The antidiuretic hormone, vasopressin (AVP), plays an important role in the regulation of plasma volume and osmolality by triggering the reabsorption of water, urea, and sodium from the forming uring in the kidney. The isolated urinary bladder of the toad, Bufo marinus, has been used extensively to study the cellular mechanism of action of AVP (or the amphibian antidiuretic hormone, vasotocin (AVT), which is similar to AVP). It is not known whether different receptors and (or) different target cells are involved in the three transport functions of the hormone. We plan to approach this problem by isolating and purifying AVT receptors from isolated toad bladders by Percoll- and sucrose- density gradient centrifugation, affinity chromatography with a biotinyl derivative of AVT linked to an avidin column, and photoaffinity labeling of receptors with radio-active AVT analogues followed by SDS-PAGE. Both agonistic and antagonistic radio-labeled photoaffinity analogues will be synthesized for this purpose. After AVT receptor proteins have been purified from toad bladder, antibodies will be raised in rabbits to these receptors, and the action of antireceptor antibodies to inhibit binding of radio-labeled hormone to one or more classes of receptors in membranes will be compared with their effect on adenylate cyclase activity and on water, urea, and sodium transport across the bladder epithelium. AVT analogues containing fluorescein or rhodamine moieties in positions 7 (agonists) or 4 (antagonists) will be synthesized and their localization in frozen tissue slices of toad bladder studied by quantitative fluorescence microscopy. Displacement of fluorescent AVT by different receptor antibodies will be used as a way of matching different cell types with different transport functions. Similarly, a second antibody with a fluorescent label will be used to localize the distribution of anti-receptor antibodies. It is hoped that these studies will shed light on the question of how a single hormone can differentially regulate three independent transport functions in the same target organ.
