Characterization of 5‐HT3 and ‘atypical’ 5‐HT receptors mediating guinea‐pig ileal contractions in vitro

Abstract

1 Neuronal 5-hydroxytryptamine (5-HT) receptors mediating contraction of guinea-pig ileal segments have been characterized in vitro by the use of methysergide to block 5-HT₁-like and 5-HT₂ receptors. Concentration-response curves to 5-HT were biphasic (first phase, defined as those responses occurring between 1 nm and 0.32 μm 5-HT, –log EC₅₀ = 7.15 ± 0.08; second phase, defined as these responses occurring between 0.32 μm and 32 μm 5-HT, –log EC₅₀ = 5.32 ± 0.03) but monophasic to 5-methoxytryptamine (−log EC₅₀ = 7.0 ± 0.08) and 2 methyl 5-HT (−log EC₅₀ = 5.2 ± 0.13). The maximal response of the first phase to 5-HT and the maximal response to 5-methoxytryptamine were 30 ± 4% and 35 ± 5% respectively of the maximum response to the second phase of the 5-HT concentration-effect curve (set at 100%). In contrast, the maximal response to 2-methyl-5-HT equalled that obtained with 5-HT (second phase). 2 The responses comprising the second phase of the concentration-effect curve to 5-HT were antagonized by 1 μm ICS 205–930, ondansetron, granisetron, quipazine, N-methyl-quipazine and (R,S)-zacopride and the following pK_B values, with 5-HT as the agonist, were obtained at the 5-HT₃ receptor: ICS 205–930 7.61 ± 0.05, ondansetron 6.90 ± 0.04, granisetron 7.90 ± 0.04, (S)-zacopride 8.11 ± 0.06, (R,S)-zacopride 7.64 ± 0.11, and (R)-zacopride 7.27 ± 0.06. 3 Under conditions of 5-HT₁-like, 5-HT₂ and 5-HT₃ receptor blockade, the following rank order of agonism was observed: 5-HT > 5-methoxytryptamine = renzapride > (S)-zacopride > (R,S)-zacopride > 5-carboxamidotryptamine > BRL 24682 > (R)-zacopride > metoclopramide > 2-methyl-5-HT ≫ sulpiride. 8-Dihydroxydiphenylaminotetralin (8-OHDPAT), GR 43175, N,N-dipropyl-5-carboxamidotryptamine, ondansetron, ICS 205–930, granisetron, quipazine and N-methyl-quipazine were inactive as agonists and antagonists. Relative to 5-HT, (R,S)-zacopride acted as a partial agonist (intrinsic activity, α = 0.80; –log EC₅₀ = 6.3 ± 0.12; –log K_A = 6.1 ± 0.03) as did (R)-zacopride (α = 0.4, –log EC₅₀ 5.7 ± 0.08, –log K_A = 5.5 ± 0.11). (S)-zacopride acted as a full agonist (−log EC₅₀ = 6.9 ± 0.03). ICS 205–930 (3 μm) antagonized competitively responses to 5-HT, 5 methoxytryptamine, (R,S)- and (S)-zacopride and 5-carboxamidotryptamine yielding –log K_B estimates ranging from 6.1–6.5. 4 It is concluded that two different 5-HT receptors mediate excitatory neuronal responses in the guinea-pig ileum. 5-HT₃ receptors mediate the second phase of the biphasic concentration-response curve, whereas a receptor with properties distinct from the 5-HT₁-like, 5-HT₂ and 5-HT₃ subtypes mediates the initial phase of the concentration-response curve. This receptor, which exhibits a close similarity to the 5-HT₄ subtype is: (1) stimulated by 5-methoxytryptamine but not 2-methyl-5-HT; (2) stimulated selectively by certain substituted benzamides; (3) recognizes the optical isomers of zacopride and (4) is blocked by relatively high concentrations ICS 205–930 (pK_B = 6.0–6.5) but not ondansetron, granisetron, quipazine or N-methyl-quipazine.