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The luminal (apical) border of the epithelium of the bladder in the well-hydrated toad is relatively impermeable, so the bladder usually stores hyposmotic urine. When antidiuretic hormone (ADH) increases apical membrane osmotic permeability dramatically, water is resorbed from hyposmotic mucosal solution; in the presence of hyposmotic or isosmotic mucosal solutions, ADH concomitantly induces exocytosis at the apical border of granule-rich (G) cells. Then ADH induces endocytosis at this border. We describe how an osmotic gradient affects ADH-induced endocytosis and hydroosmosis in vitro. We can assess ADH-induced endocytosis in gradient and no-gradient bladders by applying a double-marker technique that distinguishes among endocytosis, completed internalization of previously surface-attached membrane, and surface invagination by comparing the number of horseradish peroxidase (HRP) uptake bodies (endocytosis) with the number of ruthenium red (RR)-delineated bodies (surface invaginations). With this approach we find that gradient bladders have approximately six times more ADH-induced endocytosis than no-gradient bladders during 45-60 min of ADH stimulation. Furthermore, at 60 min approximately 50% of the HRP-containing structures in no-gradient bladders remain surface connected compared with approximately 1% in gradient bladders. In parallel physiological studies, no-gradient bladders reach and maintain higher induced osmotic permeabilities than gradient bladders. These findings support the hypothesis that endocytosis plays an active role in reestablishing impermeable apical surface characteristics in toad bladder.

The electron-dense granules that lie just below the apical plasma membrane of granular epithelial cells of toad urinary bladder contribute glycoproteins to that apical membrane. Also, exocytosis of granules (and tubules) elicited by antidiuretic hormone potentially doubles that apical surface, during the same period the transport changes characteristic of the hormonal response occur. Granules separated from other membrane systems of the cells provide the material to assess the importance of the granules as glycocalyx precursors and in hormone action. We used isosmotic media to effect preliminary separations by differential centrifugation. Then granules were isolated by centrifugation on self-forming gradients of Percoll of decreasing hypertonicity. We find qualitative and quantitative changes in protein composition and enzymic activities in the isolated fractions. The primary criterion for granule purification was electron microscopic morphology. In addition, polypeptide species found in the granule fraction are limited in number and quantity. The granules are enzymically and morphologically not lysosomal in nature. Granules may provide the glycoproteins of the apical glycocalyx but they differ from the isolated plasma membrane fraction enzymically, in protein composition and in proportion of esterified cholesterol. We conclude that the granules are not "average" plasma membrane precursors. Their role in the membrane properties of the toad urinary bladder may now be evaluated by characterizing permeability and other properties of the isolated organelles.

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