PHARMACOLOGICAL AND FUNCTIONAL-CHARACTERIZATION OF BRADYKININ RECEPTORS IN CANINE CULTURED TRACHEAL EPITHELIAL-CELLS

1 A direct [H-3]-bradykinin ([H-3]-BK) binding assay has been used to characterize the BK receptors in canine cultured tracheal epithelial c ells (TECs). Based on receptor binding assay, TECs have specific, satu rable, high-affinity binding sites for [H-3]-BK. 2 The specific [H-3]- BK binding was time- and temperature-dependent. Equilibrium of associa tion of [H-3]-BK with the BK receptors was attained within 30 min at r oom temperature and 1 h at 4 degrees C, respectively. 3 Analysis of bi nding 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 [H-3]-BK bindin g was 1.00 +/- 0.02. The association (K-1) and dissociation (K--1) rat e constants were (7.6 +/- 1.1) x 10(6) 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 K-1, was 1.2 +/- 0.3 nM, a value close to that calculated from S catchard plots of binding isotherms. 4 Neither a B-1 receptor selectiv e agonist (des-Arg(9)-BK, 0.1 nM-10 mu M) nor antagonist ([Leu(8), des -Arg(9)]-BK, 0.1 nM-10 mu M) significantly inhibited [H-3]-BK binding to TECs, which excludes the presence of B-1 receptors in canine TECs. 5 The specific binding of [H-3]-BK to canine TECs was inhibited by the B-2 receptor selective antagonists ([D-Arg(0), Hyp(3), Thi(5), D-Tic( 7), Oic(8)]-BK (Hoe 140, 0.1 nM-10 mu M) and [D-Arg(0), Hyp(3), Thi(5, 8), D-Phe(7)]-BK, 0.1 nM-10 mu M) and agonists (BK and kallidin, 0.1 n M-10 mu M) with a best fit by a one-binding site model. The order of p otency for the inhibition of [H-3]-BK binding was kallidin=BK=Hoe 140> [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK. 6 BK and kallidin significa ntly induced concentration-dependent accumulation of IPs with a half-m aximal response (EC(50)) at 17.6 +/- 3.5 and 26.6 +/- 5.3 nM, respecti vely, while the B-1-selective agonist, des-Arg(9)-BK did not stimulate IPs accumulation and the B-1-selective antagonist [Leu(8), des-Arg(9) ]-BK did not inhibit BK-induced IPs accumulation. Two B-2-selective an tagonists, Hoe 140 and [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK, inhi bited 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 pha rmacological characteristics of the BK receptors in canine cultured TE Cs are primarily of the B-2 receptor subtype which might regulate the function of tracheal epithelium through the activation of this recepto r subtype coupling to PI hydrolysis.