A variety of indirect data exist which, in aggregate, suggest that exocytosis can alter tight junction structure and/or permeability. These experiments are designed to assess the feasibility of utilizing a homogenous epithelia monolayer derived from a human intestinal cell line to study the effects of exocytosis on tight junction structure and permeability. To accomplish this goal we will utilize the 18N2 clone of the HT29 cell line which was derived from a human colonic carcinoma. HT 29-18N2 cells differentiate as goblet cells, grow as monolayers on coverslips, and respond to cholinergic stimulation with exocytosis of mucin granule content. We will first attempt to identify conditions under which HT2918N2 cells can be grown on permeable supports as confluent monolayers from which electrophysical and flux data can be obtained. Using physiological techniques we will then ascertain if stimulated exocytosis perturbs tight junction barrier function. Using electron microscopy and freeze fracture techniques we will define the effects which exocytosis exert on tight junction structure. Lastly we will define and inter-relate physiological and structural events which occur as monolayers recover from stimulated exocytosis. These studies may yield insights into how local events such as exocytosis influence tight junction barrier function. These studies may also define methods whereby electrophysiological studies can be performed on homogeneous, flat, easily manipulated monolayers composed entirely of one of the major subtypes of cells in the intestinal epithelium - the goblet cell.
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