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Development of system for analyzing membrane proteins of taste cells ender physiological condition.

开发用于分析味觉细胞膜蛋白的系统以实现生理条件。

基本信息

批准号：
07557117
负责人：
SATO Toshihide
金额：
$ 7.68万
依托单位：
Ngasaki University
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 1997
项目状态：
已结题

项目摘要

We deveoled a device for automatically sampling substances liberated from the isolated lingual epithelium containing taste buds during repetitive taste stimulations. The device consists of three part : (1) Chamber for isolating the mucosal side from the basolateral side of the lingual epithelium ; (2) Programming and driving apparatus for repeated stimulation of the mucosal side with taste stimuli and deparate perfusion of the basolateral side with normal Tyrode solution. The number and duration of stimulations are programd ; (3) Sampling apparatus for tanspoting the solution containing substances liberated from the epithelium into the sample cups, which are cooled down up to-20ﾟC.Using this device, we detected two peaks of peptide-like substances by a high performance liquid chromatography (HPLC) in the sample liberated from rat lingual epithelium during gustatory stimulations. Two peaks detected in the sample botained from rat are roughly consistent with the peaks for Met-enkephalin … More and CCK8, respectively. Secretion of serotonin which is observed in only type III taste bud cells was also detected during salt and acid stimulations. These results suggest that the automatic sampling device for substances liberated from living epithelial preparation developed here is useful for sampling, identification and quantification of the physiologically active substances under the physiological condition.On the other hand, using liposome with an artificial lipid, D_<14>DPC enabled us to directly extract membrane proteins from the lingual epithelial tissue. Using this technique, it was evident that the protein in the extracting membrane with liposome containting D_<14>DPC represents a high affinity to L-alanine. The gustatory neural responses to amino acids were suppressed by the liposomal treatment with D_<14>DPC of the tongue surface, but were not affected by the liposomal teatment without D_<14>DPC.Therefore, we concluded that the liposomal treatment with D_<14>DPC of the tongue epithelium in a powerful technique to extract the membrane proteins which have high affinity to tastants. Less

我们开发了一种设备，用于在重复的味觉刺激过程中，自动采样从包含味蕾的孤立舌上皮中释放出来的物质。该装置由三部分组成：(1)舌上皮基底侧与粘膜侧隔离的小室；(2)用味觉刺激反复刺激粘膜侧，并用正常泰氏液对基底侧进行分离灌流的编程和驱动装置。设定刺激次数和刺激时间；(3)进样装置，用于将含有从上皮释放出来的物质的溶液送入样品杯，冷却至-20ﾟC。使用该装置，我们在味觉刺激时从大鼠舌上皮释放的样品中用高效液相色谱法检测到两个多肽类物质的峰。在大鼠样本中检测到的两个峰与甲硫氨酸脑啡肽…的峰大致一致More和CCK8。在盐和酸的刺激下，也检测到只在III型味蕾细胞中观察到的5-羟色胺的分泌。这些结果表明，我们研制的活体上皮释放物质自动采样器可用于生理条件下生理活性物质的采样、鉴定和定量。另一方面，利用含有人工脂质的脂质体，D_&lt；14&gt；DPC使我们能够直接从舌上皮组织中提取膜蛋白。结果表明，含D_(14)&gt；DPC的脂质体提取膜中的蛋白质对L-丙氨酸有较高的亲和力。含D&lt；14&gt；DPC的舌上皮脂质体对氨基酸的味觉神经反应有抑制作用，但不受不含D&lt；14&gt；DPC的脂质体处理的影响。因此，D&lt；14&gt；DPC是一种提取与味觉亲和力较高的膜蛋白的有效方法。较少