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1997 Fiscal Year Final Research Report Summary

Relationship between the development of the nerve cell and the expression of the neurotransmitter transporter

Research Project

Project/Area Number 07680815
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Nerve anatomy/Neuropathology
Research InstitutionChiba University

Principal Investigator

KADOTA Tomoko  Chiba Univ.Sch.Med.Anatomy Associate Professor, 医学部, 助教授 (00089864)

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)
Project Period (FY) 1995 – 1997
Keywordsneurotransmitter transporter / dopamine transporter / growth cone / PC12 cells / immunohistochemistry / nerve growth factor / monoclonal antibody
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 … More 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

  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] Tomoko Kadota: "ntipeptide antibody of dopamine transporter and its expression in the PC12 cells." Acta Anatomica Nipponica. 70. 248-251 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 門田 朋子: "Na^+依存性伝達物質トランスポーター" 細胞. 27. 349-352 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tomoko Kadota: "Expression of dopamine transporter at the tips of growing neurites of PC12 cells." Journal of Histochemistry and Cytochemistry. 44. 989-996 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tomoko Kadota: "Synaptic spinules attendent on post-tetanic potentiation in cat sympathetic ganglion." Proceedings of Japan Academy. 72,SerB. 48-51 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mitsukai Fujita: "Developmental profiles of synaptophysin in granule cells of rat cerebellum: an immunohistocytochemical study." Journal of Electron Microscopy. 45. 185-194 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 門田 朋子: "シナプス機能と膜再循環" 生体の化学. 47. 287-291 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 門田 朋子: "シスプスの基本構造" Clinical Neuroscience. 14. 1-20 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 門田 朋子: "シナプスの形はこんなにも変化する" ミクロスコピア. 14(3). 174-175 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 門田 朋子: "シナプス発見物語-I仮想的シナプスから実在のシナプスへ" ミクロスコピア. 14(4). 240-245 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kadota, T., Yamaai, T., Saito, Y., Kawashima, S., Moroi, K.and Kadota, K.: "Antipeptide antibody of dopamine transporter and its expression in the PC12 cells." Acta Anat.Nipponica. 70. 248-251 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kadota, T.: "Na^+-dependent neurotransmitter transporter." Saibou. 27. 349-352 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kadota, T., Yamaai, T., Saito, Y., Akita, Y., Kawashima, S., Moroi, K., Inagaki, N.and Kadota, K.: "Expression of dopamine trans-porter at the tips of growing neurite of PC12 cells." J.Histochem.Cytochem. 44. 989-996 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kadota, T., Mizote, M.and Kadota, K.: "Synaptic spinules attendant on post-tetanic potentiation in cat sympathetic ganglion." Proc.Japan Acad. 72, Ser B. 48-51 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Fujita, M., Kadota, T.and Sato, T.: "Developmental profiles of synaptophysin in granule cells of rat cerebellum : an immuno-histocytochemical study." J.Electron Microsc.45. 185-194 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kadota, T.: "Synaptic function and the membrane recycling." Seitai no kagaku. 47. 287-291 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kadota, T.: "The ultrastructure of the synapse." Clinical Neuroscience. 14. 16-20 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kadota, T.: "Morphological changes of the synapse during LTP." Microscopia. 14(3). 174-175 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kadota, T.: "Discovery of the synapse." Microscopia. 14(4). 240-245 (1997)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-16  

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