A fluorescent version of the bradykinin B2 receptor antagonist B-9430: pharmacological characterization and use in...

B-9430 (d-Arg-[Hyp3, Igl5, D-Igl7, Oic8]-bradykinin), where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine and Oic is (3as, 7as)-octahydroindol-2-yl-carbonyl is a high affinity bradykinin B2 receptor antagonist with effects extended to the B1 receptors at high concentrations. The N-terminus of B-9430 has been extended with d-biotinyl (B-10330) or 5(6)-carboxyfluorescein-epsilon-aminocaproyl (B-10380) to derive fluorescent receptor probes. The pharmacological profile of B-10380 was similar to that of B-9430 with a minor loss of potency (a competitive antagonist of bradykinin at the B2 receptors of the human isolated umbilical vein, pA2 6.83; an insurmountable antagonist at the B2 receptors in the rabbit jugular vein; a weak competitive antagonist of the B1 receptors in the rabbit aorta, pA2 5.95). B-10330 and B-10380 displaced the binding of [3H]bradykinin from rabbit B2 receptors with a potency slightly inferior to that of B-9430 (larger gap at the rat B2 receptor). Treatment with B-10330 and fluorescent streptavidin did not support imaging of recombinant B2 receptors. However, the plasma membrane of HEK 293a cells that transiently expressed recombinant rabbit B2 receptors, but not B1 receptors, was labeled with 5-50 nM B-10380 (epifluorescence microscopy). B-10380 staining was not observed in nontransfected cells and was abolished by co-treating receptor-expressing cells with a nonpeptide antagonist. The N-terminal extension of a potent peptide antagonist of the bradykinin B2 receptor with a fluorophore produced a fluorescent probe suitable for live cell imaging and other applications at the expense of a minor loss of affinity.