Purinergic signaling in taste buds

描述（由申请人提供）：虽然在识别味觉转导的受体和下游信号机制方面取得了实质性进展，但对味觉细胞如何将这些信息传递给神经系统知之甚少。最近，我们发现ATP是连接味觉细胞激活味觉传入纤维的关键递质。使用缺乏嘌呤能受体P2X2和P2X3的小鼠，我们发现这些小鼠对所有味觉刺激都缺乏味觉反应。我们的实验室和其他人的进一步实验表明，表达苦味、甜味和鲜味味觉受体的味觉细胞通过非囊泡机制释放ATP，可能是由Pannexin1组成的半通道。此外，释放的ATP被外链核苷酸酶NTPDase2分解为ADP，在味蕾的胶质样支持细胞的膜上表达。然而，关于ATP在味蕾中作为一种传递器的作用，仍有几个问题有待解决。首先，Pannexin1是ATP释放和味觉事件完全激活所必需的吗？这还没有在体内测试过。此外，为什么在P2X2/3双敲除小鼠中没有酸和咸的反应，而已知唯一释放ATP的味觉细胞是对苦、甜和鲜味刺激作出反应的细胞？最后，外ATP酶在味蕾释放ATP的持久性中起什么作用？它是如何影响味觉功能的？在这项新的拨款提案中，我们将使用现有的基因敲除小鼠，从基因上消除味蕾中嘌呤能信号传导的关键元素。我们将使用综合系统神经科学方法-从ATP释放的细胞分析到味觉神经记录和行为-来回答以下问题：(1)Pannexin1是ATP释放和味觉神经纤维激活所必需的吗？我们将使用Pannexin1的整体和条件敲除来解决该通道是否介导味觉细胞中的ATP释放，以及它是否必要和充分激活味觉传入纤维和味觉引导行为。(2) NTPDase2在ATP释放持久性中的作用是什么？我们将利用NTPDase2敲除小鼠来确定NTPDase2如何影响释放的ATP的大小和持久性，以及它在传入神经纤维对味觉刺激的敏感性中起什么作用。这些实验结果将为ATP在味觉功能中的作用提供重要的新数据。通过了解味觉细胞如何与传入神经纤维沟通，有可能开发出干扰这一过程并调节味觉功能的药物。这可能对食物摄入失调导致的健康问题产生重要影响。

DESCRIPTION (provided by applicant): While substantial progress has been made in identifying the receptors and downstream signaling mechanisms involved in taste transduction, much less is known about how taste cells transmit this information to the nervous system. Recently we identified ATP as a key transmitter linking taste cells to activation of gustatory afferent fibers. Using mice that lack the purinergic receptors P2X2 and P2X3, we showed that these mice lack gustatory responses to all taste stimuli. Further experiments by our labs and others have shown that the taste cells that express bitter, sweet, and umami taste receptors release ATP via non-vesicular mechanisms, likely hemichannels composed of Pannexin1. Further, the ATP that is released is broken down to ADP by the ecto- nucleotidase NTPDase2, expressed on the membranes of the glial-like support cells in the taste bud. However, several questions remain about the role of ATP as a transmitter in taste buds. First, is Pannexin1 required for ATP release and full activation of gustatory afferents? This has not been tested in vivo. Further, why are sour and salty responses absent in the P2X2/3 double knockout mice when the only taste cells known to release ATP are the cells that respond to bitter, sweet, and umami stimuli? Finally, what is the role of the ecto-ATPase in the perdurance of the ATP released from taste buds and how does this impact gustatory function? In this new grant proposal we will use existing knockout mice to genetically eliminate key elements of purinergic signaling in taste buds. We will use an integrated systems neuroscience approach-- from cellular assays of ATP release to gustatory nerve recording and behavior-- to answer the following questions: (1) Is Pannexin1 required for ATP release and activation of gustatory nerve fibers? We will use both a global and a conditional knockout of Pannexin1 to address whether this channel mediates ATP release in taste cells, and whether it is necessary and sufficient for full activation of gustatory afferent fibers and taste guided behavior. (2) What is the role of NTPDase2 in the perdurance of ATP release? We will utilize NTPDase2 knockout mice to determine how NTPDase2 affects the magnitude and perdurance of the ATP that is released and what role this plays in the sensitivity of the afferent nerve fibers to taste stimuli. Results from these experiments will provide important new data about the role of ATP in gustatory function. By understanding how taste cells communicate with afferent nerve fibers, it may be possible to develop pharmaceutical agents that will interfere with this process and modulate taste function. This could have an important impact on health problems that result from disorders of food intake. PUBLIC HEALTH RELEVANCE: The sense of taste evolved to allow discrimination of nutritionally important compounds from toxic substances. However, overconsumption of palatable foods can lead to obesity and Type II diabetes, now a major world heath problem. Conversely, the off-taste of a medicine can be a limiting factor in patient compliance. A better understanding of the mechanisms used by taste cells to detect chemicals and transmit taste information to the nervous system may lead to the development of pharmaceuticals that can control overconsumption and limit the bitter tastes of many drugs.