Pharmacological and functional characterization of the guinea-pig B2 bradykinin receptor stably expressed in CHO-K1 cell line

1 In the present study, pharmacological properties of a bradykinin B, receptor amplified either from guinea-pig ileum or lung and homologous to the previously reported sequence except two amino-acid changes L-124-->P and N-227-->Y in the receptor protein were characterized. 2 Tritiated bradykinin ([H-3]-BK) specifically bound to the cloned guinea-pig B, bradykinin receptor stably expressed in Chinese hamster ovary cells (CHO-K1) with a K-D value of 0.29 +/- 0.07 nM. In competition experiments, bradykinin (BK) affinity constant value was 0.21 +/- 0.05 nM while the two specific kinin B-1 ligands, des-Arg(9)-bradykinin (DBK) and des-Arg(9)-Leu(8)-bradykinin (DLBK) were unable to compete with [H-3]-BK. As the specific peptide antagonist D-Arg-[Hyp(3),Thi(6),D-Tic(7),Oic(8)]- bradykinin (HOE140), (E)-3-(6-acetamido-3-pyridil)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinoliny-1)oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide (FR173657) and 1-[[3-[2,4-dimethyl-quinolin-8-yl)oxymethyl] -2,4-dichloro-phenyl]sulfonyl]-2(S)-[[4-[4-(aminoiminomethyl)-phenylcarbonyl]piperazin-1-yl]carbonyl]pyrrolidine (LF16-0335C) exhibited a high affinity for this receptor with K-i values of 7.34 +/- 2.45 mM and 8.54 +/- 1.55 nM respectively. 3 BK and kallidin (KD) increased inositol phosphates (IPs) levels with EC50 values of 0.44+/-0.12 nM and 6.88+/-0.28 nM, respectively. Neither DLBK nor DBK (0.01 nM to 10 muM) stimulated or inhibited IPs turnover and as expected HOE140 did not raise IPs production. HOE140 (0.1 muM) and LF 16-0335c (1 muM) right shifted the BK response curve with pK(B), values of 9.24+/-0.4 and 8.4+/-0.3, respectively. 4 The results indicate that this cloned guinea-pig receptor displayed typical pharmacological properties of a bradykinin B, receptor and support the existence of a single B, receptor in this species.