| Project/Area Number |
14570650
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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 |
Circulatory organs internal medicine
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| Research Institution | GIFU UNIVERSITY |
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Principal Investigator |
TSUCHIYA Kunihiko Gifu University, Hospital, Lecture, 医学部附属病院, 助手 (30313878)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Tomoyuki Gifu University, School of Medicine, Lecture, 医学部, 助手 (20332687)
FUJIWARA Hisayoshi Gifu University, Graduate School of Medicine, Professor, 大学院・医学研究科, 教授 (80115930)
小戝 健一郎 岐阜大学, 医学部, 助教授 (90258418)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | embryonic stem cell / differentiation and induction / myocyte / metabolic inhibition / ATP-sensitive K channel / イオンチャネル / パッチクランプ法 / L型Caチャネル / 超微形態 |
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| Research Abstract |
(1)Examination of ion channel property
(Method)
Using embryonic stem (ES) cells of mice, beating cells were initially differentiated and induced. In these cells, dual metabolic inhibition both glycolytic and oxidative ATP production were employed to activate surface ATP-sensitive K (K_<ATP>) channel. Therefore, K_<ATP> channel expression was confirmed and the amount of currents was also recorded in cell-attached patch clamp technique. Additional experiments were performed to examine the effects of K_<ATP> channel opener and inhibitor on ES cells.
(Results)
Myocytes obtained from ES cells were difficult to activate K_<ATP> channels even by using dual metabolic inhibition. Therefore, stable recordings of K_<ATP> current were impossible because K_<ATP> channels were not activated until cells got shrinkage and patch membrane was broken. Although in short time recording, the current activated successfully was thought to be K_<ATP> channel according to its current-voltage relationship and contin
… More
uous activation pattern. However, this current was rarely suppressed by glibenclamide, a K_<ATP> channel inhibitor. The reason why glibenclamide could not suppress this current was that most myocytes obtained from ES cells were firstly activated in a critical state. Maximal channel numbers included in patch membrane with 5MΩ resistance were only 2. In a few case that K_<ATP> currents were activated during cell structure kept almost intact, these currents were dose-dependently inhibited by glibenclamide as same as adult myocyte. A response of ES cells for pinacidil, a K_<ATP> channel opener was similar to one of adult myocyte. Finally, it was concluded that ES cells were rather resistant to ischemia in this experiment.
(2)Observation of ultrastructure
(Method)
After fixation by glutaraldehyde and osmium, ultrastructure of myocyte including myofibril and sarcomere obtained from ES cell were observed by using both transmission and scanning electron microscopy. Thereby the appearance and distribution of K_<ATP> channels were evaluated in ES cells
(Results)
Myocyte obtained from ES cell showed few myofibril and sarcomere compared with adult cell, this finding was well consistent with the result that the expression of K_<ATP> channel was very rare in ES cells. Less
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