Functional interactions between muscarinic M2 receptors and 5‐hydroxytryptamine (5‐HT)4 receptors and β‐adrenoceptors in isolated oesophageal muscularis mucosae of the rat

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Abstract
Relaxations of isolated oesophageal muscularis mucosae of rat are mediated by 5‐hydroxytryptamine (5‐HT), acting at 5‐HT4 receptors, and isoprenaline, principally acting via β3‐adrenoceptors. The aim of this study was to investigate the hypothesis that muscarinic M2 receptors, also present in this tissue, functionally oppose 5‐HT and β‐adrenoceptor‐relaxant effects in this preparation. Contractions of rat oesophageal muscularis mucosae were induced, in a concentration‐dependent manner, by the muscarinic receptor agonist, oxotremorine m (pEC50 = 6.7 ± 0.1). The contractile responses to oxotremorine m were surmountably antagonized by the following compounds, (pKB values in parentheses): atropine (9.1 ± 0.2), 4‐DAMP (4‐diphenylacetoxy‐N‐methyl piperidine methiodide, 8.7 ± 0.1), p‐F‐HHSiD (para‐fluoro‐hexa‐hydro‐siladifenidol, 7.5 ± 0.1), zamifenacin (8.6 ± 0.3), himbacine (7.2 ± 0.2), pirenzepine (6.8 ± 0.3) and methoctramine (6.2 ± 0.2). These data are consistent with a role for muscarinic M3 receptors mediating contractions to oxotremorine M. The contractile response was associated with a low receptor reserve, since the responses were shifted to the right and virtually abolished by the alkylating agent, 4‐DAMP mustard (4‐diphenylacetoxy‐N‐(2‐chloroethyl) piperidine, 40 nM; 60 min equilibration). In tissues precontracted with U46619 (0.7 μm; approx. EC90), isoprenaline (pEC50 = 8.0 ± 0.1) and 5‐HT (pEC50 = 7.5 ± 0.2) induced concentration‐dependent relaxations. The isoprenaline potency was slightly, but significantly, different in tissues precontracted with oxotremorine m (isoprenaline, pEC50 = 7.4 ± 0.2). In contrast, the potency of 5‐HT (pEC50 = 7.5 ± 0.2), in tissues that were precontracted with 1 μm (EC90) oxotremorine M, was identical. When these experiments were repeated in the presence of the muscarinic M2 receptor antagonist, methoctramine (1 μ), there was no effect on the relaxant potencies to either 5‐HT or isoprenaline. Collectively, these data suggest that muscarinic M2 receptors do not, under these conditions, modulate relaxant potencies to either 5‐HT or isoprenaline. In a second protocol, tissues were pre‐contracted with U46619 (0.7 μm) and relaxed with either 5‐HT (0.1 μm) or isoprenaline (0.1 μm). In these tissues (in which the muscarinic M3 receptor population was extensively depleted by alkylation), oxotremorine m caused concentration‐dependent re‐contractions (i.e. reversal of relaxations). In tissues relaxed with 5‐HT, the potency of oxtremorine m was 5.9 ± 0.2, while in tissues relaxed with isoprenaline, the potency (pEC50) = 5.6 ± 0.3. These re‐contractions were antagonized, in a surmountable fashion, by methoctramine (1 μm; pKB = 7.6 ± 0.1). Similar observations were seen when relaxations were induced by isoprenaline (1 μm; pKB = 7.5 ± 0.2). Under these conditions, therefore, the pKB values are consistent with activation of muscarinic M2 receptors, and inconsistent with activation of M3 receptors. It is concluded that in isolated oesophageal muscularis mucosae of rat, muscarinic M3 receptors mediate direct contractions and are associated with a low receptor reserve. When this population is depleted, and the tissues relaxed via activation of receptors that augment adenylyl cyclase activity, a functional role for muscarinic M2 receptors is revealed.