Vesicular traffic in the ADH-sensitive toad urinary bladder

Abstract

Water channels and fluid-phase markers are retrieved from the apical membrane of the toad bladder upon removal of antidiuretic hormone (ADH). These cells maintain high ADH responses even after repeated stimulation, suggesting a high recycling rate of vesicles carrying water channel proteins. Alternatively, there could be a large population of vesicles with only a small fraction responding. The number of retrieved labeled vesicles with single or multiple markers were counted by custom image analysis programs that extracted novel information for statistical analysis. The programs included median and brightness thresholds and an algorithm that counted vesicles by color and size. Recycling was evaluated by loading the retrieved vesicles with Texas Red dextran or biotin/avidin-fluorescein and evaluating the loss of these vesicles due to ADH restimulation. Tissue was stimulated with ADH and then surface proteins were covalently labeled with NHS-SS-Biotin followed by avidin-fluorescein. The tissue was then exposed to Texas Red dextran for 10 min during washout of ADH. ADH restimulation caused a significant reduction in vesicle number with a maximal drop of 22%. Biotin-labeled vesicles were also significantly reduced by 25% after ADH restimulation but did not show preferential sorting and recycling back to the membrane in comparison to vesicles with fluid-phase marker. To study marker retrieved from both the apical and basolateral membranes, samples were exposed to FITC dextran in the serosal bath for 60 min, followed by addition of ADH and Texas Red dextran to the mucosal bath. Cells were examined for colocalization of both markers at various time points following ADH washout. No significant colocalization was seen at the 0 time point but colocalization increased by 60 and 105 mins. After ADH restimulation, vesicles with marker only from the basolateral or apical bath were reduced but vesicles with both markers remained unchanged. Additionally, the basolateral marker uptake was stimulated by a transepithelial osmotic gradient compared to ADH stimulation alone. This work provides evidence that in native ADH-responsive tissue the majority of vesicles labeled from the apical membrane do not respond to ADH. Additionally, apical marker can enter ADH-insensitive vesicles with marker retrieved from the basolateral membrane.