Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells

1. A direct [³H]-bradykinin ([³H]-BK) binding assay has been used to characterize the BK receptors in canine cultured tracheal epithelial cells (TECs). Based on receptor binding assay, TECs have specific, saturable, high-affinity binding sites for [³H]-BK. 2. The specific [³H]-BK binding was time- and temperature-dependent. Equilibrium of association of [³H]-BK with the BK receptors was attained within 30 min at room temperature and 1 h at 4°C, respectively. 3. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (K(D)) of 1.5 ± 0.2 nM and a maximum receptor density (B(max)) of 53.2 ± 5.2 fmol mg^-1 protein. The Hill coefficient for [³H]-BK binding was 1.00 ± 0.02. The association (K₁) and dissociation (K_-1) rate constants were (7.6 ± 1.1) x 10⁶ M^-1 min^-1 and (9.2 ± 1.5) x 10 M^-3 min^-1, respectively. K(D), calculated from the ratio of K_-1 and K₁, was 1.2 ± 0.3 nM, a value close to that calculated from Scatchard plots of binding isotherms. 4. Neither a B₁ receptor selective agonist (des-Arg⁹-BK, 0.1 nM-10 μM) nor antagonist ([Leu⁸, des-Arg⁹]-BK, 0.1 nM-10 μM) significantly inhibited [³H]-BK binding to TECs, which excludes the presence of B₁ receptors in canine TECs. 5. The specific binding of [³H]-BK to canine TECs was inhibited by the B₂ receptor selective antagonists ([D-Arg(O), Hyp³, Thi⁵, D-Tic⁷, Oic⁸]-BK (Hoe 140, 0.1 nM-10 μM) and [D-Arg(O), Hyp³, Thi^5,8, D-Phe⁷]-BK, 0.1 nM-10 μM) and agonists (BK and kallidin, 0.1 nM-10 μM) with a best fit by a one-binding site model. The order of potency for the inhibition of [³H]-BK binding was kallidin= BK = Hoe 140 > [DArg(O), Hyp³, Thi^5,8, D-Phe7(])-BK. 6. BK and kallidin significantly induced concentration-dependent accumulation of IPs with a half-maximal response (EC₅₀) at 17.6 ± 3.5 and 26.6 ± 5.3 nM, respectively, while the B₁-selective agonist, des-Arg⁹-BK did not stimulate IPs accumulation and the B₁-selective antagonist [Leu⁸, des-Arg⁹]-BK did not inhibit BK-induced IPs accumulation. Two B₂-selective antagonists, Hoe 140 and [D-Arg(O), Hyp³, Thi^5,8 D-Phe⁷]-BK, inhibited BK-stimulated IPs accumulation with apparent pK(B) values of 8.8 ± 0.3 and 7.0 ± 0.3, respectively. 7. It is concluded that the pharmacological characteristics of the BK receptors in canine cultured TECs are primarily of the B₂ receptor subtype which might regulate the function of tracheal epithelium through the activation of this receptor subtype coupling to PI hydrolysis.