FUNCTIONAL INTERACTIONS BETWEEN MUSCARINIC M(2) RECEPTORS AND 5-HYDROXYTRYPTAMINE (5-HT)(4) RECEPTORS AND BETA(3)-ADRENOCEPTORS IN ISOLATEDESOPHAGEAL MUSCULARIS MUCOSAE OF THE RAT

1 Relaxations of isolated oesophageal muscularis mucosae of rat are me diated by 5-hydroxytryptamine (5-HT), acting at 5-HT4 receptors, and i soprenaline, principally acting via beta(3)-adrenoceptors. The aim of this study was to investigate the hypothesis that muscarinic M(2) rece ptors, also present in this tissue, functionally oppose 5-HT and beta- adrenoceptor-relaxant effects in this preparation. 2 Contractions of r at oesophageal muscularis mucosae were induced, in a concentration-dep endent 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-meth yl piperidine methiodide, 8.7 +/- 0.1), p-F-HHSiD (para-fluoro-hexa-hy dro-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 shif ted to the right and virtually abolished by the alkylating agent, 4-DA MP 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 isop renaline 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 tissu es that were precontracted with 1 mu M (EC(90) oxotremorine M, was ide ntical. When these experiments were repeated in the presence of the mu scarinic M(2) receptor antagonist, methoctramine (1 mu M), there was n o effect on the relaxant potencies to either 5-HT or isoprenaline. Col lectively, these data suggest that muscarinic M(2) receptors do not, u nder these conditions, modulate relaxant potencies to either 5-HT or i soprenaline. 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 isoprenal ine (0.1 mu M). In these tissues (in which the muscarinic M(3) recepto r population was extensively depleted by alkylation), oxotremorine M c aused concentration-dependent re-contractions (i.e. reversal of relaxa tions). 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 isopre naline (1 mu M; PKB = 7.5 +/- 0.2). Under these conditions, therefore, the pK(B) values are consistent with activation of muscarinic M(2) re ceptors, and inconsistent with activation of M(3) receptors. 5 It is c oncluded that in isolated oesophageal muscularis mucosae of rat, musca rinic M(3) receptors mediate direct contractions and are associated wi th a low receptor reserve. When this population is depleted, and the t issues relaxed via activation of receptors that augment adenylyl cycla se activity, a functional role for muscarinic M(2) receptors is reveal ed.