Selective fixation with glutaraldehyde of ADH-induced urea permeability sites in toad bladder

Toad bladders exposed to vasopressin (ADH) and then fixed on the mucosal surface with 1% glutaraldehyde were highly permeable to water and to urea compared to control bladders fixed in the absence of hormone. When identical conditions of fixation were were used, but the concentration of glutaraldehyde was decreased to 0.25%, the ADH-induced increase in membrane permeability to urea was preserved whereas water permeability was not. About 74% of the hormone-induced urea permeability sites were preserved by glutaraldehyde and were stable to changes in temperature as suggested by a constant value for the activation energy of urea movement of 5.4 kcal/mole (4-33 degrees C). In other studies bladders were exposed at low temperatures to 0.17% glutaraldehyde applied either to the serosal or the mucosal surface. The ADH-induced increase in membrane permeability to urea, bulk water, and tritiated water was well preserved with serosal fixation, but not with mucosal fixation. The observation that the urea pathway can be selectively preserved with 0.25% glutaraldehyde applied to the mucosa indicates that this structure is more accessible and (or) more sensitive to low-dose glutaraldehyde than is the ADH-induced water pathway. The observation that glutaraldehyde is more effective in stabilizing the ADH-induced urea channels from the serosal than from the mucosal surface indicates that these channels are not fixed at the extracellular surface of the apical plasma membrane. It appears, rather, that glutaraldehyde exerts its effects from an intracellular position, where it cross-links components of the urea channels at the cytoplasmic surface of the apical membrane and (or) inactivates the intracellular machinery responsible for the removal or dispersal of the ADH-induced urea permeability sites.