Dynamic Cell Biological Approach to the Mechanism of Hormone Containing Granule Release from Pancreatic B-cells.

动态细胞生物学方法研究胰腺 B 细胞释放含激素颗粒的机制。

• 批准号: 63480264
• 负责人: KANAZAWA Yasunori
• 金额: $ 3.58万
• 依托单位: Jichi Medical School (1989)The University of Tokyo (1988)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480264/
• 关键词: 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

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 … More es.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

为了定量分析激素释放过程中细胞质内发生的事件顺序，阐明激素释放的主要途径--含激素颗粒的分泌机制，本研究测定了细胞质内游离Ca^<++>，它被认为是激素释放过程中最重要的因素。所用的细胞称为RINm 5 F细胞，其是从大鼠内分泌胰腺恶性肿瘤中克隆的。细胞在K^+或氨甲酰胆碱的刺激下释放胰岛素。用24 nMK ^++>或60 μ M氨甲酰胆碱孵育15分钟，可以确认该细胞的功能。使用Fura 2作为指示剂和配备有相差系统的Argus 100（Nikon-Hamamatsu Photonics）Ca^<++>分析系统测量细胞质中的游离Ca^<++>。该系统使我们能够测量胞浆内游离Ca^<++>分布及其时间依赖性变化，并与相差细胞图像平行。 ...更多信息 细胞外K ^+浓度升高到24 mM时，胞浆内Ca^++的积累在10秒内就被激活，之后在120秒内达到稳定状态。游离Ca^<++>在细胞质中分布均匀。细胞外Ca^<++>的耗竭和在培养基中加入60 μ M EGTA完全抑制了细胞内游离Ca^<++>的积累，而细胞外K ^+的升高则抑制了细胞内游离Ca^<++>的积累。这一观察结果表明，钾依赖性Ca^++通道是钾刺激胰岛素释放的最重要机制，已知刺激蛋白激酶-C系统的氨甲酰胆碱在1秒内刺激胞浆内游离Ca^++增加，并在2-3秒内恢复到初始水平。Ca^<++>的分布是不均匀的。主要的隆起发生在富含内质网的区域。与钾离子实验中的观察结果相反，细胞外Ca^<++>的缺失并不影响氨甲酰胆碱刺激的胞浆内Ca^<++>升高。这些结果表明，氨甲酰胆碱刺激细胞质中至少某些细胞器（可能是内质网系统）释放Ca^++，证实了胰岛素释放机制的生化研究假说。特别是对不同刺激条件下胞浆内游离Ca^<++>随时间变化的观察，使我们对胰岛素释放机制有了更深的认识，从而有助于对激素释放机制的分析。少

项目成果

野田光彦/金澤康徳: 糖尿病. 33. (1989)

野田光彦/金泽康典：糖尿病 33。（1989）

Mitsuhiko,NODA/Yasunori,KANAZAWA: Diabetes. 38. (1989)

野田光彦/金泽康典：糖尿病。