| Project/Area Number |
07680815
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Nerve anatomy/Neuropathology
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| Research Institution | Chiba University |
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Principal Investigator |
KADOTA Tomoko Chiba Univ.Sch.Med.Anatomy Associate Professor, 医学部, 助教授 (00089864)
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| Co-Investigator(Kenkyū-buntansha) |
MIZOTE Muneaki Teikyo-Heisei Univ.Eng.Inform.Professor, 教授 (70009645)
SAITO Yumiko Tokyo Metro.Inst.Med.Sci.Mol.Biol.Researcher, 研究員 (00215568)
TOYAMA Yoshiro Chiba Univ.Sch.Med.Anatomy Lecturer, 医学部, 講師 (70009637)
MOROI Kayoko Chiba Univ.Sch.Med.Mol.Biol.Research Assistant, 医学部, 助手 (80110352)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
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| Keywords | neurotransmitter transporter / dopamine transporter / growth cone / PC12 cells / immunohistochemistry / nerve growth factor / monoclonal antibody / ノルアドレナリン・トランスポーター / PCR反応 / ウエスタンプロッティング |
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| Research Abstract |
In the present study we have produced monoclonal antibodies (MAbs) against oligopeptides of a dopamine transporter (DAT) protein. Using the antipeptide antibody, we have examined the expression of DAT in the neuron, especially the changes in subcellular localization of DAT during development. In this study we used PC12 cells, which are a clonal line of rat pheochromecytoma cells that secrete dopamine, epinephrine and acetylcholine and develop as neuron-like cells after treatment with nerve growth factor (NGF).
An attempt to produce antipeptide antibodies against the oligopeptides specific in amino acid sequence to a rat dopamine transporter was made with an in vitro immunization method. Two monoclonal antibodies, Mabs H-1a and H-1b, were produced. Western blot analysis confirmed that the above antibodies recognized a -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 c
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onfirmed by an antibody absorption test using two kinds of 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 NGF.The antibody labeled the surface of PC12 cells in control. When the cells were treated with NGF,the expression of DAT was significantly emphasized first in the cytoplasm and then on the surface of growth cones from the beginning of neurite outgrowth. 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-polmerase chain reaction (RT-PCR). DAT mRNA significantly increased in the NGF-treated cells after 7 days of incubation, whereas NET mRNA markedly decreased. Less
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