The proposed research will be an investigation of the role of cytoplasmic vesicles and their influence on ion transport regulation across ion transporting epithelia. Conventional, ion specific microelectrodes, capacitance measurements, fluctuation analysis and """"""""patch"""""""" clamp methods will be used to measure apical and basolateral membrane potentials, conductances, areas, area changes and single ionic channel properties as well as intracellular ion activities as a function of vesicle fusion effects and pharmacological manipulations to alter vesicle translocation. Measurement of the effect of microfilament and microtubule blocking agents and volume perturbation methods on membrane potentials, resistances and ion activities will be determined to access possible regulation of vesicle translocation. Combined use of cytochalasin B (a microfilament disrupting agent) and colchicin (a microtubule blocking agent) in conjunction with capacitance measurements will allow a determination of these two cytoskeletal structures in vesicle translocation into the apical membrane. Dietary manipulations will be used to determine whether cytoplasmic vesicle density is under hormonal control. Vesicle density is being measured using a newly developed electrical method. Possible vesicle fusion into the basolateral membrane will be measured using antibiotics to reduce apical resistance and volume and stretch regimes to stimulate fusion into the basolateral membrane. An alternate epithelial preparation (the ureters) will be developed to study vesicle translocation. Although the mammalian bladder epithelium translocates vesicles in response to mechanical stimuli other epithelia respond through hormonal interaction. Measurement of toad urinary bladder vesicle translocation and comparison with mammalian bladder will allow a modeling of active and passive vesicle translocation in ion regulation. The results will contribute to the understanding of cytoplasmic vesicles in the regulation of ion transport across epithelia.
