CHARACTERIZATION OF BRADYKININ-B(2) RECEPTORS ON HUMAN IMR-90 LUNG FIBROBLASTS - STIMULATION OF CA-45(2+) EFFLUX BY D-PHE(7) SUBSTITUTED BRADYKININ ANALOGS

[H-3]Bradykinin binds to intact human IMR-90 fetal lung fibroblasts in a time and dose-dependent manner. Binding equilibrium was attained by 120 minutes at 4-degrees-C. [H-3]Bradykinin binding was saturable; Scatchard analysis of saturation binding data demonstrated a single binding site having a K(D) = 1.8 +/- 0.2 nM and a receptor concentration of 17.4 +/- 4.0 fmol/10(5) cells. The calculated value for K(D)(k-1/k1) from the association (k1 = 4.71 x 10(6) mol-1 min-1) and dissociation (k-1 = 1.13 x 10(-2) min-1) rate constants was 2.4 nM. The rank order of potency observed for bradykinin peptide agonists, bradykinin > Lys-bradykinin > Met,Lys-bradykinin > Ile,Ser-bradykinin >> des-Arg9-bradykinin, is consistent with that of a bradykinin B2 receptor. Bradykinin stimulated efflux of Ca-45(2+) from IMR-90 cells dose dependently with an EC50 = 331 +/- 50 pM. Ca-45(2+) efflux was also demonstrated with Lys-bradykinin and Met-Lys-bradykinin but not by des-Arg10-kallidin (100 nM) or NKA (1 muM). Hoe-140 inhibited bradykinin-induced Ca-45(2+) efflux (IC50 = 3 +/- 2 nM). D-Phe7-substituted bradykinin analogues stimulated Ca-45(2+) efflux dose dependently and this stimulation of Ca-45(2+) efflux was inhibited by Hoe-140. These results suggest that D-Phe7 substituted bradykinin analogues are agonists at the bradykinin B2 receptor in IMR-90 cells.