MOLECULAR MECHANISMS OF GUT BARRIER DYSFUNCTION

虽然脓毒症和肠出血症中促进肠损伤的机制 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了 产生多能介质一氧化氮（NO-）。尽管一些 在文献中存在线索，NO调节的机制 肠上皮屏障功能在炎症条件下，如 脓毒症或炎症性肠病，知之甚少。因此，委员会认为， 本文提出的研究目标是提高我们对 改变的基本细胞和分子机制 在由NO-和/或其他相关的 活性氮中间体（RNI）。该项目已组织 四个具体目标。目的1：工程化四环素调节的 表达质粒以允许控制转录调节 诱导型一氧化氮合酶（iNOS）基因表达的研究 分化的肠细胞系，Caco-2，以测试 假设过量内源性产生NO-是足够的（甚至 在不存在其它促炎介质的情况下）以增加肠 上皮通透性目的2：使用（i）细胞因子刺激的培养的 肠细胞（Caco-2和T84细胞），（ii）工程化细胞系 （iii）用于监测肠道的体内模型系统， 大鼠的粘膜呼吸;和（iv）来自内毒素血症的粘膜样品 野生型或iNOS "敲除"小鼠，测试上调 肠上皮细胞产生的NO导致细胞ATP 基于线粒体功能障碍和/或激活的消耗 聚腺苷二磷酸核糖聚合酶。目的3：描述的细胞系 根据目标2，将被用来检验假设， 或内源性产生的NO-促进磷酸化， 关键细胞骨架蛋白的去磷酸化和NO介导的 蛋白质酪氨酸磷酸化或去磷酸化的改变 关键细胞骨架蛋白和NO介导蛋白质改变 酪氨酸磷酸化导致细胞骨架完整性的变化， 上皮通透性目的4：检验假设， 供应的或内源性产生的NO-促进单（ADP）-核糖基化;和 确定造成这种现象的NO敏感元素。 拟议的实验将提供强大的新工具（例如，的caco-2 以Tc调节的方式表达iNOS的细胞系）用于研究 NO-对上皮细胞功能的影响。此外，拟议的研究 应该开辟富有成效的调查路线， 机制[例如，细胞骨架蛋白的单（ADP）-核糖基化] 肠上皮细胞通透性的调节 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.