Functional characterization of bradykinin analogues on recombinant human bradykinin B-1 and B-2 receptors

We have examined the activity of a range of kinins on recombinant human bra dykinin receptors, using a high throughput functional assay which measures intracellular Ca2+ responses. The most potent agonist for Chinese hamster o vary (CHO) cells stably expressing recombinant human bradykinin B-1 recepto rs were Des-Arg(9)-bradykinin (EC50 = 7.9 nM) and Des-Arg(10)-kallidin (EC5 0 = 8.6 nM), while the most potent agonist for CHO cells expressing human b radykinin B-2 receptors was bradykinin (EC50 = 2.0 nM). These findings conf irm the validity of the recombinant system and the microtitre plate imaging -based characterization system when compared to known agonist properties of the native receptors. The concentration-response relationship for bradykin in at bradykinin B-2 receptors was potently inhibited by [D-Arg(0),Hyp(3),b eta-(2-thienyl)-Ala(5),D-Tic(7),Oic(8)]-bradykinin (Hoe140) (IC50 = 71 nM), which was 500-fold more potent against the B-2-expressing cells than the B -1 cells. Bradykinin B-1 receptor-mediated responses activated by Des-Arg(1 0)-kallidin were fully antagonized by Des-Arg(9)-[Leu(8)]bradykinin (IC50 = 59 nM), Des-Arg(10)-Hoe140 (IC50 = 211 nM) and most potently by Lys-Lys-Ar g-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic (B9858) (IC50 = 14 nM), none of which displ ayed any activity against the bradykinin B-2 receptor cell line up to 3 mu M. None of the antagonists displayed partial agonism activity in these cell lines. All bradykinin B-1 and B-2 receptor antagonists tested acted in an apparently non-competitive manner that is likely to be due in part to their kinetics and to the nature of the functional assay used. (C) 2000 Elsevier Science B.V. All rights reserved.