Neural Control of Large Intestinal Mucosa

肠道神经内的神经反射控制适当的消化功能所必需的分泌和运动模式。机械刺激使肠嗜铬细胞释放5-羟色胺(5-HT)，并与反射中粘膜下传入神经元上的5-HT1P/4受体结合，引起上皮氯离子分泌。总体目标是了解施加在结肠上的机械刺激转化为氯化物分泌的机制。将使用三种机械刺激：粘膜抚摸、旋转抖动和细胞变形。第一个目的是使用一种综合的方法来检测豚鼠、人类或核苷酸受体缺陷小鼠肌肉剥离的结肠对机械刺激的反应以及类癌肿瘤细胞系Bon的5-羟色胺释放和氯离子分泌。释放的核苷酸将被识别，嘌呤受体亚型根据激动剂/拮抗剂的效力进行分类，质膜信号站被称为小凹，通过电子显微镜识别。第二个目的是确定在机械刺激过程中，相关的嘌呤受体是否依赖于小窝结构来传递适当的信号。第三个目的是确定GQ家族的G蛋白是否与小窝中的嘌呤受体共定位，以介导机械诱导的Bon细胞中5-羟色胺的释放。在这些和随后的研究中，免疫沉淀和免疫印迹技术将鉴定富含小窝的部分中是否存在信号分子。第四个目的是研究单个Bon细胞机械变形的机制，并确定磷脂酶C和细胞内钙储存在机械诱导的5-羟色胺释放中的作用。第五个目的是通过检测钙储存和磷脂酶C的磷酸化来确定蛋白激酶C调节机械诱导的5-羟色胺释放的机制。这项研究以其从整体系统到单细胞的机制方法，将为正常人类和动物组织中5-羟色胺释放的调节提供新的见解。这将为类癌的基本生理学和5-羟色胺的释放提供新的信息，可能有助于类癌综合征的治疗。了解5-羟色胺的调节可能是治疗与肠嗜铬细胞数量过多或减少相关的功能性肠病的关键。

Neural reflexes within the enteric nervous control secretion and motility patterns necessary for appropriate digestive function. Mechanical stimulation release 5- hydroxytryptamine (5-HT) from enterochromaffin cells and binds to 5-HT1P/4 receptors on submucosal afferent neurons in the reflex to cause epithelial chloride secretion. The overall goal is to understand the mechanisms by which a mechanical stimulus applied to the colon is transduced into chloride secretion. Three mechanical stimuli will be used: mucosal stroking, rotational shaking and cell deformation. The 1st aim uses an integrative approach to examine 5-HT release and chloride secretion in muscle-stripped colon from guinea pig, human or nucleotide- receptor deficient mice n response to mechanical stimulation or in a carcinoid tumor cell line, BON. Nucleotides released will be identified, the purinoreceptor subtype classified by agonist/antagonist potencies and the plasma membrane signaling stations known as caveolae identified by electron microscopy. The 2nd aim is to determine whether the purinoceptor in question is dependent on caveolae structure for appropriate signaling during mechanical stimulation. The 3rd aim is to determine if G proteins of the Gq family co-localize with purinoceptors in caveolae to mediate mechanically-evoked 5-HT release in BON cells. For these and subsequent studies immunoprecipitation and immunoblotting techniques will identify the presence of signaling molecules in fractions enriched in caveolae. The 4th aim examines the mechanism involved in mechanical deformation of single BON cells, and determines the role of phospholipase C and intracellular calcium stores in mechanically-evoked 5-HT release. The 5th aim identifies mechanisms by which protein kinase C in modulates mechanically-evoked 5-HT release by examining calcium stores and phosphorylation of phospholipase C. This study with its mechanistic approach from examining integrated systems to the single cell will provide new insights into the regulation of 5-HT release in normal human and animal tissue. It will provide new information about the basic physiology of carcinoid tumors and 5- HT release that may benefit the management of carcinoid syndrome. Understanding the regulation of 5-HT may be a key to management of functional bowel disease associated with an excess or reduced number of enterochromaffin cells.