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CARDIOVASCULAR

B-9972 (D-Arg-[Hyp³,Igl⁵,Oic⁷,Igl⁸]-bradykinin) Is an Inactivation-Resistant Agonist of the Bradykinin B₂ Receptor Derived from the Peptide Antagonist B-9430 (D-Arg-[Hyp³,Igl⁵,D-Igl⁷,Oic⁸]-bradykinin): Pharmacologic Profile and Effective Induction of Receptor Degradation

Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec and Department of Medicine, Université Laval, Quebec City, Quebec, Canada (M.-T.B., G.M., F.M.); and Department of Biochemistry, University of Colorado Health Sciences Center, Denver, Colorado (L.G., J.M.S.)

The bradykinin B₂ receptor is a heptahelical receptor regulated by a cycle of phosphorylation, endocytosis, and extensive recycling at the cell surface following agonist stimulation. B-9430 (D-Arg-[Hyp³,Igl⁵,D-Igl⁷,Oic⁸]-bradykinin) is a second generation peptide antagonist found to be competitive at the human B₂ receptor and insurmountable at the rabbit B₂ receptor (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B-10344 (D-Arg-[Hyp³,Igl⁵,Oic⁷,D-Igl⁸]-bradykinin; inverted sequence Oic⁷, D-Igl⁸) and B-9972 (D-Arg-[Hyp³,Igl⁵,Oic⁷,Igl⁸]-bradykinin); they are low- and high-potency agonists, respectively, in vascular preparations. The potency gap between bradykinin and B-9972 is narrow in contractility assays, despite the fact that B-9972 affinity is 7-fold inferior at the rabbit B₂ receptor (radioligand binding competition assay). The effects of agonists on receptors were compared using two chimerical constructions based on rabbit B₂ receptors: conjugate of the B₂ receptor with green fluorescent protein (B₂R-GFP) and the N-terminally tagged conjugate of the myc epitope with the B₂ receptor. Imaging and immunoblotting showed that B-9972 induced a persistent endocytosis of cell surface B₂ receptors in human embryonic kidney 293 cells with slow receptor degradation (weak after 3 h of treatment, important at 12 h) and B₂R-GFP desensitization ([³H]bradykinin endocytosis and extracellular signal-regulated kinase 1/2 phosphorylation assays). Bradykinin was not active in this respect but when combined with captopril, induced some degradation. B-9430 reduced the endocytosis and degradation of B₂ receptors by the agonists. The results illustrate the agonist-antagonist transition in B₂ receptor peptide ligands with a constrained C-terminal structure, the importance of species in their pharmacological profile, and the possibility of selectively degrading receptors using a peptidase-resistant agonist.

Address correspondence to: Dr. François Marceau, Centre de Recherche en Rhumatologie et Immunologie, Room T1–49, Centre Hospitalier Universitaire de Québec, 2705 Laurier Boulevard, Quebec, Canada G1V 4G2. E-mail: francois.marceau{at}crchul.ulaval.ca

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