The scientific goal of this proposal is to elucidate the role of endocytosis and transepithelial transport in the Cholera toxin (CT) induced activation of adenylate cyclase in intestinal epithelial cells. CT binds to ganglioside GM1 in the intestinal apical membrane and the A1 subunit eventually gains access to adenylate cyclase on the cytoplasmic face of the basolateral membrane. Available evidence from studies on non-polarized cells indicates that CT is internalized by receptor mediated endocytosis, and that the A1 subunit may not diffuse freely in the cytoplasm but very likely remains membrane associated and ribosylates Gs in a lipid environment. By applying biochemical, biophysical, immunoelectroscopic, and other methods to cultured epithelial monolayers and intestinal segments in vivo an in vitro, the following hypotheses will be tested: A. The Cholera toxin A/B complex assures its own delivery to the basolateral membrane of intestinal cells by opportunistically binding to vesicular endocytic and transepithelial transport pathways. B. Interaction of CT/ganglioside complexes with a subclass of endogenous membrane proteins play a role in cellular processes--presumably, internalization, endocytosis and/or transport--which are necessary for toxin activity. Dr. Lencer is a fully trained gastroenterologist on the faculty of the Combined Program in Pediatric G.I. and Nutrition at The Children's Hospital, MGH, and Harvard Medical School. He has three years of laboratory experience during which he developed an interest in the field of directed membrane traffic within polarized epithelia. He has two original publications: 1) on the differential effect of development on the binding of CT to intestinal cell surfaces (a study which led him to put forth the hypotheses in this grant request) and 2) on the membrane traffic responsible for the action of vasopressin on renal collecting duct epithelia. He is currently extending the use of fluorescein labeled dextran to further characterize the vesicular cycling of the ADH- sensitive water channel and has completed a collaboration with Dr. Neutra on the direct effects of CT on intestinal goblet cells (manuscript prepared). The research in Dr. Neutra's laboratory focuses in part on intracellular membrane systems involved in uptake, sorting, and degradation or transepithelial transport of luminal macromolecules and in part on the development and maintenance of epithelial cell polarity. Most of the experimental approaches and techniques used in these studies are directly applicable to Dr. Lencer's project. Dr. Neutra has proven skills as an educator as well as a scientist. Drs. Walker and Ausiello will act as co-sponsors and will offer expertise and training in biochemical, biophysical, and immunochemical techniques not available in Dr. Neutra's laboratory. In addition, the setting of these laboratories makes the resources at Harvard Medical School, its Digestive Diseases Center and Center for the Study of Kidney Diseases, and the Boston biomedical community available to Dr. Lencer for academic studies.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Clinical Investigator Award (CIA) (K08)
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Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
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Children's Hospital Boston
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Madara, J L; Parkos, C; Colgan, S et al. (1992) Cl- secretion in a model intestinal epithelium induced by a neutrophil-derived secretagogue. J Clin Invest 89:1938-44