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1993 Fiscal Year Final Research Report Summary

Three dimentional assay of cyclic nucleotide in cultured cells with use of a confocal system

Research Project

Project/Area Number 03557034
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 内科学一般
Research InstitutionTHE UNIVERSITY OF THE AIR

Principal Investigator

KITO Shuzo  The University of the Air Div.of Health Sciences Professor, 教養学部, 教授 (00010140)

Co-Investigator(Kenkyū-buntansha) MIYOSHI Rie  Tokyo Women's Sch.of Medicine Dept.of Pharm.Instructor, 助手 (80209965)
SEMBA Jun'ichi  The University of the Air Div.of Health Sciences Associate Professor, 教養学部, 助教授 (30183429)
Project Period (FY) 1991 – 1993
Keywordscalcium ion / fluorometry / cyclic AMP / A kinase / regulatory subunit / catalytic subunit / isothiocyanate / D1 and D2 receptors
Research Abstract

The Method of intracellular calcium ion assay in which fura 2 and other calcium chelating reagents are used for fluorometry, has been well established. As for an qually important bioactive substance, cyclic AMP assay in living cell has not been successful.
In this study, we tried to assay cyclic AMP in living cells by fluorometry and to observe three dementionally by means of motor-driven focus controller in its image analysis.
cDNA of regulatory subunit of cyclic AMP dependent protein kinase (A kinase) was inserted into escherichiacoli and the protein was prepared.
For fluorescence labelling, tetramethylrhodamine was used for regulatory subunit and fluorescein isothyanate for catalytic subunit. These labelled proteins were microinjected into cells.
When 2 molecules of each of regulatory and catalytic subunit are in tetramer, fluorescence light from catalytic subunit can be the excitation light to regulatory subunit. When these subunit molecules are separated, fluorescence light from catalytic subunit itself is detected. This is the principle of fluorescence assay of cyclic AMP.We used mixed culture of the rat striatum in which D1 and D2 receptors coexisted. As widely known, D1 and D2 receptors act in opposite directions as regards to cyclic AMP level within cells. Activated D1 receptor decrease cyclic AMP, while D2 receptor increase it. For these opposite directions of action to cyclic AMP, it was not always possible to assay cyclic AMP at detectable level.

  • Research Products

    (36 results)

All Other

All Publications (36 results)

