Enteric neural regulation of barrier function in the intestine

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The single layer of epithelial cells that line the gastrointestinal (GI) tract is an essential barrier between the external environment and the inside of the body; it is also an important sensor of the luminal environment. Because of the critical nature of this barrier, complex and sophisticated defenses have evolved to prevent or mitigate injury. The nature of the defenses reflects the burden placed on the gut, which must protect itself from the processes of digestion, as well as from the ingestion of bacteria, parasites and other agents found in food. At the same time, the epithelium allows the passage of nutrients, electrolytes, specific antigens and water. Hence, the epithelial barrier is selectively permeable and tightly regulated. The ability to regulate permeability resides at the level of the tight junction, a network of proteins that joins adjacent epithelial cells and controls movement of material between the cells. Local host defense functions are coordinated and regulated by neurons of the enteric nervous system. However, little is known about the fundamental mechanisms that underlie neural control of the apical tight junctional complex of the intestine. We aim to address this significant gap in our knowledge of fundamental regulatory mechanisms of the GI tract through a collaborative research program based on our extensive experience with the study of the neural regulation of intestinal function. We will test the general hypothesis that the enteric innervation regulates epithelial barrier function by altering the expression and localization of tight junction proteins. In this proposal, we will address three specific aims to test this hypothesis: 1. Do enteric nerves control epithelial permeability? Using techniques routine to our labs, we will measure transepithelial permeability to ions and specific permeability markers in segments of mouse colon studied in vitro, which will be exposed to neuronal stimulation and to drugs that block the responses to neurotransmitters. At the end of the experiments, tissues will be processed for determination of tight junction protein distribution using immunohistochemistry and confocal microscopy. 2. Which enteric neurotransmitters regulate epithelial permeability in animal and cell models? Human epithelial cell lines that develop normal tight junction structures in culture, will be exposed to known enteric neurotransmitters: acetylcholine, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide. Permeability and tight junction protein localization will be determined as in Aim 1. Changes in tight junction protein gene expression will be determined using standard techniques. 3. What are the cellular mechanisms whereby enteric neurotransmitters regulate epithelial permeability? We will determine the intracellular biochemical pathways that mediate the effects of neurotransmitters on tight junction protein expression and trafficking. Using epithelial cell lines as above, we will employ cell biology approaches to assess the signaling pathways that couple neurotransmitter receptor activation with tight junction protein gene expression and trafficking to or from the tight junction. Training of high qualified personnel. Our research program has an excellent track record for training graduate students and post-doctoral fellows in a vibrant and stimulating research environment. The proposed studies have been designed to engage two graduate students, supported by experienced research technicians. Significance. By systematically studying the neural regulation of the apical junctional complex, these studies will provide a better understanding of the fundamental mechanisms that underlie the physiological control of the intestinal barrier.