MOLECULAR MECHANISMS OF GUT BARRIER DYSFUNCTION

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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.