Enteric neural regulation of barrier function in the intestine

单层上皮细胞排列在胃肠道（GI）是外部环境和体内之间的重要屏障；它也是一个重要的光环境传感器。由于这一屏障的关键性质，复杂和复杂的防御措施已经发展到防止或减轻伤害。这种防御的性质反映了肠道承受的负担，它必须保护自己免受消化过程的影响，以及防止摄入食物中的细菌、寄生虫和其他病原体。同时，上皮允许营养物质、电解质、特定抗原和水通过。因此，上皮屏障是选择性可渗透的，并受到严格调控。调节通透性的能力存在于紧密连接水平，紧密连接是连接相邻上皮细胞并控制细胞间物质运动的蛋白质网络。局部宿主防御功能是由肠神经系统神经元协调和调节的。然而，关于肠顶端紧密连接复合体的神经控制的基本机制知之甚少。我们的目标是通过一个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项目来解决我们在胃肠道基本调节机制方面的这一重大空白。我们将检验肠神经支配通过改变紧密连接蛋白的表达和定位来调节上皮屏障功能的一般假设。在本提案中，我们将针对三个具体目标来验证这一假设：1。肠神经是否控制上皮通透性？使用我们实验室常规的技术，我们将在体外研究的小鼠结肠片段中测量经上皮对离子的渗透性和特定渗透性标记物，这些片段将暴露于神经元刺激和阻断对神经递质反应的药物中。在实验结束时，组织将被处理，以确定紧密连接蛋白分布使用免疫组织化学和共聚焦显微镜。2. 在动物和细胞模型中，哪种肠内神经递质调节上皮通透性？人类上皮细胞系在培养中形成正常的紧密连接结构，将暴露于已知的肠道神经递质：乙酰胆碱、血管活性肠多肽、P物质和降钙素基因相关肽。通透性和紧密连接蛋白的定位将在Aim 1中确定。紧密连接蛋白基因表达的变化将使用标准技术来确定。3. 肠内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。如上所述，使用上皮细胞系，我们将采用细胞生物学方法来评估神经递质受体激活与紧密连接蛋白基因表达以及往返紧密连接的运输的信号通路。培养高素质人才。我们的研究项目在培养研究生和博士后方面有着良好的记录，在一个充满活力和刺激的研究环境中。拟议的研究由两名研究生参与，并由经验丰富的研究技术人员提供支持。的意义。通过系统地研究顶端连接复合体的神经调节，这些研究将更好地理解肠屏障生理控制的基本机制。

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.