Antagonist-Induced Intracellular Sequestration of Rabbit Bradykinin B 2 Receptor

Abstract

Abstract —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) B 2 receptors (B 2 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 B 2 R–green fluorescent protein (B 2 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 B 2 R. The stably expressed construction in HEK-293 cells was functionally active (phospholipase A 2 assay), and the antagonists mentioned above retained their respective surmountable or insurmountable behavior. Competition of [ 3 H]BK binding to B 2 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. B 2 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 B 2 R-GFP into cells over 24 hours, whereas LF 16.0687 had no effect on the subcellular distribution of B 2 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 B 2 R expressed in an heterologous system.