Iodinated photoreactive vasopressin antagonists: labelling of hepatic vasopressin receptor subunits

To identify and characterize V 1 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 V 1 antagonists: [Mca1 1 , d ‐Tyr 2 ,MeAla 7 ,Lys 8 ]VP (l), [Mca 1 ,MeAla 7 ,Arg 8 ,Lys 9 ]VP (2), [Mca 1 ,MeAla 7 ,Arg 8 , d ‐Lys 9 ]VP (3). Introduction of the photoreactive 4‐azidophenylamidino group into the side‐ chain of Lyss in analogue 1 or into Lys 8 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 Tyr 2 with 125 I resulted in photoreactive antagonists of high specific radioactivity, which had roughly the same binding affinity as vasopressin for the rat hepatic V 1 receptor (K d = 0.9—1.8 nM). In photoaffinity labelling experiments with purified rat liver membranes, containing 2–3 pmol V 1 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 3 H‐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 V 1 receptor. Furthermore the radioiodinated photoreactive V 1 antagonists should be helpful to identify V 1 receptor proteins in membranes of other cell types.