Vasopressin-induced transfer via light vesicles of receptors and water channels from basolateral to apical membrane of toad bladder

Toad bladder epithelial cells were homogenized and fractionated by Percoll density-gradient centrifugation. Binding of tritium-labeled vasopressin ([³H]AVP) was measured in surface membranes (SM), microsomes (M), and a 100,000 g (60-min) micrsomal supernatant fraction (S). More than two-thirds of the total receptors were in S. Receptors in SM - but not in S - were tightly coupled to G-protein as suggested by inhibition of [³H]AVP binding by GTP. GTP-sensitivity of [³H]AVP binding was not altered by vasotocin (AVT) stimulation, although the distribution of receptors shifted from SM to S. Intact bladders, exposed on the serosal side to 1-desamino, 7-lysine-(4-azidobenzoyl), 8-arginine vasotocin (d7-N3-AVT) in the presence of UV light, exhibited a persistent hydroosmotic response compared to controls stimulated with photoaffinity analog in the dark. Cell fractions from the irradiated bladders showed a reduction in [³H]AVP binding in SM and S. Intact bladders, exposed on the mucosal side to d7-N3-AVT in the presence of UV light (while stimulated from the serosal side with AVP) exhibited a decrease in [³H]AVP binding in SM compared to controls without d7-N3-AVT. These findings suggest the following working hypothesis for the hydroosmotic action of vasopressin: (1) Vasopressin binds to receptors at the basolateral membrane, which is water-permeable in the unstimulated state; (2) The hormone-receptor complex is endocytosed and, together with other components of the basolateral membrane containing "water channels", appears in "light vesicles"; (3) These light vesicles (and/or similar vesicles in a cytoplasmic reservoir) move to the apical membrane where they fuse with the water-impermeable apical membrane and thereby render the rate-limiting barrier permeable to water.