2002 Fiscal Year Annual Research Report

開口分泌素過程の分子機構とその制御機構に関する研究

Research Project

Project/Area Number	12480232
Research Institution	SOPHIA UNIVERSITY
Principal Investigator	熊倉鴻之助上智大学, 理工学部, 教授 (70129790)
Co-Investigator(Kenkyū-buntansha)	今泉美佳杏林大学, 医学部, 助手 (40201941) 前川昌平神戸大学, 大学院・自然科学研究科, 教授 (40173695) 笹川展幸上智大学, 理工学部, 助教授 (20187107)
Keywords	クロマフィン細胞 / 開口分泌 / 開口キネティクス / 顆粒運動 / PKC / 多顆粒開口分泌 / アクチン-ミオシン相互作用 / アクチン細胞骨格
Research Abstract	1)微小炭素繊維電極アンペロメトリー法により、高カリウム刺激で引き起こされる個々のクロマフィン顆粒の開口キネティクスに対するPKC活性化の影響を調べた。TPA処理によって、シングルイヴェントのRise timeならびにMiddle widthが増大した。PKC阻害によりその効果は消失したことから、PKC活性化は個々の分泌顆粒の開口速度を調節することが明らかとなった。同様にして、アクチン-ミオシン相互作用ならびにアクチン細胞骨格が開口速度の調節に関わることが明らかとなった。 2)分泌顆粒の可視的測定解析を進めた結果、これまで活発な顆粒運動が観察された位置よりも焦点を形質膜方向に近づけると、開口分泌を示すと考えられる蛍光強度の増大が観察された。高カリウム刺激による開口像の中に、いわゆるCompound exocytosis(多顆粒開口分泌)を示すと考えられる持続性の蛍光強度変化が観察された。走査型電子顕微鏡による細胞内の微細構造観察から、複数の顆粒鎖と形質膜の融合像が得られ、クロマフィン細胞においてCompound exocytosisが行なわれる事が明らかになった。この成果は、世界初の分泌顆粒の走査電顕像であり、またCompound exocytosisの走査電顕像である。 3)神経細胞のシナプス小胞とプレシナプス膜に局在するタンパク質NAP-22は、PC12細胞にも発現している。NAP-22の脂質結合能を解析するためC末よりの欠損体を大腸菌で発現させ、リポソームとの結合を解析した。脂質結合にはN端のミリスチル化が必須であること、フォスファチジルコリン存在下でコレステロールやフォスファチジルエタノールアミンが存在すると膜結合能を持つことを見い出した。続いて培養細胞への発現と脂質染色により培養細胞においてもタンパク質と脂質の局在が見られることを観察した。NAP-22が局在するラフト領域が分泌顆粒のリサイクリングに関わる可能性が考えられる。

Research Products

(19 results)

All Other

All Publications (19 results)

