MOLECULAR MECHANISMS OF GUT BARRIER DYSFUNCTION

尽管脓毒症和肠易激综合征促进肠道损伤的机制 内毒素血症仍有待阐明，我们小组获得了大量数据 以及其他人认为，一个重要的因素可能已经结束- 产生多能介质，一氧化氮(NO-)。尽管有些人 线索存在于文献中，即NO调节的机制 肠道上皮屏障在炎症条件下的功能，如 败血症或炎症性肠病，人们对此知之甚少。因此， 本文提出的研究的目的是提高我们对 改变背后的基本细胞和分子机制 NO和/或其他相关物质对肠上皮通透性的影响 活性氮中间体(RNI)。这个项目已经组织好了。 在四个具体目标下。目标1：设计一种受四环素调控的 允许受控转录调控的表达载体 诱导型一氧化氮合酶(INOS)基因的表达 分化的肠上皮细胞系Caco-2，以检测 假设内源性产生过量的NO-就足够了(甚至 在没有其他促炎介质的情况下)增加肠道 上皮通透性。目的2：使用(I)细胞因子刺激的培养 肠上皮细胞(Caco-2和T84细胞)，(II)工程化细胞系 在目标1下描述的(3)活体监测肠道的模型系统 大鼠的粘膜呼吸；以及(Iv)内毒素血症的粘膜样本 野生型或诱导型一氧化氮合酶“敲除”小鼠，测试上调表达的假设 肠上皮中NO的产生导致细胞内的ATP 以线粒体功能障碍和/或激活为基础的衰竭 这种酶，聚(ADP)-核糖聚合酶。目标3：所描述的细胞系 在目标2下，将用来检验外源供应的假设 或内源性产生的NO-促进磷酸化或 关键细胞骨架蛋白的去磷酸化及其介导的NO 蛋白酪氨酸磷酸化或去磷酸化的改变 关键的细胞骨架蛋白和NO介导的蛋白质变化 酪氨酸磷酸化导致细胞骨架完整性和 上皮通透性。目标4：检验外生性假设 供应或内源性产生的NO促进单核苷酸(ADP)核糖化；以及 找出造成这一现象的非敏感因素。 拟议的实验将提供强大的新工具(例如，Caco-2 以TC调节方式表达iNOS的细胞株)用于研究 NO-对上皮细胞功能的影响此外，拟议的研究 应该开辟关于根本问题的富有成效的调查路线 机制[例如，细胞骨架蛋白的单(ADP)-核糖化] 肠黏膜上皮通透性的调节机制 生理和病理生理条件。

Although the mechanisms promoting intestinal injury in sepsis and endotoxemia remain to be elucidated, extensive data obtained by our group as well as others suggest that one important factor is probably over- production of the pluripotent mediator, nitric oxide (NO-). Although some clues exist in the literature, the mechanisms whereby NO-modulates intestinal epithelial barrier function in inflammatory conditions, such as sepsis or inflammatory bowel disease, are poorly understood. Accordingly, the goal of the studies proposed herein is to improve our understanding of the fundamental cellular and molecular mechanisms underlying alterations in intestinal epithelial permeability induced by NO- and/or other related reactive nitrogen intermediates (RNIs). The project has been organized under four Specific Aims. Aim 1: Engineer a tetracycline-regulated expression plasmid to permit controlled transcriptional regulation of inducible nitric oxide synthase (iNOS) gene expression in a well- differentiated enterocytic cell-line, Caco-2, in order to test the hypothesis that excessive endogenous generation of NO- is sufficient (even in the absence of other pro-inflammatory mediators) to increase intestinal epithelial permeability. Aim 2: Using (i) Cytokine-stimulated cultured enterocytes (Caco-2 and T84 cells), (ii) the engineered cell line described under Aim 1, (iii) an in vivo a model system for monitoring gut mucosal respiration in rats; and (iv) mucosal samples from endotoxemic wild-type or iNOS "knock-out" mice, test the hypothesis that up-regulation of NO production in the intestinal epithelium leads to cellular ATP depletion on the basis of mitochondrial dysfunction and/or activation of the enzyme, poly(ADP)-ribose polymerase. Aim 3: The cells lines described under Aim 2 will be used to test the hypothesis that exogenously supplied or endogenously produced NO- promotes the phosphorylation or dephosphorylation of key cytoskeletal proteins and that NO-mediated alterations in protein tyrosine phosphorylation or dephosphorylation of key cytoskeletal proteins and that NO-mediated alterations in protein tyrosine phosphorylation lead to changes in cytoskeletal integrity and epithelial permeability. Aim 4: Test the hypothesis that exogenously supplied or endogenously produced NO-promotes mono(ADP)-ribosylation; and identify the NO-sensitive elements responsible for this phenomenon. The proposed experiments will provide powerful new tools (e.g., the Caco-2 cell line expressing iNOS in a Tc-regulated fashion) for studying the effects of NO- on epithelial function. In addition, the proposed studies should open up fruitful lines of investigation regarding the fundamental mechanisms [e.g., mono(ADP)-ribosylation of cytoskeletal proteins] underlying the regulation of intestinal epithelial permeability under physiologic and pathophysiologic conditions.