Antagonist-Induced Intracellular Sequestration of Rabbit Bradykinin B 2 Receptor

—In a contractility assay based on the rabbit jugular vein, the structurally related drugs NPC 17731 or icatibant (1 to 3 nmol/L) were insurmountable antagonists of bradykinin (BK) B2 receptors (B2 Rs). After ample washing (3 hours), the antagonism exerted by these peptides was not reversible. By contrast, the antagonist LF 16.0687 (30 to 100 nmol/L) was competitive and reversible. A rabbit B2 R–green fluorescent protein (B2 R-GFP) conjugate was expressed in mammalian cells. In COS-1 cells, it exhibited an affinity for [3 H]BK (K D =1.61 nmol/L) similar to that of the wild-type rabbit B2 R. The stably expressed construction in HEK-293 cells was functionally active (phospholipase A2 assay), and the antagonists mentioned above retained their respective surmountable or insurmountable behavior. Competition of [3 H]BK binding to B2 R-GFP by the antagonists or BK was largely reversible after a 3-hour washout period at 0°C; at 37°C, icatibant or NPC 17731 effects were not reversible. B2 R-GFP was visualized in the plasma membranes of HEK-293 cells and rapidly internalized in response to BK. NPC 17731 or icatibant slowly translocated B2 R-GFP into cells over 24 hours, whereas LF 16.0687 had no effect on the subcellular distribution of B2 R-GFP. Cell extract immunoblotting with anti-GFP antibodies revealed a 101- to 105-kDa protein that was not significantly degraded on 24 hours of cell treatment with any of the ligands but was translocated in part to the 15 000-g pellet of the extract on treatment with BK or the noncompetitive antagonists. NPC 17731 and icatibant are noncompetitive, nonequilibrium antagonists that promote the cellular sequestration of rabbit B2 R expressed in an heterologous system.