Development of super-sensitive assay for hormone secretion from single cells

单细胞激素分泌超灵敏检测方法的开发

• 批准号: 07557070
• 负责人: SEINO Susumu
• 金额: $ 6.91万
• 依托单位: Chiba University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557070/
• 关键词: ATP; Insulin; Calcium; ATP receptor; Exocytosis

ATP is costored with neurotransmitters in the synaptic vesicles of neuronal cells and with hormones in the secretory vesicles. ATP is known to be coreleased with various hormones, including insulin from pancreatic beta-cells and catecholamine from chromaffin cells. We have developed the sensitive assay which can moniter the released hormone, using pancreatic beta-cells as a model system. To monitor insulin secretion from single beta-cells, a single beta-cell was surrounded in culture by fura-2 loaded calf pulmonary artery endothelium (CPAE) cells, which can detect the ATP.CPAE cells did not respond with a rise in cytoplasmic calcium concentration ([Ca^<2+>]i) to either tolbutamide or kainate, but did respond with a rise in [Ca^<2+>]i to ATP without desensitization and in a dose-dependent manner. A brief application of tolbutamide (10mM) increased [Ca^<2+>]i in both the beta-cell and the adjacent CPAE cells in coculture. Suramin, an ATP receptor blocker, inhibited the tolbutamide-induce … More d elevation in [Ca^<2+>]i in the CPAE cells, but did not inhibit the elevation in [Ca^<2+>]i in the beta-cell, confirming that the insulin secretagogue-induced Ca^<2+> response in CPAE cells in coculture is mediated by ATP released from the beta-cell. The coculture system using CPAE cells as reporter cells is, therefore, useful for monitoring insulin secretion from single intact beta-cells.We have also shown by this system that upon stimulation of pancreatic beta-cells, they respond to the realesd ATP,suggesting that ATP may act on pancreatic beta-cells to stimulate insulin secretion through P_2 purinoreceptors (P_<2X> receptor). We isolated cDNA encoding a fourth member (P_<2X-4>) of the P_<2X> family by screening a rat pancreatic islet cDNA library. Rat P_<2X-4> is a protein of 388 amino acids and has two putative transmembrane segments. P_<2X-4> mRNA is widely expressed in brain and peripheral tissues, including various endocrine tissues and it is also expressed in various hormone-secreting cell lines. We have heterologously expressed the cloned P_<2X-4> in Xenopus laevis oocytes and have characterized its pharmacological properties ATP,its analogs and ADP activate cation-selective ion channels. The order of agonist potency is ATP>ADP>2-methyl-thioATP (2MeSATP) >>alphabeta-methelene-ATP (alphabetameATP). These results suggests that P_<2X-4> mediates extracellular ATP-induced biological effects in non-neuronal cells including, endocrine cells, as well as in neuronal cells. Less

ATP与神经递质共同储存在神经元细胞的突触囊泡中，与激素共同储存在分泌囊泡中。已知ATP与各种激素共同释放，包括来自胰腺β细胞的胰岛素和来自嗜铬细胞的儿茶酚胺。我们以胰岛β细胞为模型系统，建立了一种灵敏的激素释放检测方法。为了监测单个β细胞的胰岛素分泌，在培养的单个β细胞周围加入Fura-2负载的小牛肺动脉内皮细胞（CPAE），该细胞可以检测ATP。（[Ca^2+]i），但对ATP的反应是[Ca^2+]i的升高，而不发生脱敏，且呈剂量依赖性。短暂应用甲苯磺丁脲（10 mM）可增加共培养的β细胞和相邻CPAE细胞中的[Ca^2+]i。ATP受体阻断剂苏拉明可抑制甲苯磺丁脲诱导的细胞凋亡。 ...更多信息 d CPAE细胞[Ca^2+]i的升高，但不能抑制β细胞[Ca^2+]i的升高，这证实了在共培养的CPAE细胞中胰岛素促分泌素诱导的Ca^2+反应是由β细胞释放的ATP介导的。用CPAE细胞作为报告细胞的共培养系统可用于监测单个完整胰岛β细胞的胰岛素分泌，并证实胰岛β细胞对ATP有反应，提示ATP可能通过P_2嘌呤受体（P_2 receptor，P_1 receptor）作用于胰岛β细胞，刺激胰岛β细胞分泌胰岛素<2X>。我们从大鼠胰岛cDNA文库中筛选出了编码P_家族第四个成员（P_）的cDNA<2X-4><2X>。大鼠P_<2X-4>1蛋白由388个氨基酸组成，具有两个跨膜区。PmRNA<2X-4>广泛表达于脑和外周组织，包括各种内分泌组织，也表达于多种肿瘤分泌细胞系。我们在非洲爪蟾卵母细胞中异源表达了克隆的<2X-4>P_1，并对其药理学性质进行了研究。激动剂作用强度顺序为ATP&gt;ADP&gt;2-甲硫基ATP（2 MeSATP）&gt;&gt; α-亚甲基-ATP（α-ATP）。这些结果表明，P_<2X-4>2不仅在神经细胞中，而且在非神经细胞（包括内分泌细胞）中介导细胞外ATP诱导的生物学效应。少

项目成果

難波範行 他: "ホルモンと臨床" 医学の世界社, 7 (1996)

Noriyuki Namba 等：“激素与临床实践”Igaku no Sekaisha，7 (1996)

Kuromi,H.et al.: "A monitor of secretion from single pancreatic β-cells." Diabetes. 44. 1213-1217 (1995)

Kuromi, H. 等人：“单个胰腺 β 细胞分泌的监测。”44. 1213-1217 (1995)

Inagaki,N.et al.: "Expression and role of ionotropic glutamate receptors in pancreatic islet cells." FASEB. J.9. 686-691 (1995)

Inagaki，N.等人：“胰岛细胞中离子型谷氨酸受体的表达和作用。”

Marqueze, B.et al.: "Cellular localization of Synapototagmin I,II,and III mRNAs in the central nervous system, pituitary and adrenal glands of the rat" J.Neurosci.15. 4906-4917 (1995)

Marqueze, B.等人：“大鼠中枢神经系统、垂体和肾上腺中突触结合蛋白 I、II 和 III mRNA 的细胞定位”J.Neurosci.15。

Inagaki, N.et al.: "Reconstitution of I_<KATP> : An inward rectifier subunit plus the sulfonylurea receptor" Science. 270. 1166-1170 (1995)

Inagaki, N.等人：“I_<KATP> 的重构：内向整流亚基加上磺酰脲受体”科学。