Salt responses in isolated taste cells

分离的味觉细胞中的盐反应

• 批准号: 8871429
• 负责人: Brian Lewandowski
• 金额: $ 5.6万
• 依托单位: MONELL CHEMICAL SENSES CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8871429
• 关键词: Amiloride; Anions; Cell Communication; Cell membrane; Cells; Characteristics; Code; Communication; Data; Detection; Environment; Epithelial; Feedback; Flavoring; Frequencies; Fungiform Papilla; Genes; Goals; Harvest; Health; Hypertension; Image; Label; Link; Measures; Mediating; Methodology; Modeling; Molecular; Morbidity - disease rate; Mus; Paracrine Communication; Pathway interactions; Physiological; Play; Population; Populations at Risk; Preparation; Prevalence; Process; Recruitment Activity; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Salts; Shapes; Signal Transduction; Sodium; Sodium Channel; Sodium Chloride; Solutions; Stimulus; Structure; Taste Buds; Taste Perception; Techniques; Testing; Time; Transgenic Mice; Type I Epithelial Receptor Cell; Type II Epithelial Receptor Cell; Type III Epithelial Receptor Cell; Uncertainty; Validation; base; cell preparation; cell type; epithelial Na+ channel; improved; insight; receptor; receptor expression; research study; response; salt sensitive; taste stimuli; taste transduction; theories

DESCRIPTION (provided by applicant): Salt overconsumption has been linked to excess morbidity from hypertension. In order to develop solutions to counteract salt overconsumption without sacrificing palatability, it is important to fully understand how salt taste is processed i taste buds. There are at least two pathways in taste buds that encode salt taste. These two pathways are distinguished by their sensitivity to the epithelial sodium channel (ENaC) blocker amiloride. The amiloride-sensitive (AS) pathway is selective for Na+ and Li+ salts and involves epithelial sodium channels (ENaCs). The amiloride-insensitive (AI) pathway is broadly responsive to sodium and non-sodium salts. Although the types of taste cells that express AS and AI salt taste pathways have been proposed, the available data are limited and often inconsistent. Furthermore, it is not clear how selective are salt-responsive cells, and whether their responses to other taste qualities are due to co-expression of different taste receptors in the same taste cell or indirect activation through cell-to-cell communication. In Aim 1 these uncertainties will be resolved by analyzing breadth of tuning and the expression of cell-type marker genes in physiologically identified AS and AI salt taste cells isolated from the fungiform papillae. Fura2 Ca2+ imaging will be used to measure responses to salts (with and without amiloride) and other taste stimuli. Single-cell RT-PCR will be used to test for expression of marker genes for type I, II and III cells, as well as for the expression of channels and receptors that have been implicated in taste transduction Because isolated taste cells should respond only to stimuli for which they express receptors, interpretation of these experiments will not be confounded by the possibility that cell responses to multiple taste qualities are caused by cell-to cell signaling. Thus, the responses of isolated AS and AI salt-responsive taste cells should answer whether salt receptors are ever co-expressed with receptors for other tastes. The influence of the protected microenvironment of the taste bud on salt taste will also be examined by comparing the characteristics of isolated salt-sensitive cells with characteristics of the same population of cells in an intact taste bud. This will be achieved in Aim 2 by comparing salt-responsive PKD2L1-expressing type III cells from PKD2L1-GFP transgenic mice in dissociated and intact taste bud preparations. Tastant-evoked Ca2+ responses will be measured to analyze breadth of tuning, the time-course of taste responses, and the effect of anion on salt taste. These experiments will provide information about the local network interactions used by the taste bud to help encode and accurately transmit taste information. The proposed studies will provide insight into the mechanisms of salt taste. Results from these studies may have significant implications for models of taste coding at the periphery, particularly labeled-line models. Finally, research into cell-to-cell communication in the taste bud may provide insights into the mechanisms underlying practically important perceptual interactions, such as the suppression of bitter by salty taste.

描述（由申请人提供）：盐摄入过量与高血压发病率过高有关。为了开发解决方案来抵消盐的过度消耗而不牺牲适口性，充分了解盐味在味蕾中的加工过程非常重要。味蕾中至少有两条编码咸味的通路。这两种途径的特点是对上皮钠通道 (ENaC) 阻断剂阿米洛利的敏感性。阿米洛利敏感 (AS) 途径对 Na+ 和 Li+ 盐具有选择性，并涉及上皮钠通道 (ENaC)。阿米洛利不敏感 (AI) 途径对钠盐和非钠盐广泛敏感。尽管表达 AS 和 AI 盐味通路的味觉细胞类型已被提出，但可用数据有限且常常不一致。此外，尚不清楚盐反应细胞的选择性如何，以及它们对其他味觉质量的反应是否是由于同一味觉细胞中不同味觉受体的共表达或通过细胞间通讯的间接激活所致。在目标 1 中，这些不确定性将通过分析从菌状乳头中分离出的生理学鉴定的 AS 和 AI 盐味细胞中的调节广度和细胞类型标记基因的表达来解决。 Fura2 Ca2+ 成像将用于测量对盐（含或不含阿米洛利）和其他味觉刺激的反应。单细胞 RT-PCR 将用于测试 I、II 和 III 型细胞的标记基因的表达，以及与味觉转导有关的通道和受体的表达，因为分离的味觉细胞应该只对由于它们表达受体的刺激，对这些实验的解释不会被以下可能性所混淆：细胞对多种味觉质量的反应是由细胞与细胞之间的相互作用引起的。 细胞信号传导。因此，分离的 AS 和 AI 盐反应味觉细胞的反应应该可以回答盐受体是否与其他味觉受体共表达。通过将分离的盐敏感细胞的特征与完整味蕾中相同细胞群的特征进行比较，还将检查受保护的味蕾微环境对盐味的影响。这将在目标 2 中通过比较解离和完整味蕾制剂中来自 PKD2L1-GFP 转基因小鼠的盐响应性 PKD2L1 表达 III 型细胞来实现。将测量促味剂诱发的 Ca2+ 反应，以分析调节的广度、味觉反应的时间进程以及阴离子对盐味的影响。这些实验将提供有关味蕾使用的本地网络交互的信息，以帮助编码和准确地传输味道信息。拟议的研究将深入了解盐味的机制。这些研究的结果可能对外围的味觉编码模型，特别是标记线模型具有重要意义。最后，对味蕾细胞间通讯的研究可能有助于深入了解实际上重要的知觉相互作用的潜在机制，例如咸味抑制苦味。