Differential modulation of endothelin ligand-induced contraction in isolated tracheae from endothelin B (ET(B)) receptor knockout mice

Abstract

The role of endothelin B (ET(B)) receptors in mediating ET ligand-induced contractions in mouse trachea was examined in ET(B) receptor knockout animals. Autoradiographic binding studies, using [(125)I]-ET-1, confirmed the presence of ET(A) receptors in tracheal and bronchial airway smooth muscle from wild-type (+/+) and homozygous recessive (-/-) ET(B) receptor knockout mice. In contrast, ET(B) receptors were not detected in airway tissues from (-/-) mice. In tracheae from (+/+) mice, the rank order of potencies of the ET ligands was sarafotoxin (Stx) S6c>ET-1>ET-3; Stx S6c had a lower efficacy than ET-1 or ET-3. In tissues from (-/-) mice there was no response to Stx S6c (up to 0.1 microM), whereas the maximum responses and potencies of ET-1 and ET-3 were similar to those in (+/+) tracheae. ET-3 concentration-response curve was biphasic in (+/+) tissues (via ET(A) and ET(B) receptor activation), and monophasic in (-/-) preparations (via stimulation of only ET(A) receptors). In (+/+) preparations SB 234551 (1 nM), an ET(A) receptor-selective antagonist, inhibited the secondary phase, but not the first phase, of the ET-3 concentration-response curve, whereas A192621 (100 nM), an ET(B) receptor-selective antagonist, had the opposite effect. In (-/-) tissues SB 234551 (1 nM), but not A192621 (100 nM), produced a rightward shift in ET-3 concentration-response curves. The results confirm the significant influence of both ET(A) and ET(B) receptors in mediating ET-1-induced contractions in mouse trachea. Furthermore, the data do not support the hypothesis of atypical ET(B) receptors. In this preparation ET-3 is not an ET(B) receptor-selective ligand, producing contractions via activation of both ET(A) and ET(B) receptors.

Figure 1

Montage of darkfield (b – e) autoradiograms showing the distribution of total binding of [¹²⁵I]-ET-1 in murine tracheal smooth muscle from (+/+) (wildtype, panel b; panel a=brightfield image), (+/−) (panel d) and (−/−) (panel e) ET_B receptor knockout mice. Non-specific binding, determined in the combined presence of BQ-123 (1 μM) and Stx S6c (100 nM), is shown in panel c. Bar=100 μm.

Figure 2

Montage of darkfield (b – h) autoradiograms showing the distribution total binding of [¹²⁵I]-ET-1 in murine bronchial smooth muscle from (+/+) (wildtype, panel c; panel a=brightfield image) and (−/−) (homozygous ET_B receptor knockout; panel d) mice. Nonspecific binding, determined in the combined presence of the ET_A receptor-selective ligand BQ-123 (1 μM) and the ET_B receptor-selective ligand Stx S6c (100 nM), is shown in panel b. The impact of Stx S6c (100 nM) on binding is shown in panels e (+/+) and f (−/−) and that of BQ-123 (1 μM) is shown in panels g (+/+) and h (−/−). Bar=100 μm.

Figure 3

Histogram showing quantitative autoradiographic data for the binding of [¹²⁵I]-ET-1 (% total binding) in mouse tracheal airway smooth muscle from wild-type (+/+; filled columns), heterozygous ET_B receptor knockout (+/−; unfilled columns) and homozygous ET_B receptor knockout mice (−/−; hatched columns). Data are presented as total binding, and binding detected in the presence of the ET_A receptor-selective ligand BQ-123 (1 μM), or the ET_B receptor-selective ligand Stx S6c (100 nM) or in the combined presence of BQ-123 (1 μM) and Stx S6c (100 nM) (nonspecific binding).

Figure 4

ET-1, Stx S6c or ET-3 concentration-response curves in trachea from (a) wild-type (+/+); (b) heterozygous (+/−) and (c) homozygous (−/−) ET_B receptor knockout mice. The results are presented as a percentage of the contraction to 10 μM carbachol and are the mean±s.e.mean; (a) n=6 – 12; (b) n=8 – 9; (c) n=4 – 12.

Figure 5

Effects of SB 234551 (ET_A receptor-selective antagonist; 100 nM) or A192621 (ET_B receptor-selective antagonist; 100 nM), alone and in combination, against ET-1 concentration-response curves in trachea from (a – c) wild-type (+/+) or (d – f) homozygous (−/−) ET_B receptor knockout mice. The results are presented as a percentage of the contraction to 10 μM carbachol and are the mean±s.e.mean; (a) n=6, (b) n=3, (c) n=3, (d) n=3, (e) n=3, (f) n=3.

Figure 6

Effects of (a) SB 234551 (ET_A receptor-selective antagonist; 100 nM) or (b) A192621 (ET_B receptor-selective antagonist; 100 nM) against Stx S6c concentration-response curves in trachea from wild-type (+/+) mice. The results are presented as a percentage of the contraction to 10 μM carbachol and are the mean±s.e.mean; (a) n=4, (b) n=4. Note, no response to Stx S6c (up to 0.1 μM) is observed in homozygous (−/−) ET_B receptor knockout mice.

Figure 7

Effects of (a, c) SB 234551 (ET_A receptor-selective antagonist; 1 nM) or (b, d) A192621 (ET_B receptor-selective antagonist; 100 nM) against ET-3 concentration-response curves in trachea from (a, b) wild-type (+/+) or (c, d) homozygous (−/−) ET_B receptor knockout mice. The results are presented as a percentage of the contraction to 10 μM carbachol and are the mean±s.e.mean; (a) n=4, (b) n=3, (c) n=7, (d) n=5. Note, there was evidence of a biphasic ET-3 concentration-response curve; the dashed lines represent the theoretical extension of the second component of the control curve.