Covalent labeling of hydrosmotic toad bladder receptors with an antagonist of vasotocin.
A photoreactive analogue of vasotocin, [1-desamino,4-lysine(azidobenzoyl),8-arginine]vasotocin (4-N3-AVT), has been examined in the isolated toad urinary bladder for biological activity and binding to hormonal receptors. Although 4-N3-AVT induced only a small increase in bladder permeability to water, it behaved as a potent inhibitor of hydrosmotic action of [8-arginine]vasotocin (AVT) and [8-arginine]vasopressin (AVP). The inhibitory action of 4-N3-AVT was readily reversed on removal of the analogue from the serosal bathing solution. On the other hand, when bladders were exposed to 4-N3-AVT in the presence of long wavelength UV light (365 nm), the inhibition by 4-N3-AVT was not reversed on washout of the analogue. The dose of vasopressin required for a half-maximal response (ED50 value) was increased from 5 X 10(-9) to 1.3 X 10(-7) M in bladders photolabeled with 4-N3-AVT and the maximal response capacity of the tissue (intrinsic activity) was reduced to 79% of nonphotolabeled controls. A crude membrane preparation derived from bladders photolabeled with 4-N3-AVT contained 72 fmol of specific binding sites for tritium-labeled vasopressin per milligram protein, whereas nonphotolabeled controls had 136 fmol of specific binding sites per milligram protein. These observations suggest that 4-N3-AVT forms a covalent bond with hydrosmotic receptors in the presence of UV light. This is the first antagonistic photoaffinity analogue observed in the toad bladder and it may serve as a useful tool for analyzing the cellular mechanism of action of antidiuretic hormone.
D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001681:Biological Assay; D002024:Bufo marinus; D002463:Cell Membrane Permeability; D004305:Dose-Response Relationship, Drug; D011945:Receptors, Angiotensin / Q000378:metabolism*; D017483:Receptors, Vasopressin; D013997:Time Factors; D014466:Ultraviolet Rays; D001743:Urinary Bladder / Q000378:metabolism* / Q000528:radiation effects; D014668:Vasotocin / Q000031:analogs & derivatives* / Q000378:metabolism*
D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; C034862:vasotocin receptor; C052826:vasotocin, 1-deamino-4-Lys(azidobenzoyl)-8-Arg-; D001127:Arginine Vasopressin; D014668:Vasotocin