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Visualization of spatio-temporal pattern of IP3/Ca2+ signaling in salivary glands

唾液腺 IP3/Ca2 信号传导时空模式的可视化

基本信息

批准号：
17591946
负责人：
TOJYO Yosuke
金额：
$ 2.24万
依托单位：
Health Sciences University of Hokkaido,
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591946/
关键词：
salivary glands calcium ion inositol 1, 4, 5-trisphosphate calcium signal fluorescent imaging GFP FRET LIBRA

项目摘要

1. The Ca^<2+> signaling in salivary ductal cells was visualized by multi-photon microscopy. The results indicated that ducts contain a certain subpopulation of cells, which exhibits particularly high sensitivity to these Ca^<2+>-mobilizing agonists. Also, low concentrations of phenylephrine, carbachol and ATP activated different subpopulations of ductal cells.2. To analyze IP_3 production in salivary cells, variants of the FRET-based biosensor LIBRA were constructed. To obtain the pH-stable LIBRA, we replaced the FRET acceptor EYFP to Venus, a more bright and pH-stable mutant. Next, we optimized the linkers between ECFP and IP_3-binding domain and between Venus and IP_3-binding domain. Furthermore, we replaced Venus and ECFP to their circularly permuted types.3. We expressed the variants of LIBRA to cultured cells and measured the changes in LIBRA fluorescence in saponin- permeabilized cells. Using the improved LIBRA, change in the ratio increased to about 140%.4. Dispersed rat parotid acinar cells were prepared by enzymatic digestion and cultured in DMEM-F12 medium. At 6 h after dispersion, the cultured acinar cells showed a similar morphology to that of the cells immediately after dispersion. The polarity seemed to maintain at least for 6 h. At 48 h after dispersion, the polarity was lost in most of the cells, but some cells maintained it. Intracellular Ca^<2+> concentrations ([Ca^<2+>]i) were measured in fura-2-loaded cells. Stimulation with agonists induced significant increases in [Ca^<2+>]i even in the cells cultured for 48 h.5. Dispersed rat parotid acinar cells were transfected with cDNA for GFP and PKC-GFP. The optimal condition for tranfection was examined using various reagents and medium. The used of Lipofectoamine 2000 and Opti MEM was most effective for tranfection. Stimulation with ionomycin induced translocation of PKC-GFP from cytoplasm to plasma membrane.

1.通过多光子显微镜观察唾液导管细胞中的Ca 2+ 信号传导。结果表明，导管含有特定的细胞亚群，其对这些Ca 2+ -动员激动剂表现出特别高的敏感性。此外，低浓度的去氧肾上腺素、卡巴胆碱和ATP可激活不同的导管细胞亚群。2．为了分析唾液细胞中 IP_3 的产生，构建了基于 FRET 的生物传感器 LIBRA 的变体。为了获得 pH 稳定的 LIBRA，我们将 FRET 受体 EYFP 替换为更亮、pH 稳定的突变体 Venus。接下来，我们优化了 ECFP 和 IP_3 绑定域之间以及 Venus 和 IP_3 绑定域之间的链接器。此外，我们将Venus和ECFP替换为它们的循环排列类型。3．我们向培养细胞表达了 LIBRA 的变体，并测量了皂苷透化细胞中 LIBRA 荧光的变化。使用改进后的LIBRA，变化比例增加到140%左右。4．通过酶消化制备分散的大鼠腮腺腺泡细胞并在DMEM-F12培养基中培养。分散后6小时，培养的腺泡细胞表现出与分散后立即细胞相似的形态。极性似乎维持至少6小时。分散后48小时，大多数细胞失去极性，但一些细胞保持极性。在负载fura-2的细胞中测量细胞内Ca 2+ 浓度([Ca 2+ ]i)。即使在培养48小时的细胞中，用激动剂刺激也诱导[Ca 2+ ] i 显着增加。5。用GFP和PKC-GFP的cDNA转染分散的大鼠腮腺腺泡细胞。使用各种试剂和培养基检查转染的最佳条件。使用Lipofectoamine 2000 和Opti MEM 进行转染最有效。离子霉素刺激诱导 PKC-GFP 从细胞质易位至质膜。