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

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 323; no. 2; pp. 534 - 546
• Main Authors: Bawolak, Marie-Thérèse, Gera, Lajos, Morissette, Guillaume, Stewart, John M, Marceau, François
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.11.2007
• Subjects: Animals; Bradykinin - analogs & derivatives; Bradykinin - metabolism; Bradykinin - pharmacology; Cercopithecus aethiops; COS Cells; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases - metabolism; Humans; In Vitro Techniques; Male; Oligopeptides - pharmacology; Phosphorylation; Rabbits; Radioligand Assay; Receptor, Bradykinin B2 - agonists; Receptor, Bradykinin B2 - analysis; Receptor, Bradykinin B2 - metabolism; Vasoconstriction - drug effects
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.107.123422

The bradykinin B 2 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-[Hyp 3 ,Igl 5 , d -Igl 7 ,Oic 8 ]-bradykinin) is a second generation peptide antagonist found to be competitive at the human B 2 receptor and insurmountable at the rabbit B 2 receptor (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B-10344 ( d -Arg-[Hyp 3 ,Igl 5 ,Oic 7 , d -Igl 8 ]-bradykinin; inverted sequence Oic 7 , d -Igl 8 ) and B-9972 ( d -Arg-[Hyp 3 ,Igl 5 ,Oic 7 ,Igl 8 ]-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 B 2 receptor (radioligand binding competition assay). The effects of agonists on receptors were compared using two chimerical constructions based on rabbit B 2 receptors: conjugate of the B 2 receptor with green fluorescent protein (B 2 R-GFP) and the N-terminally tagged conjugate of the myc epitope with the B 2 receptor. Imaging and immunoblotting showed that B-9972 induced a persistent endocytosis of cell surface B 2 receptors in human embryonic kidney 293 cells with slow receptor degradation (weak after 3 h of treatment, important at 12 h) and B 2 R-GFP desensitization ([ 3 H]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 B 2 receptors by the agonists. The results illustrate the agonist-antagonist transition in B 2 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.