[Publications] Noyama, K.: "Localization of cyclic nucleotide phosphodiesterase 2 in thebrain-derived Triton-insoluble low-density fraction(raft)"Neurosci.Res.. 45. 141-148 (2003)
[Publications] 熊倉鴻之助: "開口分泌の分子機構"上智大学生命科学研究所紀要. 21. 53-75 (2002)
[Publications] Konosuke Kumakura: "Multiple Roles of Actin Cytoskeleton in Catecholamine Release from Chromaffin Cell"Catecholamine Research : From Molecular Insights to Clinical Medicine Eds. T.Nagatsu, T.Nabeshima, R.McCarty, D.S.Goldstein. 57-60 (2002)
[Publications] M.-F.Bader: "Exocytosis : The Chromaffin Cell As a Model System"The Chromaffin Cell ; Transmitter Biosynthesis, Storage, Release, Actions, and Informatics. Daniel T.O'Connor and Lee E.Eiden (eds.). 178-183 (2002)
[Publications] S.Quetglas: "Calmodulin and lipid binding to synaptovrevin regulates calcium-dependent exocytosis"The EMBO Journal. 21. 1-10 (2002)
[Publications] Ohyama, K.: "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)
[Publications] Terashita, A.: "The lipid binding activity of a neuron specific protein NAP-22 studied in vivo and in vitro"J.Neurosci.Res.. 70. 172-179 (2002)
[Publications] Nobuyuki Sasakawa: "Roles of actin filaments and actin-myosin interaction in the regulation of exocytosis in chromaffin cells"The Chromaffin Cell ; Transmitter Biosynthesis, Storage, Release, Actions, and Informatics. Daniel T.O'Connor and Lee E.Eiden (eds.). 273-274 (2002)
[Publications] Maekawa, S.: "Localization of neuronal growth-associated microtuble-destabilizing factor SCG10 in Brain-derived rat membrane microdomains"J.Biochem.. 129. 691-697 (2001)
[Publications] Orito, A: "Calcium-dependent association of annexin VI, protein kinase C α and neurocalcin α on the raft fraction derived from synaptic plasma membrain of rat brain"J.Neurosci.Res.. 64. 235-241 (2001)
[Publications] Kumanogoh, H.: "Biochemical and morphological analysis on the localization of Rac1 in neurons"Neurosci.Res.. 39. 189-196 (2001)
[Publications] 伊藤尚: "Increase in N-terminal hydrophobicity in mastoparan molecule enhanced the original hemolytic and catecholamine releasing activities from chromaffin cells"東京保健科学学会誌. 3(2). 190-117 (2000)
[Publications] Uematsu, A.: "Specific localization of the vacuolar H+-ATPase in the Triton insoluble low-density fraction (raft) of bovine chromaffin granule membrane"J.Biochem Mol Biol Biophys.. 4. 395-400 (2000)
[Publications] Ohira, K: "Absence of Trk B and Insulin receptor β in the Triton insoluble low-density fraction (raft) of rat brain"NeuroReport. 11. 1307-1311 (2000)
[Publications] 伊藤尚: "Increase in N-terminal hydrophobicity in mastoparan molecule enhanced the original hemolytic and catecholamine releasing activities from chromaffin cells"東京保健科学学会誌. 3(2). 109-117 (2000)
[Publications] Uematsu, A.: "Specific localization of the vacuolar H+-ATPase in the Triton insoluble low-density fraction (raft) of bovine chromaffin granule membrane"J.Biochem Mol Biol Biophys.. 4. 395-400 (2000)
[Publications] Ohira, K.: "Absence of Trk B and Insulin receptor β in the Triton insoluble low-density fraction (raft) of rat brain"NeuroReport. 11. 1307-1311 (2000)
[Publications] Funatsu, N.: "Identification of gelsolin as a action regulatory activity of a Triton insoluble low density fraction of bovine brain"Neurosci Res. 36. 311-317 (2000)
[Publications] 熊倉鴻之助: "シナプス小胞exocytosisの分子メカニズム"BRAIN MEDICAL. 12(3). 18-24 (2000)

2002 Fiscal Year Annual Research Report

開口分泌素過程の分子機構とその制御機構に関する研究

Principal Investigator

熊倉 鴻之助 上智大学, 理工学部, 教授 (70129790)

Research Products

[Publications] Noyama, K.: "Localization of cyclic nucleotide phosphodiesterase 2 in thebrain-derived Triton-insoluble low-density fraction(raft)"Neurosci.Res.. 45. 141-148 (2003)

[Publications] 熊倉鴻之助: "開口分泌の分子機構"上智大学 生命科学研究所 紀要. 21. 53-75 (2002)

[Publications] Konosuke Kumakura: "Multiple Roles of Actin Cytoskeleton in Catecholamine Release from Chromaffin Cell"Catecholamine Research : From Molecular Insights to Clinical Medicine Eds. T.Nagatsu, T.Nabeshima, R.McCarty, D.S.Goldstein. 57-60 (2002)

[Publications] M.-F.Bader: "Exocytosis : The Chromaffin Cell As a Model System"The Chromaffin Cell ; Transmitter Biosynthesis, Storage, Release, Actions, and Informatics. Daniel T.O'Connor and Lee E.Eiden (eds.). 178-183 (2002)

