title: Characterization of a novel binding site for 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]bradykinin on epithelial membranes of guinea pig ileum.
abstract: We have recently shown that (a) [125I-Tyr8]bradykinin (BK) recognized bradykinin binding sites in guinea pig epithelium membranes with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg protein, and (b) B2 agonists and some B2 antagonists, such as D-Arg-[Hyp3,D-Phe7,Leu8]BK, inhibited this specific binding with a Ki value of 32 nM. In the present study, we have radioiodinated the B2 antagonist Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK and have performed a full characterization of the binding properties of this tracer in the same membrane preparation. Equilibrium experiments performed in the absence or presence of an excess of BK (10(-5) M) showed that 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK specifically labelled two different sites. One of these is the same as the site labelled by [125I-Tyr8]BK, and this indicates that 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK interacts specifically with kinin B2 receptors. Equilibrium experiment performed in the presence of an excess of BK (10(-5) M) indicated that specific binding of 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK to the second site is also saturable and Scatchard analysis showed that the site is of high affinity with a Kd of 16.8 nM and a Bmax of 2.08 pmol/mg protein. Surprisingly, unlabelled B2 agonists such as bradykinin, [Tyr8]BK, [Leu8]BK, [Hyp3,Tyr8(OMe)]BK, D-Arg-[Hyp3]BK and kallidin were found to be inactive on this second site. A series of B2 receptor antagonists, Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK, D-Arg-[Hyp3,D-Phe7,Leu8]BK, D-Arg-[Hyp3,Leu5,8,D-Phe7]BK, D-Arg-[Hyp3,Gly6,D-Phe7,Leu8]BK and D-Arg-[Hyp3,Thi5,8,D-Phe7]BK inhibited 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK binding with Ki values of 25.0, 20.9, 15.8, 64.6 and 6606.9 nM respectively. On the other hand, [Thi5,8,D-Phe7]BK did not interfere with 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK but was found to be a potent inhibitor of [125I-Tyr8]BK binding (Ki = 53.7 nM). As expected, B1 receptor agonists, antagonists and peptides non-related to BK such as substance P, neurokinin A, neurokinin B, angiotensin II, bombesin, vasopressin and the calcitonin gene related peptide were unable to compete with 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK. The results show that 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK is interacting with two distinct binding sites in the guinea pig epithelium: one is the well known bradykinin B2 receptor and the other is a new, non-characterized binding site that interacts exclusively with some bradykinin receptor antagonists.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Que., Canada.
authors.forename: C
authors.initials: C
authors.lastname: Tousignant
authors.forename: D
authors.initials: D
authors.lastname: Regoli
authors.forename: N E
authors.initials: NE
authors.lastname: Rhaleb
authors.forename: D
authors.initials: D
authors.lastname: Jukic
authors.forename: G
authors.initials: G
authors.lastname: Guillemette
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006168:Guinea Pigs; D007082:Ileum / Q000378:metabolism; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000032:analysis; D013329:Structure-Activity Relationship
chemical_list: D007457:Iodine Radioisotopes; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; C026201:bradykinin, Tyr-; C076380:bradykinin, Tyr-Arg-(Hyp(3)-Phe(7)-Leu(8))-; D001920:Bradykinin