Existence of Three Subtypes of Bradykinin B2Receptors in Guinea Pig

Abstract:We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high‐affinity site in brain, lung, and ileum (KD= 70–200 pM), which constitutes ∼20% of the bradykinin binding, and to a second, lower‐affinity site (0.63–0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high‐ and lower‐affinity sites in each tissue and to suggest the existence of three subtypes of B2receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2breceptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower‐affinity site in our binding studies. Receptor subtype B2cconstitutes ∼20% of binding sites in the brain and lung and is equivalent to the high‐affinity site in brain and lung. We suggest that a third subtype of B2receptor (high‐affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities. The potency of bradykinin analogues for the three B2subtypes is as follows: for B2a, bradykinin = D‐Arg[Hyp3,Thi5,D‐Tic7,Oic8]bradykinin (HOE 140) > [Hyp3]bradykinin > kallidin = D‐Argo[Hyp3,Thi5,8,D‐Phe7]bradykinin (NPC 349) = [Thi5,8,D‐Phe7]bradykinin (NPC 431) > D‐Arg[Hyp3,D‐Phe7]bradykinin (NPC 567) = [D‐Phe7]bradykinin (NPC 361); for B2b, bradykinin = HOE 140 > kallidin > NPC 349 = [Hyp3]bradykinin > NPC 431 = NPC 567 = NPC 361; and for B2c, bradykinin > NPC 349 = NPC 431 > kallidin > [Hyp3]bradykinin > HOE 140. We thought that the binding characteristics of the bradykinin analogues in lung were not sufficiently different to warrant the classification of a separate class of receptors (B3) as has been proposed previously. We suggest that, based on the structure of the bradykinin analogues, that amino acid residues 3, 5, and 8 of the bradykinin sequence are important for the differentiation of binding at the three different B2subtypes.