The binding of antidiuretic hormone (ADH) to the basolateral (blood-facing) membrane of selected cells in tight epithelia (mammalian collecting duct, anuran skin and bladder) increases water, sodium and urea transport across the apical (exterior facing) membrane of these cells. A large body of evidence strongly suggests that the ADH-induced increase in apical membrane water permeability is due to insertion of membrane particle aggregates into the apical membrane as visualized by freeze-fracture electron microscopy. However, neither the composition nor putative water channel characteristics of membrane particle aggregates are presently known. In the toad urinary bladder, ADH stimulation causes the fusion of cytoplasmic vesicles called aggrephores which deliver particle aggregates into the apical membrane of granular cells. A large transepithelial osmotic gradient, either during continuous hormone stimulation or following removal of ADH, causes the endocytosis of portions of apical membrane which are enriched for membrane particle aggregates. I have used the specific nature of apical membrane retrieval to undertake studies of the ADH-regulated water channel. Using a combination of intracellular and apical membrane iodination techniques, I have identified several proteins which appear on the apical surface of toad bladder only after ADH stimulation and are present in vesicles shown to be enriched for membrane particle aggregates. Fractionation of toad bladder epithelial cell homogenates yielded a purified vesicle fraction enriched for the same proteins identified by iodination studies. Recently, a panel of monoclonal antibodies has been created which are directed at components of this vesicle fraction. The proposed study is divided into 4 parts. First, each of these monoclonal antibodies will be characterized by a combination of biochemical fractionation and immunocytochemistry. The existence of similar proteins in the mammalian collecting duct and anuran skin will be probed using immunolocalization and Western blot analysis. Second, the size, composition and topology of these apical membrane vesicles will be studied. Third, I will measure the water and proton permeabilities of these apical membrane vesicles and study the effects of various antibodies and perturbants to identify components of the water channel. Fourth, using monoclonal FAB fragments, I will study the role of selected proteins in the ADH-induced water permeability response.
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