PHYSIOLOGY OF ISOLATED HUMAN TASTE RECEPTOR CELLS

人类味觉细胞的转导机制尚不清楚。本申请的目的是加强从人类志愿者的真菌状乳头中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人类正常味觉反应开辟新的重要途径，并为确定人类化学感觉疾病的病程提供动力。本研究的三个目的是：制定方案，以最大限度地提高分离的人类味觉细胞的活力；表征刺激调控下这些细胞内钙离子活性和膜电位的变化；并对这些细胞进行松散补丁分析，以识别这些孤立细胞中的刺激调节电流。迄今为止，研究人员根据这一建议开发的分离人类味觉细胞的程序尚未得到优化，导致各种制剂之间存在不可接受的差异。为了使表征研究有效地进行，需要更好地定义分离方案。将进行酶类型/量、营养、培养基和保存条件的实验。使用Fura 2和di-8-ANEPPS染料进行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。对主要是甜的和苦的刺激的反应将被评估。松散贴片研究将用于描述单个细胞的味道形态和化学特异性。人类味觉细胞尚未成为生理实验研究的对象。人类味觉的独特特性可以用来测试单个细胞。志愿者可以在去除真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化学感觉障碍的治疗方案的可能性。这项研究的成功完成将允许在味觉的心理物理测量和受体细胞的生物物理特性之间建立相关性。更全面地了解味觉的外围和中枢过程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.