CHARACTERIZATION OF THE INTERACTION BETWEEN MUSCARINIC M(2) RECEPTORS AND BETA-ADRENOCEPTOR SUBTYPES IN GUINEA-PIG ISOLATED ILEUM

1 Contraction of guinea-pig ileum to muscarinic agonists is mediated by M(3) receptors, even though they account for only 30% of the total muscarinic receptor population. The aim of this study was to characterize the biochemical and functional effects of stimulation of the predominant M(2) muscarinic receptor (70%) and to investigate the hypothesis that M(2) receptors specifically oppose beta-adrenoceptor-mediated effects in the ileum. 2 In guinea-pig ileal longitudinal smooth muscle slices, isoprenaline, a non-selective beta-adrenoceptor agonist, and BRL 37344 (sodium-4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl]-phenoxyacetate sesquihydrate), a beta(3)-adrenoceptor selective agonist, increased cyclic AMP accumulation with -log EC(50) values of 6.6 +/- 0.1 and 5.8 +/- 0.1 respectively. Maximal stimulation by BRL 37344 (10 mu M) was 26.4 +/- 5.2% of that observed with isoprenaline (10 mu M). Isoprenaline (10 mu M)-stimulated cyclic AMP accumulation was significantly, but not completely, inhibited by propranolol (5 mu M), With a propranolol-resistant component of 28.2 +/- 6.8% of the maximal stimulation to isoprenaline. In contrast, basal and BRL 37344 responses were resistant to this antagonist. These data provide evidence that both beta(1)- and beta(3)-adrenoceptors activate adenylyl cyclase in guinea-pig ileum. 3 Isoprenaline (10 mu M)-stimulated cyclic AMP accumulation was inhibited (67.4 +/- 0.9%) by the muscarinic agonist (+)-cis-dioxolane (-log EC(50) = 7.3 +/- 0.1). The rank order of antagonist affinities against the (+)-cis-dioxolane response was (-log K-B values in parentheses): atropine (9.0 +/- 0.2) > methoctramine (7.1 +/- 0.1) >p-fluoro-hexa-hydrosilaphenidol (p-F-HHSiD; 6.5 +/- 0.2) greater than or equal to pirenzepine (6.3 +/- 0.2). (+)-cis-dioxolane also significantly inhibited BRL 37344 (10 mu M; 56.5 +/- 2.4%) stimulated cyclic AMP accumulation. These data suggest that M(2) receptors mediate inhibition of cyclic AMP accumulation in response to both beta(1)- and beta(3)-adrenoceptor stimulation in guinea-pig ileum. 4 5-Hydroxytryptamine (5-HT), vasoactive intestinal peptide, prostaglandins E(2) and E(1), all at 10 mu M, significantly increased cyclic AMP accumulation. (+)-cis-Dioxolane (10 mu M) inhibited both basal and agonist-induced cyclic AMP accumulation. Thus the inhibitory effect of M(2) receptor agonism does not appear to be restricted to beta-adrenoceptor-stimulated cyclic AMP accumulation. 5 The potential for involvement of activation of M(2) receptors on responses to beta-adrenoceptor agonists was also studied functionally. Selective M(3) receptor alkylation was achieved by pretreatment of tissues with 4-DAMP mustard (40 nM), in the presence of methoctramine (1 mu M; to protect M(2) receptors). After washing, tissues were pre-contracted with histamine (0.3 mu M) and relaxed with isoprenaline (0.6 mu M). Under these conditions, oxotremorine M caused concentration-dependent contractions (-log EC(50) of 7.8 +/- 0.1), that were surmountably antagonized by methoctramine (1 mu M) With a -log K-B estimate of 7.4 +/- 0.1. Similar observations were seen versus relaxation produced by BRL 37344 (1 mu M), where the -log K-B value for methoctramine was 7.8 +/- 0.2. These data suggest that M(2) receptors mediate a functional inhibition of relaxant responses to isoprenaline and BRL 37344. 6 These findings are consistent with beta(1)- and beta(3)-adrenoceptors coupling to stimulation of adenylyl cyclase in guinea-pig ileum; a response that is inhibited by M(2) receptor stimulation. Concordantly, Mt receptor stimulation also inhibits relaxation to both beta(1),- and beta(3),-adrenoceptor stimulation. These results implicate M(2) receptors in the modulation of sympathetic control of ileal motility.