Characterization of receptors mediating contraction induced by tachykinins in the guinea-pig isolated common bile duct

Abstract

1. We studied the effect of the natural tachykinins and of synthetic agonists selective for the tachykinin NK₁, NK₂ and NK₃ receptors, on the motility of guinea-pig isolated common bile duct longitudinally-oriented smooth muscle.

2. All the tachykinins tested (both natural and synthetic) produced a concentration-dependent contractile response of the guinea-pig isolated common bile duct: these effects underwent a marked tachyphylaxis, especially the responses elicited by NK₁ and NK₃ receptor-selective agonists.

3. Among the natural tachykinins neurokinin B (EC₅₀=3.2 nM; 95% c.l.=2.0–5.1; n=4) was the most potent, being about 40 and 25 fold more potent than substance P (EC₅₀=121.6 nM; 95% c.l.=94–157; P<0.01; n=4) and neurokinin A (EC₅₀=83.4 nM; 95% c.l.=62–112; P<0.01; n=4), respectively. Among the synthetic analogues the NK₃ receptor-selective agonist senktide (EC₅₀=1.1 nM; 95% c.l.=0.7–1.8; n=8) was the most potent, being about 120, 110 and 20 fold more potent than [Sar⁹]substance P sulfone (NK₁ receptor-selective) (EC₅₀=130.4 nM; 95% c.l.=99–172; P<0.01; n=8), [βAla⁸]NKA (4–10) (NK₂ receptor-selective) (EC₅₀=120.1 nM; 95% c.l.=95–151; P<0.01; n=8) and septide (NK₁ receptor-selective) (EC₅₀=22.6 nM; 95% c.l.=18–28; P<0.01; n=8), respectively. All tachykinins (natural or synthetic receptor agonists) produced a similar E_max, averaging about 50% of that produced by KCl (80 mM).

4. Atropine (1 μM) did not affect the responses to either NK₁ or NK₂ receptor-selective agonists, whereas it reduced the E_max of senktide by about 50%, without affecting its potency (EC₅₀). Tetrodotoxin (1 μM) totally blocked senktide-induced contractions, as did the combined pretreatment with atropine plus the tachykinin NK₁ and NK₂ receptor-selective antagonists GR 82334 and MEN 11420 (1 μM each), respectively.

5. GR 82334 (1 μM) blocked with apparent competitive kinetics septide- (apparent pK_B=7.46±0.10; n=5) and [Sar⁹]substance P sulfone- (apparent pK_B=6.80±0.04; n=4) induced contractions. MEN 11420 (30–300 nM), a novel potent NK₂ receptor antagonist, potently antagonized [βAla⁸]NKA (4–10), with competitive kinetics (pK_B=8.25±0.08; n=12: Schild plot slope=−0.90; 95% c.l.=−1.4; −0.35). The NK₃ receptor-selective antagonist SR 142801 (30 nM) produced insurmountable antagonism of the senktide-induced contractions (E_max inhibited by 64%). None of the above antagonists, tested at the highest concentrations employed against tachykinins, affected the concentration–response curve to methacholine (0.1–300 μM).

6. We conclude that tachykinins produce contraction of the guinea-pig isolated common bile duct by stimulating NK₁, NK₂ and NK₃ receptors. The responses obtained by activating NK₁ and NK₂ receptors are atropine-resistant. The contraction obtained by stimulating NK₃ receptors is totally neurogenic, being mediated by the release of endogenous acetylcholine and tachykinins; the latter act, in turn, on postjunctional tachykinin NK₁/NK₂ receptors. The role of the NK₃ receptor as prejunctional mediator of the excitatory transmission operated by tachykinins is discussed.

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