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 bradykinin receptors, using a high throughput functional assay which measures intracellular Ca(2+) responses. The most potent agonist for Chinese hamster ovary (CHO) cells stably expressing recombinant human bradykinin B(1) receptors were Des-Arg(9)-bradykinin (EC(50)=7.9 nM) and Des-Arg(10)-kallidin (EC(50)=8.6 nM), while the most potent agonist for CHO cells expressing human bradykinin B(2) receptors was bradykinin (EC(50)=2.0 nM). These findings confirm 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 bradykinin at bradykinin B(2) receptors was potently inhibited by [D-Arg(0),Hyp(3), beta-(2-thienyl)-Ala(5),D-Tic(7),Oic(8)]-bradykinin (Hoe140) (IC(50)=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(10)-kallidin were fully antagonized by Des-Arg(9)-[Leu(8)]bradykinin (IC(50)=59 nM), Des-Arg(10)-Hoe140 (IC(50)=211 nM) and most potently by Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic (B9858) (IC(50)=14 nM), none of which displayed any activity against the bradykinin B(2) receptor cell line up to 3 microM. 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.
Simpson
P B
PB
Neuroscience Research Centre, Merck Sharp & Dohme Research Laboratories, Terlings Park, Eastwick Road, Harlow, UK. peter_simpson@merck.com
Woollacott
A J
AJ
Hill
R G
RG
Seabrook
G R
GR
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005470:Fluorometry; D006801:Humans; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000187:drug effects*; D011994:Recombinant Proteins / Q000187:drug effects
D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011994:Recombinant Proteins; D001920:Bradykinin; D002118:Calcium