Chemosensing in the Gastrointestinal Tract

描述（由申请方提供）：胃肠道（GI）粘膜对腔内内容物的感知在调节消化功能和保护免受有害物质影响方面起着关键作用。最近发现苦味受体（T2 R）和G蛋白亚单位，？？gustducin和？？在口腔中介导味觉信号的转导蛋白也在GI粘膜中表达，这表明这些信号分子参与管腔中有害物质的功能检测，并可能启动保护性反应，包括停止食物摄入。本申请将检验以下假设：GI粘膜中苦味化学感受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以调节GI功能和食物摄入。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）使用c-fos作为神经元活性的标志物，沿着肠腔内T2 R激动剂对迷走神经传入通路的作用和作用部位，以及CCK和PYY的作用，CCK和PYY是影响GI功能和进食行为的肽，作用于迷走神经传入上的CCK 1和Y2受体，1b）T2 R激动剂对迷走神经通路的影响，使用迷走神经传入神经支配胃和十二指肠的电生理记录，和1c）T2 R激动剂诱导的传入神经元激活是否由？? gustducin和？？转导素具体目标2将通过确定2a）胃运动功能、肠分泌、食物摄入和厌恶行为对T2 R亚型激动剂的反应的变化，2b）这些作用是否由？gustducin和？？转导蛋白以及它们是否涉及CCK和PYY作用于CCK 1和Y2受体，2c）禁食和进食以及苦味刺激对？gustducin，？？转导素和选择的T2 R，和2d）体外肠内分泌STC-1细胞中由T2 R激动剂引发的信号转导途径（细胞内Ca 2+和ERK），以及使用磷酸化钙调蛋白依赖性激酶2（CAMK 2）的免疫组织化学作为细胞内Ca 2+升高的标志物，管腔内T2 R激动剂是否原位激活内分泌细胞。长期目标是了解调节管腔化学传感的机制。这是重要的，因为肠腔内容物的分子传感调节运动性、信号分子的释放和稳态维持，并且它还负责检测可能引发对生存至关重要的反应的摄入的有害药物和毒素。公共卫生相关性：胃肠道（GI）粘膜对腔内内容物的感知在消化功能的调节和对有害物质的保护中起着关键作用。目前的应用将集中在不同的味觉受体的不同亲和力的促苦味剂激活的途径，以及这些激动剂对胃肠道功能和摄食行为的影响。长期目标是了解调节管腔化学传感的机制，这是一个重要的生理过程，控制GI功能反应，影响进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.