The rate-limiting step in hydrosmotic response of frog urinary bladder.

The ADH-induced water fluxes and the associated appearance of intramembranous particle aggregates in the luminal membrane of frog urinary bladders have been correlated in a time course study. Plots of the onset and reversal of the oxytocin-induced hydrosmotic response were sigmoidal in shape, symmetrical and slowed by low temperature to the same degree. Parallel freeze-fracture studies showed that the mean size distribution of the aggregates was constant at different temperatures and at different times during hormonal stimulation and washout. No qualitatively different picture of aggregate formation was detected at low temperature: this suggests that the insertion and removal of individual aggregates into or from the apical plasma membrane is a rather rapid process, both at 20 and at 6.5 degrees C. As in the case of water permeability, both aggregate appearance and disappearance were similarly slowed by lowering the temperature. A similar time-course study of the inhibition of the hydrosmotic response by acidification of the medium was also made. In this case, lowering the incubation temperature induced a clear dissociation between net water flow and the surface area occupied by the aggregates. For the first time, a low water permeability was found associated with a high aggregate surface area in the apical membrane, indicating that cellular acidification induces an impairment of aggregate function rather than a reduction of surface area.