These experiments are designed to define in detail the structure of small intestinal and T-84 intestinal epithelial cell line permeability barriers, with emphasis on tight junctions, in normal and perturbed model states and to relate variations in structure with permeability. To accomplish these goals, a variety of electron microscopic, freeze fracture, ionic and macromolecular tracer, and electrophysiological experiments will be conducted in parallel. First, morphometric quantitation of parameters of junctional structure along the crypt villus axis of the jejunum will be performed and, using known structure function relationships and circuit analysis, a hypothesis describing the partitioning of passive ion permeation along this axis and contrasting it with ileal passive ion permeation will be formulated. We will also apply biophysical, detergent extraction, ultrastructural, and newer freeze fracture techniques to normal intestinal epithelium to better characterize the structural and physical states of junctional """"""""pores"""""""". Similar analysis will be performed on T-84 monolayers in the steady state and during junctional development. Fourthly, T-84 monolayers will be utilized to examine the effects of cell cycle on junctional permeability. Such monolayers will also be utilized to examine the importance of cell surface proteins in the structural and functional expression of junctions. As a sixth goal, we will examine in detail the effect of cytochalasin D on ileal and T-84 junctional permeability since this agent appears to exert its effect by stimulating contraction of the apical actinmyosin ring and thus may represent a model for studying the relationships between cytoskeletal contraction and permeability. We will also study a flounder intestinal model which affords us the opportunity to examine junctional structure and function in the absence of cytoskeletal influence. As an eighth goal, we will examine in further detail the structural and functional alterations in junctions induced by a physiologically relevant perturbation--short term mucosal osmotic stress. Our last two efforts will be geared toward analysis of junctional structure and function in model states related to disease. These studies will examine villus epithelial restitution after injury, and the effects on epithelial cells of Clostridium difficile toxins. These experiments designed to correlate epithelial and specifically tight junction structure in normal and abnormal states with mucosal permeability may yield insights into small intestinal epithelial function.
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