This proposal describes combined physiological and ultrastructural studies of the toad urinary bladder designed to gain insight into the mechanism of action of the hormone vasopressin. Considerable evidence now indicates that the incidence of intramembrane particle aggregates visualized by freeze-fracture electron microscopy are closely correlated with the water permeability response and that a membrane shuttle system plays a role in this response. However, direct evidence that the aggregates are the site of water channels is lacking and important features of the proposed shuttle system remain uncertain. In order to clarify the role of aggregates and microfilaments in the action of vasopressin, the incidence of intramembrane particle aggregates and the water permeability response to vasopressin will be examined in cytochalasin B treated bladders. Ultrastructural localization of actin, calmodulin, clathrin and fodrin with specific antibodies will be carried out to determine which cytophasmic elements are associated with aggregate containing membrane. Antibodies will be developed to apical membrane isolated from the bladder and screened for antibodies which bind to the apical surface only following vasopressin stimulation (AVP+). The correlation between AVP+ antibody binding and vasopressin-induced physiological changes will be used to assess whether antibodies bind to components specifically associated with the change in water permeability. Immunofluorescence and electron microscopic localization of antibody will be used to assess binding of antibody to the intramembrane particle aggregates. Antibodies will also be screened for specific inhibition of vasopressin-induced changes in water permeability and sodium transport. Localization of antibodies by thin section and fracture-label electron microscopy will be used to determine the source of the aggregates involved in the vasopressin response. Electron microscope localization studies will also be used to determine the fate of vasopressin-induced apical membrane structures during withdrawal of vasopressin and to evaluate the extent to which they recycle in subsequent responses.
