| Project/Area Number |
12557011
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
General pharmacology
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| Research Institution | Kumamoto University |
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Principal Investigator |
MIYAMOTO Eishichi Kumamoto University School of Medicine, Professor, 医学部, 教授 (50109659)
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| Co-Investigator(Kenkyū-buntansha) |
TAKEUCHI Yusuke Kumamoto University School of Medicine, Instructor, 医学部, 助手 (90336214)
KASAHARA Jiro Kumamoto University School of Medicine, Instructor, 医学部, 助手 (10295131)
YAMAMOTO Hideyuki Kumamoto University School of Medicine, Assistant Professor, 医学部, 講師 (60191433)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥13,400,000 (Direct Cost: ¥13,400,000)
Fiscal Year 2001: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 2000: ¥9,000,000 (Direct Cost: ¥9,000,000)
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| Keywords | CaM kivases / gene expression / Ca^<2+> signaling / dopamine D2 receptor / nuclear localization signal / NG108--15 cell / brain-derived neurotrophic factor / cell model / CaMキナーゼII / CaMキナーゼIIサブタイプ |
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| Research Abstract |
The present study intends to establish the model of the cell line in which functionally important proteins such as receptors, enzymes, ion channels are expressed. Stimulation of the established cells with neurotransmitters hormones, growth factors etc. activates the expressed proteins in the cells. These models serve as the established systems to analyze effects of drugs and to create new drugs.
The study focuses the analysis on the intracellular calcium (Ca^<2+>) and on the functional significance of CaM kinases I, II, III, IV and kinase. The cytosolic and nuclear isoforms of CaM kinase II were expressed in NG108-15 cells in which the expression of brain-derived neurotrophic factor (BDNF) was examined. The expression of BDNF was not increased by the transfection of the cytosolic isoforms such as CaM kinase II δ1, and δ2 but was increased by CaM kinase II δ3 and αB. The quantitative method of RT-PCR for the amount of the mRNA indicated the increase in Exon IV-BDNF mRNA but not Exon III-BDNF mRNA. This suggest that the nuclear isoforms only increase the mRNA and proteins of BDNF.
Dopamine D2 receptors (D2R) consist of the long chain receptor (D2RL) and the short chain receptor (D2RS). Both cDNAs were transfected into NG108-15 cells and the stable cells with the expression of each receptor were obtained. The analysis by confocal laser microscopy revealed different localization of each receptor in the cells. Stimulation of D2R increased the concentration of the intracellular Ca^<2+>. The cDNA of CaM kinase II δ3 with nuclear localization signal was transiently transfected in the stable cells of D2RL. Stimulation of D2R of the transfected cells with the agonist resulted in activation of the nuclear isoform of CaM kinase II and the increase in the expression of BDNF.
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