[Publications] S.Quetglas: "Calmodulin and lipid binding to synaptovrevin regulates calcium-dependent exocytosis"The EMBO Journal. 21. 1-10 (2002)

[Publications] Ohyama, K.: "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)

[Publications] Terashita, A.: "The lipid binding activity of a neuron specific protein NAP-22 studied in vivo and in vitro"J.Neurosci.Res.. 70. 172-179 (2002)

[Publications] Nobuyuki Sasakawa: "Roles of actin filaments and actin-myosin interaction in the regulation of exocytosis in chromaffin cells"The Chromaffin Cell ; Transmitter Biosynthesis, Storage, Release, Actions, and Informatics. Daniel T.O'Connor and Lee E.Eiden (eds.). 273-274 (2002)

[Publications] Maekawa, S.: "Localization of neuronal growth-associated microtuble-destabilizing factor SCG10 in Brain-derived rat membrane microdomains"J.Biochem.. 129. 691-697 (2001)

[Publications] Orito, A: "Calcium-dependent association of annexin VI, protein kinase C α and neurocalcin α on the raft fraction derived from synaptic plasma membrain of rat brain"J.Neurosci.Res.. 64. 235-241 (2001)

[Publications] Kumanogoh, H.: "Biochemical and morphological analysis on the localization of Rac1 in neurons"Neurosci.Res.. 39. 189-196 (2001)

[Publications] 伊藤 尚: "Increase in N-terminal hydrophobicity in mastoparan molecule enhanced the original hemolytic and catecholamine releasing activities from chromaffin cells"東京保健科学学会誌. 3(2). 190-117 (2000)

[Publications] Uematsu, A.: "Specific localization of the vacuolar H+-ATPase in the Triton insoluble low-density fraction (raft) of bovine chromaffin granule membrane"J.Biochem Mol Biol Biophys.. 4. 395-400 (2000)

[Publications] Ohira, K: "Absence of Trk B and Insulin receptor β in the Triton insoluble low-density fraction (raft) of rat brain"NeuroReport. 11. 1307-1311 (2000)

[Publications] 伊藤 尚: "Increase in N-terminal hydrophobicity in mastoparan molecule enhanced the original hemolytic and catecholamine releasing activities from chromaffin cells"東京保健科学学会誌. 3(2). 109-117 (2000)

[Publications] Uematsu, A.: "Specific localization of the vacuolar H+-ATPase in the Triton insoluble low-density fraction (raft) of bovine chromaffin granule membrane"J.Biochem Mol Biol Biophys.. 4. 395-400 (2000)

[Publications] Ohira, K.: "Absence of Trk B and Insulin receptor β in the Triton insoluble low-density fraction (raft) of rat brain"NeuroReport. 11. 1307-1311 (2000)

[Publications] Funatsu, N.: "Identification of gelsolin as a action regulatory activity of a Triton insoluble low density fraction of bovine brain"Neurosci Res. 36. 311-317 (2000)

[Publications] 熊倉 鴻之助: "シナプス小胞exocytosisの分子メカニズム"BRAIN MEDICAL. 12(3). 18-24 (2000)

熊倉鴻之助上智大学, 理工学部, 教授 (70129790)

[Publications] 熊倉鴻之助: "開口分泌の分子機構"上智大学生命科学研究所紀要. 21. 53-75 (2002)

[Publications] 伊藤尚: "Increase in N-terminal hydrophobicity in mastoparan molecule enhanced the original hemolytic and catecholamine releasing activities from chromaffin cells"東京保健科学学会誌. 3(2). 190-117 (2000)

[Publications] 伊藤尚: "Increase in N-terminal hydrophobicity in mastoparan molecule enhanced the original hemolytic and catecholamine releasing activities from chromaffin cells"東京保健科学学会誌. 3(2). 109-117 (2000)

[Publications] 熊倉鴻之助: "シナプス小胞exocytosisの分子メカニズム"BRAIN MEDICAL. 12(3). 18-24 (2000)