Covalent labeling of hydrosmotic toad bladder receptors with an antagonist of vasotocin

P. Eggena, A. Buku, C. L. Ma, L. I. Somoza, H. R. Wyssbrod, I. L. Schwartz, J. D. Glass

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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 analgoue from the serosal bathing solution. One 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 wih 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.

Original languageEnglish
Pages (from-to)C657-C662
JournalAmerican Journal of Physiology - Cell Physiology
Issue number6 (21/6)
StatePublished - 1987


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