ADH-induced recycling of fluid-phase marker from endosomes to the mucosal surface in toad bladder

In the toad urinary bladder, the reversal of antidiuretic hormone (ADH) stimulation results in the endocytosis of apical membrane water channels, along with any fluid-phase marker present in the mucosal bathing solution. We have loaded vesicles with horseradish peroxidase (HRP), then restimulated the bladders and measured the reappearance of endocytosed HRP in the mucosal bath. HRP-loaded bladders that were restimulated showed HRP release that peaked sharply within 15 min after restimulation. Varying the interval between loading and restimulation did not vary HRP release significantly. Restimulation with forskolin gave HRP release values similar to ADH. The amount of HRP released correlated with the magnitude of water permeability induced. The demonstration that fluid-phase markers can be recycled from endosomes to the apical surface in a hormone-dependent fashion indicates that endocytosed membrane, containing water channels, is able to rapidly recycle back to the surface in response to hormone restimulation. In addition, marker release declined progressively with repeated restimulation, totaling < 30% of the retrieved amount. This result indicates that a relatively large proportion of the retrieved marker reaches a nonrecycling compartment.