  • [Publications] Miyoshi,R.: "Cholecystokinin increases intracellular Ca^<2+> concentration in cultured striatal neurons." Neuropeptides. 18. 115-119 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R.: "Influence of age on N-methyl-D-aspartate antagonist binding sites in the rat brain studied by in vitro autoradiography." Synapse. 8. 212-217 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R: "Effect of age on α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA)binding sites in the rat brain studied by in vitro autoradiography." Neurochemical Research. 16. 849-854 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R.: "Effect of caerulein on expression of the immediate early genes c-fos and zif/268 in the rat brain." Neuroscience Letters. 145. 205-208 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R.: "Effect of estrogen on immediate early gene expression in the rat brain." Society for Neuroscience Abstracts. 18. 57.1 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kito S.: "Effect of glucocorticoid on in vivo release of neurotransmitters from the rat hippocampus." Society for Neuroscience Abstracts. 18. 57.4 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kito,S.: "A cytoprotective effect of nicotine on hippocampal neurons." Society for Neuroscience Abstracts. 19. 772.15 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R.: "Effect of nicotine on neurotransmitter release from the rat hippocampus." Society for Neuroscience Abstracts. 19. 384.6 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kito,S.: "Effects of glucocorticoid on hippocampal neurons:in vivo and in vitro studies." Neurochemical Research. 18. 832 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Watanabe,A.: "Measurement of glutamate,aspartate and glycine and its potential precursors in human brain using high-performance liquid chromatography by pre-column derivatization with diethylaminoazobenzene sulphonyl chloride." Journal of Chromatography. 583. 241-245 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Semba,J.: "Effect of milacemide on extracellular and tissue concentrations of dopamine and 5-hydroxytryptamine in rat frontal cortex." Br.J.Pharmacol.105. 59-62 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Patsalos,P.N.: "A freely moving and behaving rat model for the chronic and simultaneous study of drug pharmacokinetics(blood)and neuropharmacokinetics(cerebrospinal fluid):hematological and biochemical characterization and kinetic evaluation using carbamazepine." JPM. 28. 21-28 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Semba,J.: "Antiepileptic drug pharmacokinetics and neuropharmacokinetics in individual rats by repetitive withdrawal of blood and cerebrospinal fluid:milacemide." Br.J.Pharmacol.108. 1117-1124 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Semba,J.: "Milacemide effects on the temporal inter-relationship of amino acids and monoamine metabolites in rat cerebrospinal fluid." European Journal of Pharmacology. 230. 321-326 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R.: "Influence of age on N-methyl-D-aspartate antagonist binding sites in the rat brain studied by in vitro autoradiography." Synapse. 8. 212-217 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R.: "Effect of age on α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA)binding sites in the rat brain studied by in vitro autoradiography." Neurochemical Research. 16. 849-854 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R.: "Effect of estrogen on immediate early gene expression in the rat brain." Society for Neuroscience Abstracts. 18. 57.1 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kitok S.: "Effect of glucocorticoid on in vivo release of neurotransmitters from the rat hippocampus." Society for Neuroscience Abstracts. 18. 57.4 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kito,S.: "A cytoprotective effect of nicotine on hippocampal neurons." Society for Neuroscience Abstracts. 19. 772.15 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R.: "Effect of nicotine on neurotransmitter release from the rat hippocampus." Society for Neuroscience Abstracts. 19. 384.6 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kito,S.: "Effects of glucocorticoid on hippocampal neurons:in vivo and in vitro studies." Neurochemical Research. 18. 832 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kito,S.: "NMDA Receptor Related Agents:Biochemistry,Pharmacology and Behavior,edited by Kameyama,T.,Nabeshima,T.,Domino,E.F." NPP Books, 9 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kito,S: "Neuroreceptor Mechanisms in Brain,edited by Kito,S.,Segawa,T.and Olsen,R.W." Plenum Press,New York, 10 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi,R.: "The Basal.Ganglia III,edited by Bernardi,G.,Carpenter,M.B.,Di Chiara,G.,Morelli,M.and Stanzione,P." Plenum Press,New York, 7 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Miyoshi, R., Kito, S.and Nomoto, T.: "Cholecystokinin increases intracellular Ca^<2+> concentration in cultured striatal neurous." Neuropeptides. 18. 115-119 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Miyoshi, R., Kito, S., Doudou, N.and Nomoto, T.: "Influence of age on N-methyl-D-aspartate antagonist binding sites in the rat brain studied by in vitro autoradiography." Synapse. 8. 212-217 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Miyoshi, R., Kito, S., Doudou, N.and Nomoto, T.: "Effect of age on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites in the rat brain studied by in vitro autoradiography." Neurochemical Research. 16. 849-854 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yoneda, Y., Ogita, K., Enomoto, R., Suzuki, T.and Kito, S.: "Identification and characterization of specific binding sites of [^3H]spermidine in synaptic membranes of rat brain." Brain Research. 563. 17-27 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Miyoshi, R., Kito, S., Muraki, T., Weiser, M.and Joh, T.H.: "Effect of caerulein on expression of the immediate early genes c-fos and zif/268 in the rat brain." Neuroscience Letters. 145. 205-208 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Miyoshi, R., Fujii, E., Muraki, T.and Kito, S.: "Effect of estrogen on immediate early gene expression in the rat brain." Society for Neuroscience Abstracts. 18. 57.1 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kito S., Miyoshi, R., Shimoyama, M., Kimura, A., Hayashi, M., Shimada, L.and Maruyama, Y.: "Effect of glucocorticoid on in vivo release of neurotransmitters from the rat hippocampus." Society for Neuroscience Abstracts. 18. 57.4 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Shimoyama, M., Miyoshi, R., Kito, S., Kimura, A., Hayashi, M., Shimada, L.and Maruyama, Y.: "Effect of mineralcorticoid on in vivo release of neurotransmitters from the rat hippocampus." Society for Neuroscience Abstracts. 18. 57.3 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kito, S.and Miyoshi, R.: "Effects of glucocorticoid on hippocampal neurons : in vivo and in vitro studies." Neurochemical Research. 18. 832 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Watanabe, A., Semba, J., Kurumaji, A., Kumashiro, S.and Toru, M.: "Measurement of glutamate, aspartate and glycine and its potential precursors in human brain using high-performance liquid chromatography by pre-column derivatization with diethylaminoazobenzene sulphonyl chloride." Journal of Chromatography. 583. 241-245 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Semba, J., Doheny, M., Patsalos, P.N., Sarna, G.and Curzon, G.: "Effect of milacemide on extracellular and tissue concentrations of dopamine and 5-hydroxytryptamine in rat frontal cortex." Bx.J.Pharmacol.105. 59-62 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Semba, J.and Patsalos, P.N.: "Milacemide effects on the temporal inter-relationship of amino acids and monoamine metabolites in rat cerebrospinal fluid." European Journal of Pharmacology. 230. 321-326 (1993)

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

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Published: 1995-03-27  

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