX处理的VM培养物。使用抗gdnf和抗bdnf的中和研究未能降低条件介质转移效应，而Millipore Ultrafree离心研究将mol.wt.置于。30 kDa左右的活度。SDS分离显示了三个潜在的感兴趣的波段。在正常培养物中存在一个35 kda的条带，在ppx培养物中增加，而在MPP+预处理/ ppx培养物中没有。这与用于生产这些凝胶的浓缩蛋白对VM培养中DA神经元生长的影响一致。由于VM培养物生长在抑制神经胶质细胞生长的神经基础培养基中，仍然以剂量依赖的方式对PPX作出反应，因此营养活性可能是DA自养因子。然而，凝胶在~ 31和55 kDa处也显示出两条因暴露于PPX而减少的条带，这些条带存在于MPP+预处理的培养物中。因此不能排除这些神经抑制因子也受PPX调节的可能性。
We previously demonstrated that media conditioned by exposure to ventral mesencephalic (VM) cultures in the presence of pramipexole (PPX) and other drugs with dopamine (DA) D3properties, increased the growth and survival of DA neurons in recipient VM cultures. This trophic activity was heat-labile and not present in parietal cortex cultures or cultures pretreated with the DA neuron toxin MPP+. In an effort to begin to identify the protein(s) responsible for this trophic effect, we compared the conditioned media from normal VM cultures, VM cultures incubated with PPX, and VM cultures pretreated with MPP+and treated with PPX. Neutralization studies using anti-GDNF and anti-BDNF failed to reduce the conditioned media transfer effect, and Millipore Ultrafree centrifugation studies placed the mol.wt. of the activity around 30 kDa. SDS separation revealed three potential bands of interest. A 35-kDa band was present in normal cultures, increased in PPX-incubated cultures, and absent in MPP+-pretreated/PPX-incubated cultures. This conforms to the effect the protein concentrates used to produce these gels had on the growth of DA neurons in VM cultures. Since VM cultures grown in neural basal media, which inhibits the growth of glia, still responded to PPX in a dose-dependent fashion, the trophic activity may be a DA autotrophic factor. However, the gels also revealed two bands at ∼31 and 55 kDa that were reduced by exposure to PPX and present in MPP+-pretreated cultures. The possibility that these are neuroinhibitory factors that are also regulated by PPX therefore cannot be ruled out.