Functional interactions between muscarinic M(2) receptors and 5-hydroxytryptamine (5-HT)(4) receptors and beta(3)-adrenoceptors in isolated oesophageal muscularis mucosae of the rat

1 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 beta(3)-adrenoceptors. The aim of this study was to investigate the hypothesis that muscarinic M(2) receptors, also present in this tissue, functionally oppose 5-HT and beta-adrenoceptor-relaxant effects in this preparation. 2 Contractions of rat oesophageal muscularis mucosae were induced, in a concentration-dependent manner, by the muscarinic receptor agonist, oxotremorine M (pEC(50) = 6.7 +/- 0.1). The contractile responses to oxotremorine M were surmountably antagonized by the following compounds, (pK(B) 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 Mg 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). 3 In tissues precontracted with U46619 (0.7 mu M; approx. EC(90)), isoprenaline (pEC(50) = 8.0 +/- 0.1) and 5-HT (pEC(50) = 7.5 +/- 0.2) induced concentration-dependent relaxations. The isoprenaline potency was slightly, but significantly, different in tissues precontracted with oxotremorine M (isoprenaline, PEC(50) = 7.4 +/- 0.2). In contrast, the potency of 5-HT (PEC(50) = 7.5 +/- 0.2), in tissues that were precontracted with 1 mu M (EC(90) oxotremorine M, was identical. When these experiments were repeated in the presence of the muscarinic M(2) receptor antagonist, methoctramine (1 mu M), there was no effect on the relaxant potencies to either 5-HT or isoprenaline. Collectively, these data suggest that muscarinic M(2) receptors do not, under these conditions, modulate relaxant potencies to either 5-HT or isoprenaline. 4 In a second protocol, tissues were pre-contracted with U46619 (0.7 mu M) and relaxed with either 5-HT (0.1 mu M) or isoprenaline (0.1 mu M). In these tissues (in which the muscarinic M(3) 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 (pEC(50)) = 5.6 +/- 0.3. These re-contractions were antagonized, in a surmountable fashion, by methoctramine (1 mu M; pK(B) = 7.6 +/- 0.1). Similar observations were seen when relaxations were induced by isoprenaline (1 mu M; PKB = 7.5 +/- 0.2). Under these conditions, therefore, the pK(B) values are consistent with activation of muscarinic M(2) receptors, and inconsistent with activation of M(3) receptors. 5 It is concluded that in isolated oesophageal muscularis mucosae of rat, muscarinic M(3) 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 M(2) receptors is revealed.