Human vascular kinin receptors of the B-2 type characterized by radioligand binding

1 The human umbilical vein responds to bradykinin (BK) with contractions that are mediated by B-2 receptors. In the present study. the corresponding vascular smooth muscle B-2 binding sites have been investigated. 2 [H-3]-BK, a full agonist labelled ligand, was used to demonstrate a single binding site giving a K-d value of 0.51 +/- 0.02 nM and a B-max of 24 +/- 1 fmol mg(-1) protein. Scatchard plots were linear (r = 0.98) in the 0.05-5 nM range of concentrations. Non-specific binding was found to be 30% of total binding. 3 Competition binding curves gave the following order of potency for various B-2 receptor agonists: BK-[Hyp(3)]-BK greater than or equal to Lys-BK much greater than[Aib(7)]-BK>>>[desArg(9)]-BK, which is typical of B-2 receptors. There was no binding to B-1 receptors since the selective B-1 receptor ligand, Lys-[desArg(9)]BK was inactive up to 10 mu M (n=4). 4 Characterization of the binding site with antagonists, performed with three chemically distinct series of peptide and non-peptide compounds, revealed a high affinity of Hoe 140 (D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-BK) (K-i 0.17 nM; n=4) which was more potent that FR 173657 ([(E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl] acrylamide]) (K-i 1.94 nM; n=4), D-Arg-[Hyp(3),D-Phe(7),Leu(8)]-BK (K-i 256 nM; n=4) and Win 64338 (phosphonium, [[4[[2[[bis(cyclohexylamino)methylene]amino]-3-(2-naphthalenyl)-1-oxopropyl]amino]phenyl]methyl]tributyl, chloride, monohydrochloride) (K-i 1,450 nM; n=4). 5 The present study describes and characterises B-2 receptor binding sites in the vascular smooth muscle of the human umbilical vein. The binding assay appears to be suitable for studying new agonists or antagonists designed to activate or block the B-2 receptor class that mediate the majority of the physiopathological effects of kinins in man.