| Project/Area Number |
15390075
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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 | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
MIWA Soichi Hokkaido Univ., Grad.School of Med., Prof., 大学院・医学研究科, 教授 (40157706)
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| Co-Investigator(Kenkyū-buntansha) |
TAKIKAWA Osamu National Institute for Longevity Sciences, Laboratory of Radiation Safety, Head, 研究所・ラジオアイソトープ管理室, 室長 (70163342)
HATTORI Yuichi Univ.of Toyama, School of Med., Prof., 医学部, 教授 (50156361)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥13,100,000 (Direct Cost: ¥13,100,000)
Fiscal Year 2005: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2004: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2003: ¥5,500,000 (Direct Cost: ¥5,500,000)
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| Keywords | Endothelin receptor / Vascular smooth muscle cell / Ca^<2+> channel / Xenopus oocyte / Ca^<2+> uptake / yeast two-hybrid / GST pull-down / cDNA cloning / cDNAクローニング / エンドセリン / 収縮 / 受容体作動性カルシウムチャネル |
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| Research Abstract |
The purpose of the present study is to isolate cDNAs encoding for receptor-operated Ca^<2+> channels activated by endothelin type A receptor (ET_AR). We found that the conventional voltage-clamp method with two electrodes cannot be used for screening Ca^<2+> influx, because expression of the receptors alone induced a large inward current resulting from Ca^<2+>-activated Cl- currents due to Ca^<2+> mobilized from the intracellular stores. Therefore, to differentiate Ca^<2+> influx through channels across the cell membrane from Ca^<2+> mobilized from the stores, we decided to use ^<45>Ca^<2+> uptake into the oocytes which were microinjected with cRNA for ET_AR and mRNA prepared from various tissues. To increase the sensitivity of the method, we developed a method to identify the injured oocytes using ^<14>C-inulin and also optimized the amount of injected cRNA for ET_AR and mRNA. Even with the improved method, we could not detect a positive fraction showing receptor-activated Ca^<2+> influx. Therefore, we decided to use a yeast-two hybrid system to isolate molecules interacting directly with ET_AR. Using this method, we have succeeded in isolating 26 clones and are now analyzing their functional roles in ET_AR-mediated Ca^<2+> signaling in the cells.
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