HIGH-AFFINITY BRADYKININ B2 BINDING-SITES SENSITIVE TO GUANINE-NUCLEOTIDES IN BOVINE AORTIC ENDOTHELIAL-CELLS

Bradykinin (BK) binding sites were studied in membranes from bovine aorta. [H-3]BK specifically bound to one high-affinity binding site (K(D) = 152 pM; B(max) = 4.6 fmol.mg-1) and was displaced by unlabled BK (K(i) = 121 pM). The B2-agonist kallidin and B2-antagonists D-Arg 0[Hyp3,Leu5,8,Gly6,D-Phe7]BK, D-Arg 0[Hyp3,D-Phe7]BK, [D-Phe7]BK, and [Thi5,8,D-Phe7]BK inhibited [H-3]BK binding with respective K(i) values of 101, 282, 678, 2000 and 6000 pM. The B1-antagonist des-Arg9[Leu8]BK had no effect. GTP, GTP-gamma-S, GDP, and GDP-beta-S but not 5'-GMP, guanosine, cyclic 3',5'-GMP, ATP, ADP, 5'-AMP, nor adenosine, inhibited [H-3]BK binding with an IC50 of 1-3-mu-M for GTP and GDP and an IC50 of 0.1-0.3-mu-M for GTP-gamma-S and GDP-beta-S. GTP and GDP at 3-mu-M decreased the B(max) value by 30-70%. Millimolar concentrations of Ca2+ and Mg2+ ions increased [H-3]BK binding and counteracted the effect of guanine nucleotides. This study demonstrates the existence of a specific high-affinity B2 BK binding site in bovine aortic endothelial cells. It suggests that this site is located on a G protein-interacting receptor.