| Project/Area Number |
15590192
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | Tokyo Women's Medical University |
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Principal Investigator |
AWAJI Takeo Tokyo Women's Medical University, Physiology, assistant professor, 医学部, 講師 (60297546)
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| Co-Investigator(Kenkyū-buntansha) |
HIRASAWA Akira Kyoto Univ.Grad.Sch., Pharm.Sci., Genomic Drug Discovery Science, associated professor, 大学院・薬学研究科・ゲノム創薬科学分野, 助教授 (70242633)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | α_1-adrenoceptor / green fluorescent protein / Ca^<2+> oscillations / subcellular localization / Hydrion density |
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| Research Abstract |
The subcellular localization and adjacent pH of the α_1-adrenoceptor (α_1-AR) subtypes conjugated with pH sensor green fluorescent protein (α_1-AR/GFpH) were simultaneously assessed using real-time imaging of living, stably expressed CHO-K1 cells. The α_<1B>-AR/GFpH fluorescence was detected predominantly on the cell surface and intracellular pH distribution showed homogeneous pattern. Stimulation of the α_<1B>-AR with norepinephrine (NE,10^<-7>M) led to an increase in intracellular pH, promoted rapid α_<1B>-AR/GFP internalization and caused Ca^<2+> oscillations. α_<1A>-AR/GFpH fluorescence was detected not only on the cell surface but also inside he cell and this localization was unaffected by exposure to NE during observation period. Intracellular pH on the cell surface was higher than intracellular compartment before NE. Stimulation of the α_<1A>-AR with NE led to a decrease in intracellular pH and caused only a Ca^<2+> transient. The α_<1D>-AR/GFpH fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to NE. Intracellular pH distribution of α_<1D>-AR/GFpH was homogeneous as in α_<1B>-AR/GFpH. NE treatment of α_<1D>-AR/GFpH expressing cells increased pH and caused Ca^<2+> oscillations. These data show that transfected α_1-AR/GFpH fusion proteins are functional and that there are differences in the cellular distribution of α_1-AR, agonist-mediated internalization, Ca^<2+> response and neighbor pH among α_1-AR subtypes. These differences could contribute to the diversity in physiologic responses regulated by the α_1-ARs. The subcellular distribution of al-subtypes is still unknown. The distribution of subcellular oragnera will be examined by immunoelectron microscopy using co-transfected subcellar marker and anti-GFP antibody.
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