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Molecular mechanisms of exocytosis: amperometric analysis of catecholamine release from single cells

Research Project

Project/Area Number 14570086
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General pharmacology
Research InstitutionSophia University

Principal Investigator

SASAKAWA Nobuyuki  Sophia Univ., Faculty of Science and Technology, Associate Prof., 理工学部, 助教授 (20187107)

Co-Investigator(Kenkyū-buntansha) KUMAKURA Konosuke  Sophia Univ., Faculty of Science and Technology, Prof., 理工学部, 教授 (70129790)
Project Period (FY) 2002 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2003: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2002: ¥3,200,000 (Direct Cost: ¥3,200,000)
Keywordsexocytosis / chromaffin cells / Catecholamine release / amperometric detection / クロマフィン細胞 / 開口放出 / カテコララミン分泌
Research Abstract

To clarify the implication of each PKC isoform in the regulation of exocytosis, we examined the effects of overexpression of PKC α and β on the kinetics of exocytosis of single vesicles using transfected cells. Amperometric detection of single exocytotic events is conducted by use of a carbon fiber electrode from a single cell. Expression of PKC isoformes was confirmed by co-expression of EGFP. In the PKCα-overexpressed cells, the frequency of exocytotic events evoked by KCI (60mM, 5min) was dramatically increased. This effect was apparently due to the increase in the later 4 min period, but not in the initial 1min. In the PKCβ-overexpressed cells, the frequency was increased both the initial and the later 4 min periods, but the effects was much evident in the initial 1 min in contrast to PKCα. The effects of overexpression was abolished by BIS, a PKC inhibitor. This might suggest that target molecules of phosphorylation by PKCα and PKCβ are different, resulting distinct regulation of exocytosis in adrenal chromaffin cells. Moreover, activation of PKCc seemd important for the recruitment of secretory vesicles to the readily releasable pool from the resetve pool. In addition, microinjection of myosinV-antiserum, inositol hexakisphosphate, inositol pentakispyrophosphate or Vamp peptide characteristically inhibited KCI-evoked event frequency. These results suggest that myosinV, highly phosphorylated inositol polyphoshates and Vamp may have important roles in the regulation of exocytosis in chromaffin cells. However, precise mechanisms remain to be elucidated.

