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

我们开发了一种装置，用于自动采样从分离的舌上皮中释放的物质，其中包含味蕾在重复的味觉刺激。该装置由三部分组成：(1)分离舌上皮基底外侧粘膜侧的腔室；(2)用味觉刺激反复刺激粘膜侧，用正常Tyrode溶液分别灌注基底外侧的编程驱动装置。对刺激的次数和持续时间进行规划；(3)取样装置，用于将含有从上皮中释放的物质的溶液转移到样品杯中，样品杯冷却至20ºC。利用该装置，我们用高效液相色谱（HPLC）检测了味觉刺激下从大鼠舌上皮中释放的样品中肽样物质的两个峰。从大鼠样品中检测到的两个峰分别与Met-enkephalin…More和CCK8的峰大致一致。在盐和酸刺激下，仅在III型味蕾细胞中观察到的血清素分泌也被检测到。这些结果表明，本发明的活上皮制剂释放物质自动取样装置可用于生理条件下生理活性物质的取样、鉴定和定量。另一方面，使用脂质体和人工脂质体D_<14>DPC可以直接从舌上皮组织中提取膜蛋白。结果表明，含D_<14>DPC的脂质体提取膜中的蛋白质与l -丙氨酸具有较高的亲和力。舌面D_<14>DPC脂质体处理后，味觉神经对氨基酸的反应受到抑制，而不受D_<14>DPC脂质体处理的影响。因此，我们得出结论，脂质体处理D_<14>的舌上皮DPC是一种强有力的技术，可以提取对味觉剂有高亲和力的膜蛋白。少

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