Irreversible Stimulation of Hydroosmotic Response in Toad Bladder by Photoaffinity Labeling with [Phe2,Phe-(p-N3)3] Vasopressin*

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Abstract
The photoreactive analogs of vasopressin, [Phe2,Phe-(p-N3)3]AVP (3a) and [Phe-(p-N3)2]AVP (2a), and the chemically reactive analogs of vasopressin, [Phe2,Phe-(p-NHCOCH2Br)3]AVP (3b) and [Phe-(p-NHCOCH2Br)2]AVP (2c), were tested in the toad bladder for irreversible stimulation or inhibition of the water permeability response. Analog 3a was an agonist with an ED50 of 4.5 .times. 10-7 M and induced a maximal osmotic water flow across bladders equivalent to 74% of that observed with the parent hormone (AVP). Photolysis of this analog of Ringer''s fluid resulted in a decrease in its biological activity, with a half-time of 7 min. However, UV irradiation of the analog in the presence of toad bladders triggered an irreversible increase in the permeability of the bladders to water. Under optimal conditions of irradiation, water permeability remained at .apprx. 60% of maximum for more than 3 h after washout of analog 3a. The addition of AVP raised the permeability of these bladders to 100%. Analog 3a did not cause irreversible stimulation without photolysis, nor did this analog induce its characteristic effect when added to the mucosal solution. Compound 2a was a potent antagonist of AVP. This inhibitory action of 2a was readily reversed in both the presence and absence of UV irradiation. Compound 3b was also a reversible inhibitor of AVP. Compound 2c was inactive as agonist or antagonist. Analog 3a apparently binds covalently at or near the toad bladder hydroosmotic receptors, resulting in a persistent increase in permeability to water of the bladder wall.