PHYSIOLOGY OF ISOLATED HUMAN TASTE RECEPTOR CELLS

人类味觉细胞所使用的转导机制知之甚少。本申请的目的是增强从人类志愿者的菌状乳头分离味觉细胞的技术，然后研究这些细胞的生理特性。这项可行性研究的成功完成将为研究正常人类味觉反应开辟新的重要途径，并推动确定人类化学感觉疾病的过程。本研究的三个目的是：开发协议，以最大限度地提高分离的人类味觉细胞的活力;表征刺激调节的细胞内钙离子活动和膜电位在这些细胞中的变化;并进行松散的补丁分析，这些细胞，以确定刺激调节电流在这些分离的细胞。迄今为止，研究人员根据这一提议开发的用于分离人类味觉细胞的程序尚未优化，导致制剂之间存在不可接受的差异。为了使表征研究能够有效进行，需要更好地定义分离方案。将进行酶类型/量、研磨、培养基和放置条件的实验。使用染料Fura 2 & di-8-ANEPPS的细胞内钙和膜电位的成像研究将允许进行基本的表征研究。这些将强调离子通道的表征和味觉细胞对药理学挑战的正常反应。将评估对主要甜和苦刺激的反应。将使用散斑研究来表征单个细胞的味觉形态和化学特异性。人类的味觉细胞还没有成为生理学实验的研究对象。人类味觉的独特性可以用来测试单个细胞。志愿者可以在去除菌状乳头之前进行味觉测试，以确定他们对特定刺激的反应。这项研究具有很高的影响力，因为它直接涉及人类的受体细胞，从而加强了开发治疗方案以治疗化学感受性疾病的可能性。这项研究的成功完成将使相关性之间的心理物理措施的味觉和生物物理特性的受体细胞。一个更完整的了解周边与中央过程的味道将是一个可能的结果。

Little is known of the transduction mechanisms used by human taste cells. It is the purpose of this application to enhance techniques for taste cell isolation from fungiform papillae of human volunteers and to then investigate the physiological characteristics of these cells. Successful completion of this feasibility study will open new important routes for studying normal human taste response and give impetus to determining the course of human chemosensory disease. The three aims of this study are: to develop protocols to maximize viability of isolated human taste cells; to characterize stimulus-regulated changes in intracellular calcium ion activities and membrane potential in these cells; and to perform loose patch analyses on these cells to identify stimulus-regulated currents in these isolated cells. Procedures developed to date by the investigators on this proposal for isolated human taste cells have yet to be optimized, resulting in unacceptable variations among preparations. In order to allow characterization studies to proceed efficiently, isolation protocols need to be better defined. Experimentation with enzyme type/amount, trituration, media and holding conditions will be performed. Imaging studies of intracellular calcium and membrane potential using the dyes Fura 2 & di-8-ANEPPS will permit basic characterization studies to be performed. These will emphasize characterization of ion channels and normal responses of taste cells to pharmacological challenges. Responses to primarily sweet and bitter stimuli will be evaluated. Loose patch studies will be used to characterize individual cells as to taste modality and chemical specificity. Human taste cells have not been the subject of study for physiological experiments. The unique specificities of human taste can be used to test individual cells. Volunteers can be taste tested prior to have fungiform papillae removed to determine their response to particular stimuli. This research is of high impact since it deals directly with the receptor cells of humans, thereby strengthening the likelihood that therapy regimens can be developed to treat chemosensory disorders. Successful completion of this study will allow correlations to be made between psychophysical measures of taste and biophysical characteristics of the receptor cells. A more complete understanding of peripheral versus central processes of taste will be a likely outcome.