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

我们研制了一种装置，用于在重复的味觉刺激过程中自动采样从含味蕾的离体舌上皮释放的物质。该装置由三个部分组成：（1）用于将舌上皮的粘膜侧与基底侧隔离的腔室;（2）编程和驱动装置，用于用味觉刺激重复刺激粘膜侧，并用正常台氏液对基底侧进行去极化灌注。（3）取样装置，用于将含有从上皮释放的物质的溶液转移到样品杯中，样品杯冷却至-20 ° C。使用该装置，我们用高效液相色谱（HPLC）检测到在味觉刺激期间从大鼠舌上皮释放的样品中的两个肽样物质峰。在大鼠样品中检测到的两个峰与甲硫氨酸脑啡肽的峰大致一致 关于我们 分别为CCK 8。在盐和酸刺激期间也检测到仅在III型味蕾细胞中观察到的血清素的分泌。这些结果表明，本文开发的活体上皮制备物释放物质自动采样装置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，使用带有人工脂质的脂质体，D_<14>DPC使我们能够直接从舌上皮组织中提取膜蛋白。结果表明，D_ DPC脂质体提取膜中的蛋白质<14>对L-丙氨酸具有较高的亲和力。用脂质体处理舌表面，D_ DPC可抑制舌上皮细胞对氨基酸的味觉神经反应<14>，而不加D_ DPC的脂质体处理舌表面，则对味觉神经反应无影响<14>。因此，D_ DPC脂质体处理<14>舌上皮细胞是一种有效的提取与促味剂有高亲和力的膜蛋白的技术。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