An osmometric method for the bioassay of vasotocin and related peptides in the toad bladder.

This study describes a new method for quantitating the antidiuretic activity of 8-arginine vasotocin (AVT) and related peptides on the isolated toad urinary bladder. The method is based on measuring changes in the osmolality of the surface fluid film of bladders that have been filled with a dilute solution and suspended in humidified air. Eight microliters of Ringer's fluid containing a known concentration of hormone are applied to small paper discs (0.7 cm in diameter), and the discs are then placed with fine forceps onto the outer surface of the bladder to which they adhere. The hormone increases the permeability to water of the epithelium that is underneath the area of the disc, and as water moves from the interior of the bladder along its osmotic gradient to the outer surface of the bladder, the Ringer's fluid in the disc becomes diluted. The magnitude of this dilution is quantitated by removing the discs to a vapor pressure osmometer at timed intervals. A supramaximal dose of AVT reduced disc fluid osmolality by 188 mosmol/kg H2O within 15 min. Similar maximal responses were observed with 8-arginine vasopressin (AVP), oxytocin, and 8-lysine vasopressin, although the potencies of these hormones diminished in the order listed above. AVT was 112-fold more potent than AVP, and AVP, in turn, was 329-fold more potent than 8-lysine vasopressin. The lower limit at which AVT was detected in this assay was 0.25 pg/disc (3 X 10(-11) M). The intra- and interassay variabilities for AVT were 14% and 28% (+/- SD), respectively. This assay is suitable for measuring the biological activity of hormone analogs lacking vasopressor activity, such as desmopressin, which was found to have a hydroosmotic activity of 8.3 +/- 2.4 U/mg. After osmotic stimulation, AVT was detected in toad plasma at a concentration of 1.4 X 10(-10) M. Therefore, this method has the requisite sensitivity for measuring this hormone in biological fluids of amphibia, reptiles, fish, and birds.