It has been hypothesized that derangements in the barrier function of the gut predispose critically ill patients to the development of bacteremia, fungemia, and endotoxicosis. The integrity of the epithelial barrier can be assessed by measuring its permeability to certain water-soluble compounds, such as 51Cr-EDTA. The permeability of the gut epithelium to hydrophilic solutes is determined by the functional status of the intercellular tight junctions (zonula occludens; ZO). Recent data suggest that the ZO is anatomically and functionally linked to the cytoskeleton. Other results indicate that the organizational integrity of the cytoskeleton depends upon the maintenance of adequate intracellular levels of ATP. Since both cellular hypoxia and oxidant stress can interfere with ATP synthesis, we have hypothesized that lipopolysaccharide (LPS)-induced mucosal hyperpermeability is caused by ATP depletion in enterocytes. Accordingly, we will investigate ischemia- and oxidant-induced mucosal ATP depletion as mechanisms for the adverse effect of endotoxin on gut epithelial permeability. The proposed studies will utilize a porcine endotoxicosis model that satisfactorily reproduces many of the hemodynamic manifestations of septic shock in resuscitated patients. Mucosal permeability will be quantitated by measuring the plasma-to-lumen clearance of 51Cr-EDTA. Mucosal oxygenation will be measured using a multiwire Clark-type surface electrode array that permits the monitoring of 02 tensions in multiple microscopic hemispheres (radius~20 uM) of tissue. Using these methods, we will assess the time-dependent effects of LPS on mucosal oxygenation, ATP content, and permeability. In addition, we will determine whether LPS-induced mucosal hyperpermeability can be prevented by maintaining normal epithelial oxygenation, either by pump-perfusing the vascular supply of the gut with oxygenated blood or perfusing the lumen of the gut with oxygenated buffer. Also, we will investigate the role of oxidants by assessing the effects of allopurinol (a xanthine oxidase inhibitor) or oxygen free-radical scavengers of mucosal permeability and ATP content in endotoxic animals. Finally, we will assess the affects of mucosal hypoxia or oxidant stress on mucosal ATP content and permeability in normal pigs. These studies should provide new insights into the mechanisms responsible for mucosal hyperpermeability in sepsis and endotoxicosis.
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