Chemosensing in the Gastrointestinal Tract

描述(由申请人提供)：胃肠道(GI)粘膜对肠腔内容物的感知在调节消化功能和保护免受有害物质伤害方面起着关键作用。最近发现，在口腔中介导味觉信号的苦味受体(T2Rs)和G蛋白亚基？？Gustducin和？？转导蛋白也在GI粘膜中表达，这表明这些信号分子参与了腔内有害物质的功能检测，并可能启动包括停止食物摄入在内的保护性反应。这一应用将检验这样一个假设，即GI粘膜中苦味化学感觉受体的激活诱导上皮细胞释放信号分子，进而激活神经元通路来调节GI功能和食物摄入量。这一假设将通过以下具体目标进行检验。具体目标1将确定1a)肠腔内T2R激动剂对迷走神经传入通路的影响和作用部位，使用c-fos作为神经元活动的标志，以及CCK和PYY，这两种影响胃肠功能和摄食行为的多肽，作用于迷走神经传入的CCK1和Y2受体，1b)T2R激动剂对迷走神经通路的影响，通过电生理记录支配胃和十二指肠的迷走神经传入通路，以及1c)T2Rs激动剂诱导的传入神经元激活是否由？Guducin和？转导蛋白介导。具体目的2将通过确定2a)胃运动功能、肠道分泌、食物摄入和厌恶行为对T2R亚型激动剂的反应的变化来确定刺激胃肠道T2R及其通过喂养调节的功能意义，2b)这些影响是否通过？？2)禁食和摄食以及苦味刺激对胃泌素、转导蛋白和部分T2Rs表达的影响；2)T2R激动剂在体外启动的肠内分泌STC-1细胞信号转导通路(细胞内钙和ERK)，以及T2R激动剂是否原位激活内分泌细胞，用免疫组织化学方法检测磷酸化钙调素依赖激酶2(CaMK2)作为细胞内钙升高的标志。长期目标是发展对管腔化学传感的调控机制的理解。这一点很重要，因为肠道内容物的分子传感调节运动、信号分子的释放和动态平衡的维持，还负责检测摄入的有害药物和毒素，这些药物和毒素可能引发对生存至关重要的反应。公共卫生相关性：胃肠道(GI)粘膜对腔内容物的感知在调节消化功能和保护其免受有害物质伤害方面起着至关重要的作用。目前的应用将集中在不同味觉受体亲和力不同的苦味剂激活的通路以及这些激动剂对胃肠功能和摄食行为的影响。长期目标是了解腔化学传感的调节机制，腔化学传感是控制胃肠道功能反应的重要生理过程，影响摄食行为和保护免受有害药物和毒素的影响。

DESCRIPTION (provided by applicant): Sensing of luminal contents by the gastrointestinal (GI) mucosa plays a critical role in the regulation of digestive functions and protection from harmful substances. The recent discovery that bitter taste receptors (T2Rs) and the G-protein subunits, ??gustducin and ??transducin, which mediate gustatory signals in the oral cavity, are also expressed in the GI mucosa suggests that these signaling molecules participate in the functional detection of harmful substances in the lumen and possibly initiate a protective response including cessation of food intake. This application will test the hypothesis that activation of bitter chemosensory receptors in the GI mucosa induces release of signaling molecules by epithelial cells that in turn activate neuronal pathways to modulate GI function and food intake. This hypothesis will be tested by the following specific aims. Specific Aim 1 will determine 1a) the effects and site of action along the gut of intraluminal T2R agonists on vagal afferent pathways using c-fos as marker of neuronal activity, and the role of CCK and PYY, peptides that affect GI function and feeding behavior, acting at CCK1 and Y2 receptors on vagal afferents, 1b) the effects of T2R agonists on vagal pathways using electrophysiological recording of vagal afferents innervating the stomach and duodenum, and 1c) whether afferent neuronal activation induced by T2Rs agonists is mediated by ??gustducin and ??transducin. Specific Aim 2 will establish the functional significance of the stimulation of gastrointestinal T2Rs and their regulation by feeding by determining 2a) the changes in gastric motor function, intestinal secretion, food intake and aversion behavior in response to T2R subtype agonists, 2b) whether these effects are mediated by ?? gustducin and ??transducin and whether they involve CCK and PYY acting at CCK1 and Y2 receptors, 2c) the effect of fasting and feeding, and of bitter stimulation on the expression of ??gustducin, ??transducin and selected T2Rs, and 2d) the signal transduction pathways (intracellular Ca2+ and ERK) initiated by T2R agonists in enteroendocrine STC-1 cells in vitro, and whether intraluminal T2R agonists activate endocrine cells in situ using immunohistochemistry for phosphorylated calmodulin dependent kinase 2 (CAMK2) as a marker for intracellular Ca2+ elevation. The long term goal is to develop an understanding of the mechanisms regulating luminal chemosensing. This is of importance since molecular sensing of gut luminal contents regulates motility, release of signaling molecules and homeostasis maintenance, and it is also responsible for the detection of ingested harmful drugs and toxins that could initiate response critical for survival. PUBLIC HEALTH RELEVANCE: Sensing of luminal contents by the gastrointestinal (GI) mucosa plays a critical role in the regulation of digestive functions and in the protection from harmful substances. The current application will focus on the pathways activated by bitter tastants with different affinity for different taste receptors and the effects of these agonists on GI function and feeding behavior. The long term goal is to develop an understanding of the mechanisms regulating luminal chemosensing, an important physiological process that controls GI functional responses that impact on feeding behavior and protection from harmful drugs and toxins.