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.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.

Non‐competitive pharmacological antagonism at the rabbit B1 receptor

“…This type of interaction was heretofore unknown for this receptor type, but several examples involving other related receptor types are known (e.g. the peptide icatibant at the rabbit BK B 2 receptor; Marceau et al, 1994;Bachvarov et al, 1995;Houle et al, 2000; the clinically used non-peptide angiotensin antagonists at the human AT 1 receptor, Vanderheyden et al, 1999).…”

Met-Lys-bradykinin-Ser-Ser, a peptide produced by the neutrophil from kininogen, is metabolically activated by angiotensin converting enzyme in vascular tissue

“…The binding of 3 nM [ 3 H]bradykinin (Perkin Elmer Life Sciences; 90 Ci/mmol) to adherent intact HEK 293 cells stably expressing B 2 R-GFP was evaluated as described [7]. The binding assay of 1 nM [ 3 H]Lys-des-Arg 9 -BK (Perkin Elmer Life Sciences; 88 Ci/mmol) to HEK 293a cells stably expressing B 1 R-YFP [8] was performed as described [9].…”

“…The mutant ACE vectors were sequenced to confirm their identity. Membranes from transfected or untransfected (control) cells were prepared as described, with some variations [7]. Briefly, cells were homogenized (2 75-cm 2 flasks) in 0.25 ml sucrose buffer (250 mM sucrose, 20 mM tricine buffer, 1 mM PMSF, 10 mg/ml leupeptin, 2 mg/ml pepstatin, and 10 mg/ml soybean trypsin inhibitor, pH 7.5).…”

“…Contractility studies were performed after 3 or 5.5 h of in vitro incubation because the response mediated by B 1 receptors is acquired in a time-and protein synthesis-dependent manner in this preparation [1]. The external jugular vein, an established bioassay for the rabbit bradykinin B 2 receptor [7], has also been isolated from the animals and mounted in organ baths as described [15]. The institutional research ethics board approved the anonymous use of human umbilical cord segments obtained after elective cesarean section deliveries.…”

Ace inhibitors - activators of kinin receptors

“…Представителем второго поколения пептидных антагонистов Б 2 Р является икатибант [38]. Его взаимодействие с Б 2 Р в значительной степени зависит от межвидовых различий [39]. Для Б 2 Р человека икатибант является сильным конкурентным антагонистом [20].…”