A Sensitive Hydroosmotic Toad Bladder Assay

Abstract

A B S T R A C T A sensitive and precise method for assaying the water permeability response evoked by neurohypophyseal hormones and their synthetic analogues on the isolated urinary bladder of the toad (Bufo marinus L.) is described. The method permits detection of 8-arginine-vasotocin at concentrations as low as 10-12M. This sensitivity, not achieved heretofore with this tissue, results largely from minimizing interference of inhibitory substances by means of an "in vitro circulation assembly." The precision of the method derives from a direct comparison between the cumulative dose-response curve of an agonist of unknown potency acting on one hemibladder and that of a reference compound acting on the contralateral hemibladder. Crystalline deamino-oxytocin is used as the reference standard in this assay. The intrinsic activity of 2-(O-methyltyrosine)-oxytocin, as defined by the maximal response, is 12 % lower than that of deamino-oxytocin. All other hormonal peptides investigated have the same intrinsic activity as deamino-oxytocin, even 5-valine-oxytocin, in spite of its extremely low affinity. A comparison of the potencies of 8-arginine-vasotocin vs. 8-argininevasopressin, 8-ornithine-vasotocin vs. 8-ornithine-vasopressin, 8-alanine-oxytocin vs. 8-alanine-oxypressin, and deamino-8-alanine-oxytocin vs. deamino-8-alanine-oxypressin suggests that an isoleucine residue in position 3 imparts a higher specificity for binding of the hormonal peptide molecule to the bladder receptor than a phenylalanine residue in this locus. I N T R O D U C T I O NT h e m a m m a l i a n antidiuretic h o r m o n e , vasopressin, modifies the p e r m e a b i l i t y to w a t e r of the distal n e p h r o n and t h e r e b y accelerates osmotic equilibration of the forming urine with the renal interstitial fluid (4). Similarly, n e u r oh y p o p h y s e a l peptides increase the rate of w a t e r m o v e m e n t along an osmotic gradient across the skins a n d bladders f r o m frogs a n d toads. In these target tissues the h o r m o n e interacts with a receptor moiety in the epithelial cell 465

Inhibition of the permeability response to vasopressin and oxytocin in the toad bladder: Effects of bradykinin, kallidin, eledoisin, and physalaemin

“…1 also shows that there was a correlation in the rate of fading of the response to the various concentrations of hormone: the higher the dose, the faster the decline in the response. These findings are essentially in concert with the results of Eggena, Schwartz and Walter (1968), despite some differences in the experimental preparation.…”

Quantitative analysis of exocytosis and endocytosis in the hydroosmotic response of toad bladder

“…The hydroosmotic response is detected as early as 5 min after ADH administration. As reported by others, we find the peak of water movement is during the 15-30 rain interval and flow declines in the 30-60 min interval (Eggena, Schwartz & Walter, 1968;Kachadorian, Casey & DiScala, 1977).…”

Threshold and Receptor Reserve in the Action of Neurohypophyseal Peptides

“…The two latter compounds had already been shown to exhibit inhibitory properties or decreased maximal effects in several other hormone-sensitive tissues (37); moreover, in an earlier investigation we had observed a diminution in the intrinsic hydroosmotic activity of [2-0-methyltyrosine]-oxytocin on the toad bladder (18).…”

“…with similar KA values. The applicability of this assumption to the receptors associated with the hydroosmotic response of the toad bladder is supported by a detailed analysis of the dose-response relationships of several neurohypophyseal hormones and synthetic analogues reported previously (18).…”