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(Hypertension. 2000;35:1319.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Antagonist-Induced Intracellular Sequestration of Rabbit Bradykinin B₂ Receptor

Steeve Houle; Jean-François Larrivée; Magdalena Bachvarova; Johanne Bouthillier; Dimcho R. Bachvarov; François Marceau

From Centre Hospitalier Universitaire de Québec, Centre de Recherche du Pavillon l’Hôtel-Dieu de Québec, Québec, Canada, G1R 2J6.

Correspondence to François Marceau, MD, PhD, Centre de Recherche, Pavillon l’Hôtel-Dieu de Québec, Centre Hospitalier Universitaire de Québec, 11 Côte-du-Palais, Québec, Canada G1R 2J6. E-mail francois.marceau{at}crhdq.ulaval.ca

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) B₂ receptors (B₂Rs). 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 B₂R–green fluorescent protein (B₂R-GFP) conjugate was expressed in mammalian cells. In COS-1 cells, it exhibited an affinity for [³H]BK (K_D=1.61 nmol/L) similar to that of the wild-type rabbit B₂R. 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 [³H]BK binding to B₂R-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. B₂R-GFP was visualized in the plasma membranes of HEK-293 cells and rapidly internalized in response to BK. NPC 17731 or icatibant slowly translocated B₂R-GFP into cells over 24 hours, whereas LF 16.0687 had no effect on the subcellular distribution of B₂R-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 B₂R expressed in an heterologous system.

Key Words: rabbits • bradykinin • veins • receptors, bradykinin

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