Kadota T, Yamaai T, Saito Y, Akita Y, Kawashima S, Moroi K, Inagaki N, Kadota K
Department of Anatomy, Chiba University Medical School, Japan.
J Histochem Cytochem. 1996 Sep;44(9):989-96. doi: 10.1177/44.9.8773564.
The four kinds of oligopeptides specific in amino acid sequence to a rat dopamine transporter (DAT), peptide-1-peptide-4, were chemically synthethized. An attempt to produce antipeptide antibodies against these oligopeptides was made with an in vitro immunization method. Two monoclonal antibodies, MAbs H-1a and H-1b, were produced against one of the oligopeptides, peptide-1. Western blot analysis confirmed that the two antibodies recognized an approximately 85,000 Da protein in a synaptosomal fraction prepared from the rat striatum but none in the fraction from the cerebellum. The specificity of the antibody to DAT was also confirmed by an antibody absorption test using two synthetic oligopeptides, one of which is specific only to DAT. These results have confirmed the specificity of the present antibody to DAT. The expression and subcellular localization of DAT were immunohistochemically examined with MAbs H-1a and H-1b in PC12 cells treated with nerve growth factor (NGF). The antibody labeled the surface of PC12 cells. When the cells were treated with NGF, the expression of DAT was significantly emphasized, first in the area mainly including the Golgi apparatus and rough endoplasmic reticulum and then on the surface of growth cones from the beginning of neurite outgrowth. DAT was detected by Western blot analysis in a microsomal fraction prepared from PC12 cells. The activity of DAT in the PC12 cells was pharmacologically confirmed by the uptake of [3H]-dopamine and blockade by uptake inhibitors. The NGF treatment doubled the dopamine uptake activity. GBR12909, a specific inhibitor of DAT, blocked the [3H]-dopamine at a concentration of 10(-7) M. The expression of DAT and norepinephrine transporter (NET) mRNA in the PC12 cells was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). DAT mRNA significantly increased in the NGF-treated cells after 7 days of incubation, whereas NET mRNA markedly decreased.
化学合成了对大鼠多巴胺转运体（DAT）氨基酸序列具有特异性的四种寡肽，即肽-1至肽-4。采用体外免疫法尝试制备针对这些寡肽的抗肽抗体。针对其中一种寡肽肽-1产生了两种单克隆抗体，即单克隆抗体H-1a和H-1b。蛋白质印迹分析证实，这两种抗体识别从大鼠纹状体制备的突触体组分中一种约85000 Da的蛋白质，但在小脑组分中未识别到。使用两种合成寡肽进行抗体吸收试验也证实了该抗体对DAT的特异性，其中一种寡肽仅对DAT具有特异性。这些结果证实了本抗体对DAT的特异性。用单克隆抗体H-1a和H-1b对经神经生长因子（NGF）处理的PC12细胞进行免疫组织化学检查，以检测DAT的表达和亚细胞定位。该抗体标记了PC12细胞的表面。当细胞用NGF处理时，DAT的表达首先在主要包括高尔基体和粗面内质网的区域显著增强，然后从神经突生长开始就在生长锥表面增强。通过蛋白质印迹分析在从PC12细胞制备的微粒体组分中检测到了DAT。通过[3H] - 多巴胺摄取和摄取抑制剂阻断，药理学上证实了PC12细胞中DAT的活性。NGF处理使多巴胺摄取活性增加了一倍。DAT的特异性抑制剂GBR12909在浓度为10^(-7) M时阻断了[3H] - 多巴胺摄取。通过逆转录 - 聚合酶链反应（RT - PCR）检测PC12细胞中DAT和去甲肾上腺素转运体（NET）mRNA的表达。孵育7天后，NGF处理的细胞中DAT mRNA显著增加，而NET mRNA明显减少。
