Previous studies have assigned distinct and rate-limiting functions to the apical and basal-lateral plasma membrane surfaces in the regulation of transepithelial transport of NaCl and water. The apical surface has been identified as a saturable site of entry of NaCl into the intracellular compartment and the basal-lateral surface as the site of the Na ion pump, responsible for active transport of salt from the cell interior into the subepithelial space. The proposed project is designed to provide information on the molecular determinants of the properties of these separate surfaces and of the mechanisms involved in hormonal regulation of trans-apical and trans-basal transport of NaCl. The urinary bladder of the toad (Bufo marinus) will be used as a model system for tight epithelia (e.g., equivalent to the distal segment of the nephron). To identify the separate surfaces in sub-fractions of the cellular organelles, the apical and basal-lateral plasma membranes will be covalently labeled with 125I, 131I, 3H, 35S, or 32P. In previous studies, we presented evidence that the apical plasma membrane can be labeled selectively with 125I or 131I by an enzymatic technique or with diazodiodosulfanilic acid (DIDISA) and the basal-lateral membranes with pyridoxal-PO4 and B3H4. 35S may be incorporated by preparing 35S-labeled DIDISA and 32P by preparing 32P-labeled pyridoxal PO4. Experiments now underway indicate that effective separation of the labeled surfaces is achieved by continuous sucrose density gradient centrifugation. Further explorations by continuous sucrose density gradient centrifugation. Further explorations of separation methods are planned, indcluding affinity techniques. The separated surfaces are being analyzed in terms of their chemical constituents and enzymatic profiles. The effects of hormones will be analyzed by studies on membrane enzymes and isotope incorporation.