Effect of Osmotic Gradient on ADH-induced Intramembranous Particle Aggregates in Toad Bladder

Paired toad urinary bladders were prepared without or with an osmotic gradient (175 mosM) across them, stimulated for 2.5 (n = 6), 5 (n = 6), 30 (n = 6) or 60 (n = 6) min with ADH (20 mU/ml), and studied by freeze-fracture electron microscopy. Water permeability at these times was assessed in additional bladders (n = 6 for each case) after tissue fixation according to the technique of Eggena. After both 60 and 30 min of ADH stimulation, the presence of a gradient compared with the absence of one was associated with fewer aggregates (242 +/- 35 vs. 382 +/- 14 x 235 micron-2 at 60 min, P less than 0.01; 279 +/- 36 vs. 470 +/- 51 x 235 micron-2 at 30 min, P less than 0.01) and lower water permeability (8.4 +/- 1.1 vs. 18.8 +/- 1.8 microgram x min-1 x cm-1 x mosM-1 at 60 min, P less than 0.005; 9.2 +/- 1.0 vs. 22.0 +/- 2.1 microgram x min-1 x cm-2 x mosM-1 at 30 min, P less than 0.001). In addition, with a gradient both maximum water permeability and maximum aggregate frequency were reached nearly together; a similar correspondence occurred without a gradient. We conclude that in the presence of an osmotic gradient both the ADH-associated aggregates and the water permeability response to ADH are prevented from reaching the higher levels observed in bladders not exposed to a gradient.