| Project/Area Number |
63480264
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
内分泌・代謝学
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| Research Institution | Jichi Medical School (1989)
The University of Tokyo (1988) |
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Principal Investigator |
KANAZAWA Yasunori Jichi Medical School Professor, 医学部, 教授 (10010399)
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| Co-Investigator(Kenkyū-buntansha) |
NODA Mitsuhiko 東京大学医学部附属病院, 医員
OKA Yoshitomo University of Tokyo, Faculty of Medicine Assistant, 医学部, 助手 (70175256)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥5,600,000 (Direct Cost: ¥5,600,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Keywords | Mechanism of insulin release / RINm5F cell / Intracytoplasmic free Ca / K dependent Ca channel / Carbamyl choline / Cell organella / インスリン分泌 / K^+刺激 / 細胞小器官 / 細胞内遊離Ca〓 / 細胞内小器管 / カルバミルコリン / 分泌現象 / Fora2 / EGTA / RINm5F |
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| Research Abstract |
The purpose of this study is to analyze the sequence of events occurred in the cytoplasm of endocrine cells during hormone release quantitatively and elucidate the mechanism of hormone containing granules secretion which is the major pathway or hormone release.
We investigated intracytoplasmic free Ca^<++> that is assumed to be the most important factor of hormone release. The cells used are called RINm5F cells which is cloned from malignant tumor of rat endocrine pancreas. The cell releases insulin by the stimulation of K^+ or of carbamyl-choline. The function of this cell was reassured by the incubation study with 24nMK^<++> or with 6OmuM carbamyl-choline for 15 minutes. Free Ca^<++> in cytoplasm is measured using Fura 2 as an indicator and Argus 100 (Nikon-Hamamatsu Photonics) Ca^<++> analyzing system equipped with phase contrast system. This system allows us to measure intracytoplasmic free Ca^<++> distribution and its time dependent changes in parallel with phase contrast cell imag
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Elevation of extacellulay K ^+up to 24 mM stimulated intracytoplasmic Ca^<++>accumulation in 10 seconds and plateaued for 120 seconds thereafter. The distribution of free Ca^<++>in cytoplasm was homogeneous. The depletion of extracellular Ca^<++>and addition of 60muM EGTA in the medium completely inhibited intracellular accumulation of free Ca^<++> by the elevation of extracellular K ^+. This observation indicates that potassium dependent Ca^<++> channel is the most important mechanism of K^+stimulated insulin release.
Carbamyl-choline which is,known to stimulate proteinkinese-C system stimulates intra cytoplasmic free Ca^<++> increase within 1 second and it returns to initial level within 2-3 seconds. The distribution of Ca^<++> is not homogeneous. The major elevation occurs in the area where is rich with endoplasmic reticulum. In contrast to the observation in the potassium experiment, the omission of the extracellular Ca^<++> do not affect carbamyl-choline stimulated intracytoplasmic Ca^<++> elevation. These observations suggest that carbamyl choline stimulates Ca^<++>release from at lease some cell organella in cytoplasm, presumably endoplasmic reticulum system.
These studies confirm the hypothesis which derived from biochemical studies on insulin release mechanism. Especially observation on time dependent events of intracytoplasmic free Ca^<++> with various stimuli gives us deep insight on the mechanism of insulin release and thus contributes the analysis of hormone release mechanism. Less
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