Functional characterization of bradykinin analogues on recombinant human bradykinin B₁ and B₂ receptors

Abstract

We have examined the activity of a range of kinins on recombinant human bradykinin receptors, using a high throughput functional assay which measures intracellular Ca²⁺ responses. The most potent agonist for Chinese hamster ovary (CHO) cells stably expressing recombinant human bradykinin B₁ receptors were Des-Arg⁹-bradykinin (EC₅₀=7.9 nM) and Des-Arg¹⁰-kallidin (EC₅₀=8.6 nM), while the most potent agonist for CHO cells expressing human bradykinin B₂ receptors was bradykinin (EC₅₀=2.0 nM). These findings confirm the validity of the recombinant system and the microtitre plate imaging-based characterization system when compared to known agonist properties of the native receptors. The concentration–response relationship for bradykinin at bradykinin B₂ receptors was potently inhibited by [d-Arg⁰,Hyp³,β-(2-thienyl)-Ala⁵,d-Tic⁷,Oic⁸]-bradykinin (Hoe140) (IC₅₀=71 nM), which was 500-fold more potent against the B₂-expressing cells than the B₁ cells. Bradykinin B₁ receptor-mediated responses activated by Des-Arg¹⁰-kallidin were fully antagonized by Des-Arg⁹-[Leu⁸]bradykinin (IC₅₀=59 nM), Des-Arg¹⁰-Hoe140 (IC₅₀=211 nM) and most potently by Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-d-Igl-Oic (B9858) (IC₅₀=14 nM), none of which displayed any activity against the bradykinin B₂ receptor cell line up to 3 μM. None of the antagonists displayed partial agonism activity in these cell lines. All bradykinin B₁ and B₂ receptor antagonists tested acted in an apparently non-competitive manner that is likely to be due in part to their kinetics and to the nature of the functional assay used.

Introduction

Kinin receptors have been classified into two distinct subtypes, bradykinin receptors B₁ and B₂, initially on the basis of distinct induction and agonist activation profiles (Regoli and Barabe, 1980). Subsequently separate gene products for B₁ and B₂-mediated responses were identified and cloned from a number of tissues and species, and subtype-specific antagonists have been developed Hess et al., 1992, Wirth et al., 1992, Menke et al., 1994, Marceau et al., 1998. Both have been suggested to play important roles in the inflammatory response to tissue injury, with the constitutively expressed bradykinin B₂ receptor likely to be important in the early stages of the response, and the inducible bradykinin B₁ receptor thought to be involved in the chronic period of inflammation and pain transduction Dray and Perkins, 1993, Marceau et al., 1998. The involvement of kinin receptors in inflammation and pain transduction has been supported by the results of studies on mice lacking bradykinin B₂ receptor expression Borkowski et al., 1995, Rupniak et al., 1997, Seabrook et al., 1997.

The nature of the bradykinin receptor modulation evoked by some kinins is not clearly established from literature reports. Distinct differences in agonist and antagonist potency and selectivity have been reported for the native kinin receptors found in different species and tissues Wirth et al., 1992, Hall, 1997, MacNeil et al., 1997, Wohlfart et al., 1997. Also, compounds with subtype selectivity in one tissue have been reported to affect both subtypes in other preparations (Wirth et al., 1992). However, to date, no genetic evidence has been reported for subtypes of either the B₁ or B₂ bradykinin receptor. It is currently unclear whether these discrepancies are due to differences in assay format, equilibration period, or receptor reserve, such that compounds with low intrinsic activity may be detected as antagonists if the physiological response requires stimulation of a large proportion of the expressed receptors (Kenakin, 1993).

The study of antagonism using functional efficacy measurement is preferable to radioligand binding for understanding the nature and consequences of the ligand–receptor interaction. As both human subtypes have been cloned, it is possible to study receptor modulation in cell lines stably expressing one or other subtype. Luminescence studies in an aequorin-human embryonic kidney cell line (aequorin-HEK-293 cells) transiently transfected with bradykinin B₁ receptors have shown that Ca²⁺-based screening is a viable approach to bradykinin receptor ligand characterization (MacNeil et al., 1997). We have, therefore, developed a Ca²⁺-based high throughput screen for bradykinin receptor activity using Chinese hamster ovary (CHO) cells expressing recombinant human kinin receptors in a 96-well real-time fluorometric imaging plate reader (FLIPR). In the present study, we have reexamined the functional effects of a range of known kinin receptor ligands for both agonist and antagonist activity.