AJP - Cell Physiology, Vol 252, Issue 6 C657-C662, Copyright © 1987 by American Physiological Society
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 and J. D. Glass
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.
