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

B-9430 (d-Arg-Hyp(3), Igl(5), D-Igl(7), Oic(8)]-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 B(2) receptor antagonist with effects extended to the B(1) receptors at high concentrations. The N-terminus of B-9430 has been extended with d-biotinyl (B-10330) or 5(6)-carboxyfluorescein-varepsilon-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 B(2) receptors of the human isolated umbilical vein, pA(2) 6.83; an insurmountable antagonist at the B(2) receptors in the rabbit jugular vein; a weak competitive antagonist of the B(1) receptors in the rabbit aorta, pA(2) 5.95). B-10330 and B-10380 displaced the binding of (3)H]bradykinin from rabbit B(2) receptors with a potency slightly inferior to that of B-9430 (larger gap at the rat B(2) receptor). Treatment with B-10330 and fluorescent streptavidin did not support imaging of recombinant B(2) receptors. However, the plasma membrane of HEK 293a cells that transiently expressed recombinant rabbit B(2) receptors, but not B(1) receptors, was labeled with 5-50nM 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 B(2) 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.