Enteric neural regulation of barrier function in the intestine

肠屏障功能的肠神经调节

• 批准号: RGPGP-2014-00074
• 负责人: MacNaughton, Wallace
• 金额: $ 1.89万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Group
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=565946
• 关键词: Enteric; neural; regulation; barrier; function

The single layer of epithelial cells that line the gastrointestinal (GI) tract is an essential barrier between the external environment and the inside of the body; it is also an important sensor of the luminal environment. Because of the critical nature of this barrier, complex and sophisticated defenses have evolved to prevent or mitigate injury. The nature of the defenses reflects the burden placed on the gut, which must protect itself from the processes of digestion, as well as from the ingestion of bacteria, parasites and other agents found in food. At the same time, the epithelium allows the passage of nutrients, electrolytes, specific antigens and water. Hence, the epithelial barrier is selectively permeable and tightly regulated. The ability to regulate permeability resides at the level of the tight junction, a network of proteins that joins adjacent epithelial cells and controls movement of material between the cells. Local host defense functions are coordinated and regulated by neurons of the enteric nervous system. However, little is known about the fundamental mechanisms that underlie neural control of the apical tight junctional complex of the intestine. We aim to address this significant gap in our knowledge of fundamental regulatory mechanisms of the GI tract through a collaborative research program based on our extensive experience with the study of the neural regulation of intestinal function. We will test the general hypothesis that the enteric innervation regulates epithelial barrier function by altering the expression and localization of tight junction proteins. In this proposal, we will address three specific aims to test this hypothesis: 1. Do enteric nerves control epithelial permeability? Using techniques routine to our labs, we will measure transepithelial permeability to ions and specific permeability markers in segments of mouse colon studied in vitro, which will be exposed to neuronal stimulation and to drugs that block the responses to neurotransmitters. At the end of the experiments, tissues will be processed for determination of tight junction protein distribution using immunohistochemistry and confocal microscopy. 2. Which enteric neurotransmitters regulate epithelial permeability in animal and cell models? Human epithelial cell lines that develop normal tight junction structures in culture, will be exposed to known enteric neurotransmitters: acetylcholine, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide. Permeability and tight junction protein localization will be determined as in Aim 1. Changes in tight junction protein gene expression will be determined using standard techniques. 3. What are the cellular mechanisms whereby enteric neurotransmitters regulate epithelial permeability? We will determine the intracellular biochemical pathways that mediate the effects of neurotransmitters on tight junction protein expression and trafficking. Using epithelial cell lines as above, we will employ cell biology approaches to assess the signaling pathways that couple neurotransmitter receptor activation with tight junction protein gene expression and trafficking to or from the tight junction. Training of high qualified personnel. Our research program has an excellent track record for training graduate students and post-doctoral fellows in a vibrant and stimulating research environment. The proposed studies have been designed to engage two graduate students, supported by experienced research technicians. Significance. By systematically studying the neural regulation of the apical junctional complex, these studies will provide a better understanding of the fundamental mechanisms that underlie the physiological control of the intestinal barrier.

排列在胃肠道（GI）的单层上皮细胞是外部环境和身体内部之间的重要屏障;它也是管腔环境的重要传感器。由于这一屏障的关键性质，复杂和精密的防御已经发展到防止或减轻伤害。防御的性质反映了肠道的负担，肠道必须保护自己免受消化过程以及食物中发现的细菌，寄生虫和其他媒介的摄入。同时，上皮允许营养物质、电解质、特异性抗原和水通过。因此，上皮屏障是选择性渗透和严格调节的。调节渗透性的能力存在于紧密连接的水平，紧密连接是连接相邻上皮细胞并控制细胞之间物质运动的蛋白质网络。局部宿主防御功能由肠神经系统的神经元协调和调节。然而，很少有人知道的基本机制，神经控制的顶端紧密连接复合体的肠。我们的目标是通过一项基于我们在肠道功能神经调节研究方面的丰富经验的合作研究计划，来解决我们对胃肠道基本调节机制的认识中的这一重大差距。我们将测试的一般假设，肠神经支配调节上皮细胞的屏障功能，通过改变紧密连接蛋白的表达和定位。在本提案中，我们将提出三个具体目标来检验这一假设：1。肠神经控制上皮细胞通透性吗？使用我们实验室的常规技术，我们将测量体外研究的小鼠结肠段中离子和特定渗透性标记物的跨上皮渗透性，这些结肠段将暴露于神经元刺激和阻断神经递质反应的药物。在实验结束时，将处理组织以使用免疫组织化学和共聚焦显微镜测定紧密连接蛋白分布。2.哪些肠道神经递质调节动物和细胞模型中的上皮通透性？在培养物中形成正常紧密连接结构的人上皮细胞系将暴露于已知的肠神经递质：乙酰胆碱、血管活性肠多肽、P物质和降钙素基因相关肽。渗透性和紧密连接蛋白定位将如目的1所述进行测定。将使用标准技术测定紧密连接蛋白基因表达的变化。3.肠神经递质调节上皮通透性的细胞机制是什么？我们将确定介导神经递质对紧密连接蛋白表达和运输的影响的细胞内生化途径。使用如上所述的上皮细胞系，我们将采用细胞生物学方法来评估将神经递质受体活化与紧密连接蛋白基因表达和运输到紧密连接或从紧密连接运输耦合的信号传导途径。培养高素质人才。我们的研究计划在充满活力和刺激的研究环境中培养研究生和博士后研究员方面有着良好的记录。拟议的研究旨在吸引两名研究生，由经验丰富的研究技术人员提供支持。意义通过系统地研究顶端连接复合体的神经调节，这些研究将提供一个更好的理解的基本机制，肠道屏障的生理控制的基础。