Pharmacological characterization of the bradykinin B-2 receptor: inter-species variability and dissociation between binding and functional responses

1 The present study addresses the differences in binding profiles and funct ional properties of the human and rat bradykinin (BK) B-2 receptor using va rious kinin receptor peptide derivatives as well as the non-peptide recepto r antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)-methyl ene]amino]-3- (2-naphtalenyl)l-oxopropyl]amino]-phenyl]-methyl]tributyl, ch loride, monohydrochloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[- N-[2,4- dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. 2 [H-3]-BK bound with a similar affinity to membranes of Chinese hamster ov ary cells (CHO-K1) expressing the cloned human (hB(2)-CHO) or rat (rB(2)-CH O) B-2 receptor, human embryonic intestine cells (INT407) expressing the na tive B-2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 6433 8 and FR173657 bound with a 3.8-6.6 fold and 7.0-16.3 fold higher affinity the rat than the human B-2 receptor, respectively. The affinity values of B K derivatives as well as non-peptide antagonists were reduced by 6-23 fold in physiological HBSS compared to low ionic strength TES binding buffer. 3 BK (0.01-3000 nM) increased inositol triphosphates (IP3) levels in hB2-CH O, rB(2)-CHO and INT407 cells. The B-2 receptor antagonist, Hoe 140 (D-Arg( 0)-[ Hyp(3), Thi(5), D-Tic(7), Oic(8)]-BK) at 10(-7) M, significantly shift ed to the right the IP3 response curves to BK giving apparent pK(B) values of 8.56, 9.79 and 8.84 for hB(2)-CHO, rB(2)-CHO and INT407 cells, respectiv ely. 4 In human isolated umbilical vein, Hoe 140, D-Arg(0)-[Hyp(3), D-Phe(7), Le u(8)]-BK and NPC 567 had a lower potency in functional assays (pK(B) 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding s tudies (pK(i) 10.52, 8.64 and 8.27, respectively). 5 FR173657 behaved as a high affinity ligand with pK(i) values of 8.59 and 9.81 and potent competitive antagonist with pK(B) values of 7.80 and 8.17 i n HUV and RU, respectively. FR173657 bound with a similar affinity the clon ed and native bradykinin BZ receptor in human (pK(i) of 8.66 and 8.59, resp ectively) and in rat (pK(i) 9.67 and 9.81, respectively). 6 In conclusion, we suggest that the binding buffer composition has to be t aken into account when screening new compounds and that inter-species diffe rences should be considered when setting up animal models with the aim of d eveloping bradykinin B-2 receptor antagonists as therapeutic agents.