Iodinated photoreactive vasopressin antagonists: labelling of hepatic vasopressin receptor subunits

Abstract

To identify and characterize V₁ vasopressin receptors, photoreactive antagonists of the glycogenolytic and vasoconstrictor activity of vasopressin have been synthesized. The following analogues with 3-mercapto- 3,3-cyclopentamethylene-propionic acid (Mca) and N-methylalanine (MeAla) in position 1 and 7 of vasopressin (VP) were effective V₁ antagonists: [Mca1¹,d-Tyr²,MeAla⁷,Lys⁸]VP (l), [Mca¹,MeAla⁷,Arg⁸,Lys⁹]VP (2), [Mca¹,MeAla⁷,Arg⁸,d-Lys⁹]VP (3). Introduction of the photoreactive 4-azidophenylamidino group into the side- chain of Lyss in analogue 1 or into Lys⁸ in analogues 2 and 3 increased the potency (for analogue 1 a tenfold increase in the antiglycogenolytic effect and a fivefold increase in the antivasopressor effect) and binding affinity for the rat hepatic V1 receptor. Mono-iodination at Tyr² with ¹²⁵I resulted in photoreactive antagonists of high specific radioactivity, which had roughly the same binding affinity as vasopressin for the rat hepatic V₁ receptor (K_d= 0.9—1.8 nM). In photoaffinity labelling experiments with purified rat liver membranes, containing 2–3 pmol V₁ receptor/ mg protein, the analogues labelled specifically two proteins with the relative molecular masses (M_I) of 30000 and 38 000. These results and the results of a recent study using ³H-labelled photoreactive vasopressin agonists [Boer, R. and Fahrenholz, F. (1985) J. Bid. Chem. 260, 15051 - 150541 provide evidence that both vasopressin agonists and antagonists can interact with the same two subunits of the heterodimeric hepatic V₁ receptor. Furthermore the radioiodinated photoreactive V₁ antagonists should be helpful to identify V₁ receptor proteins in membranes of other cell types.