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Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells
Author(s) -
Luo ShueFen,
Tsai ChuenTao,
Wu WenBin,
Pan ShiowLin,
Tsai YihJeng,
Yang ChuenMao
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb16005.x
Subject(s) - bradykinin , receptor , dissociation constant , kallidin , chemistry , agonist , binding site , antagonist , biophysics , stereochemistry , microbiology and biotechnology , biochemistry , biology , kinin
1 A direct [ 3 H]‐bradykinin ([ 3 H]‐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 [ 3 H]‐BK. 2 The specific [ 3 H]‐BK binding was time‐ and temperature‐dependent. Equilibrium of association of [ 3 H]‐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 [ 3 H]‐BK binding was 1.00 ± 0.02. The association ( K 1 and dissociation ( K ‐1 ) rate constants were (7.6 ± 1.1) × 10 6 M −1 min −1 and (9.2 ± 1.5) × 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 Scatchard plots of binding isotherms. 4 Neither a B 1 receptor selective agonist (des‐Arg 9 ‐BK, 0.1 nM‐10 μ m ) nor antagonist ([Leu 8 , des‐Arg 9 ]‐BK, 0.1 nM‐10 μ m ) significantly inhibited [ 3 H]‐BK binding to TECs, which excludes the presence of B 1 receptors in canine TECs. 5 The specific binding of [ 3 H]‐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 μ m ) and [D‐Arg 0 , Hyp 3 , Thi 5,8 , D‐Phe 7 ]‐BK, 0.1 nM‐10 μ m ) and agonists (BK and kallidin, 0.1 nM‐10 μ m ) with a best fit by a one‐binding site model. The order of potency for the inhibition of [ 3 H]‐BK binding was kallidin = BK = Hoe 140 > [D‐Arg 0 , Hyp 3 , Thi 5,8 , D‐Phe 7 ]‐BK. 6 BK and kallidin significantly induced concentration‐dependent accumulation of IPs with a half‐maximal response (EC 50 ) at 17.6 ± 3.5 and 26.6 ± 5.3 nM, respectively, 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 antagonists, Hoe 140 and [D‐Arg 0 , Hyp 3 , Thi 5,8 , D‐Phe 7 ]‐BK, inhibited BK‐stimulated IPs accumulation with apparent p K 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 B 2 receptor subtype which might regulate the function of tracheal epithelium through the activation of this receptor subtype coupling to PI hydrolysis.