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

Shue Fen Luo, Chuen Tao Tsai, Wen Bin Wu, Shiow Lin Pan, Yih Jeng Tsai, Chuen Mao Yang

Research output: Contribution to journalArticle

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Abstract

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, Thi5,8, 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, Thi5,8, 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, Thi5,8 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.

Original languageEnglish
Pages (from-to)439-445
Number of pages7
JournalBritish Journal of Pharmacology
Volume119
Issue number2
Publication statusPublished - 1996
Externally publishedYes

Fingerprint

Bradykinin Receptors
Canidae
Kallidin
Epithelial Cells
Pharmacology
Binding Sites
Temperature
Bradykinin
Hydrolysis
Epithelium

Keywords

  • Bradykinin receptor
  • Canine tracheal epithelial cells
  • Inositol phosphates
  • Kinins

ASJC Scopus subject areas

  • Pharmacology

Cite this

Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells. / Luo, Shue Fen; Tsai, Chuen Tao; Wu, Wen Bin; Pan, Shiow Lin; Tsai, Yih Jeng; Yang, Chuen Mao.

In: British Journal of Pharmacology, Vol. 119, No. 2, 1996, p. 439-445.

Research output: Contribution to journalArticle

Luo, Shue Fen ; Tsai, Chuen Tao ; Wu, Wen Bin ; Pan, Shiow Lin ; Tsai, Yih Jeng ; Yang, Chuen Mao. / Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells. In: British Journal of Pharmacology. 1996 ; Vol. 119, No. 2. pp. 439-445.
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abstract = "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, Thi5,8, 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, Thi5,8, 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, Thi5,8 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.",
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T1 - Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells

AU - Luo, Shue Fen

AU - Tsai, Chuen Tao

AU - Wu, Wen Bin

AU - Pan, Shiow Lin

AU - Tsai, Yih Jeng

AU - Yang, Chuen Mao

PY - 1996

Y1 - 1996

N2 - 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, Thi5,8, 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, Thi5,8, 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, Thi5,8 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.

AB - 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, Thi5,8, 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, Thi5,8, 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, Thi5,8 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.

KW - Bradykinin receptor

KW - Canine tracheal epithelial cells

KW - Inositol phosphates

KW - Kinins

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