Report

(3 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] Sasakawa N., Murayama N., Kumakura K: "Characteristic regulation of exocytotic events by protein kinase C isoform in single adrenal chromaffin cells"J.Pharmacol.Sci.Supplement I. 94. 191 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sasakawa N, Ohara-Imaizumi M, Okubo S, Hosaka S, Hayashi M, Kumakura K: "Roles of actin filaments and the actin-myosin interaction in the regulation of exocytosis in chromaffin cells."Ann.N.Y.Acad.Sci.. 971. 273-274 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Quetglas, S., Iborra, C., Sasakawa, N., De Haro.L., Kumakura, K., Sato, C., Seagar, M.: "Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis."The EMBO J.. 21. 3970-3979 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ohyama, A., Hosaka, K., Komiya, Y., Akagawa, K., Yamauchi, E., Taniguchi, H., Sasakawa, N., Kumakura, K: "Regulation of exocytosis through Ca2+/ATP-dependent binding of autophosphorylated Ca2+/calmodulin-activated protein kinase II to syntaxin 1A."J.Neurosci.. 22. 3342-3351 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sharif, T.R., Sasakawa, N., Sharif, M.: "Regulated expression of a dominant negative protein kinase C epsilon mutant inhibits the proliferation of U-373MG human astrocytoma cells."J.Mol.Med.. 7. 373-380 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Kumakura, K., Ohara-Imaizumi, M., Battaini, F., Sasakawa, N.: "Catecholamine Research"Kluwer Academic/Plenum Publishers. 558 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sasakawa N., Ohara-imaizumi, M., Fukuda, M., Kobayama, H., Mikoshiba, K., Ohkubo, S., Kumakura, K: "Ca2+ binding to C2A domain of synaptotagmin dissociates inositol polyphosphates from the C2B domain."Pro.Natl.Acad.Sci.USA.. (submitted for publication). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sasakawa N., Murayama N., Kumakura K: "Characteristic regulation of exocytotic events by protein kinase C isoform in single adrenal chromaffin cells"J.Pharmacol.Sci., Supplement. 1,94. 191 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Kumakura, K., Ohara-Imaizumi, M., Battaini, F., Sasakawa N.: "Multipul roles of actin cytoskeleton in catecholamine release from chromaffin cells."Catecholamine Research. 57-60 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sasakawa N., Ohara-Imaizumi M, Okubo S, Hosaka S, Hayashi M, Kumakura K.: "Roles of actin filaments and the actin-myosin interaction in the regulation of exocytosis in chromaffin cells."Ann.N.Y.Acad.Sci.. 971. 273-274 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Quetglas, S., Iborra, C., Sasakawa, N., De Haro.L., Kumakura, K., Sato, K, Leveque, C., Seagar, M.: "Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis."The EMBO J.. 21. 3970-3979 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ohyama, A., Hosaka, K., Komiya, Y., Akagawa, K., Yamauchi, E., Taniguchi, H., Sasakawa, N., Kumakura, K., Mochida, S., Yamauchi, T., Igarashi, M.: "Regulation of exocytosis through Ca2+/ATP-dependent binding of autophosphorylated Ca2+/calmodulin-activated protein kinase II to syntaxin 1A."J.Neurosci.. 22. 3342-3351 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sharif, T.R.Sasakawa, N., Sharif, M.: "Regulated expression of a dominant negative protein kinase C epsilon mutant inhibits the proliferation of U-373MG human astrocytoma cells."J.Mol.Med.. 7. 373-380 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sasakawa N., Murayama N.Kumakura K.: "Characteristic regulation of exocytotic events by protein kinase C isoform in single adrenal chromaffin cells"J.Pharmacol.Sci.Supplement I. 94. 191 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Sasakawa N, Ohara-Imaizumi M, Okubo S, Hosaka S, Hayashi M, Kumakura K.: "Roles of actin filaments and the actin-myosin interaction in the regulation of exocytosis in chromaffin cells."Ann.N.Y.Acad.Sci.. 971. 273-274 (2002)

    • Related Report
      2003 Annual Research Report
  • [Publications] Quetglas, S., Iborra, C., Sasakawa, N., De Haro, L., Kumakura, K., Sato, K., Leveque, C., Seagar, M.: "Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis."The EMBO J.. 21. 3970-3979 (2002)

    • Related Report
      2003 Annual Research Report
  • [Publications] Ohyama, A., Hosaka, K., Komiya, Y., Akagawa, K., Yamauchi, E., Taniguchi, H., Sasakawa, N., Kumakura, K., Mochida, S., Yamauchi, T., Igarashi, M.: "Regulation of exocytosis through Ca2+/ATP-dependent binding of autophosphorylated Ca2+/calmodulin-activated protein kinase II to syntaxin 1A."J.Neurosci.. 22. 3342-3351 (2002)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kumakura, K., Ohara-Imaizumi, M., Battaini, F., Sasakawa, N.: "Catecholamine Research"Kluwer Academic/Plenum Publishers. 558 (2002)

    • Related Report
      2003 Annual Research Report
  • [Publications] Sasakawa, N., et al.: "Roles of actin filaments and the actin-myosin …"Ann.N.Y. Acard Sci.. 971. 273-274 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Quetglas, S., et al.: "Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis"EMBO J.. 21. 3970-3979 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Ohyama, A., et al.: "Regulation of Exocytosis through Ca^<2+>/ATP-Dependent Binding of Autophosphorylated Ca^<2+>/Calmodulin-Activated Protein Kinase II to Syntaxin 1A"J.Neurosci.. 22. 3342-3351 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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