Evaluation of granule exocytosis in toad urinary bladder

Addition of phorbol myristate acetate (PMA) to the mucosal bathing solution induces exocytosis of the granules found in the apical cytoplasm of toad urinary bladder epithelial cells. Because mucosal application of PMA also increases epithelial water permeability in the complete absence of vasopressin, these observations have suggested that granules might have a role in mediating the permeability response to vasopressin. From electron microscopic immunolocalization studies we have identified a 70-kDa protein as a component of the apical surface glycocalyx that is stored in the granules. Using antibodies to this protein in a competitive immunoassay, we found that addition of PMA to the mucosal side results in more than a fourfold increase in antigen release into the mucosal bath. The amount of antigen detectable on the mucosal surface of the cells was doubled by mucosal PMA exposure. Serosal application of PMA or addition of the inactive analogue phorbol didecanoate to either surface failed to produce significant antigen release. These results are consistent with electron microscopic studies showing exocytosis of granules only with mucosal exposure to PMA. However, immunoassay of granule antigen after vasopressin stimulation of water permeability failed to show a detectable change in granule exocytosis. We conclude that granule exocytosis does not play an important role in mediating vasopressin-induced changes in permeability.