Selective inactivation of muscarinic M₂ and M₃ receptors in guinea‐pig ileum and atria in vitro

Abstract

1 The role of muscarinic M2 and M3 receptors in ileal smooth muscle has been evaluated by use of selective receptor alkylation. The alkylating agents, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine (4-DAMP mustard) was studied for effects against (+ )-cis-dioxolane, at muscarinic M2 and M3 receptors in guinea-pig atria or ileum, respectively. 4-DAMP mustard (10 nM, 40 min exposure) did not discriminate between these muscarinic receptors. In ileum, 4-DAMP mustard, at 100 nM, resulted in a large dextral shift (197 fold) and depression in maxima. In atria there was a smaller dextral shift (14 fold) but no depression in maxima. 2 The muscarinic antagonists, atropine (non-selective), methoctramine (M2-selective) and para-fluorohexahydro-siladiphenidol (pFHHSiD; M3 selective) were studied in protection studies against alkylation by phenoxybenzamine. Washout studies following equilibration of the tissues with atropine (30 nM), methoctramine (0.3 tiM) or pFHHSiD (3 AM), showed the compounds to be reversible. No temporal changes in sensitivity to (+)-cis-dioxolane were observed. 3 Exposure, for 20 min, of atria and ileum to phenoxybenzamine (3 and 10 JM respectively) caused dextral shifts and depressions in the maxima of the concentration-response curve to (+)-cis-dioxolane. These effects were inhibited by prior equilibration with atropine (30 nM) and methoctramine (0.1 M) in atria or atropine (30 nM) and pFHHSiD (3 JAM) in ileum. Similar results in ileum were obtained when pilocarpine was used as the agonist.

Characterization of the interaction between muscarinic M2 receptors and β‐adrenoceptor subtypes in guinea‐pig isolated ileum

“…Although no direct contractile response to M2 receptor activation can be demonstrated in guinea-pig ileum (Eglen & Harris, 1993), an indirect influence on contraction via inhibition of P-adrenoceptor-mediated relaxation remains possible.…”

“…Inactivation of M2 heterogeneous populations of muscarinic receptors (see Eglen receptors by either selective alkylation (Eglen & Harris, 1993) et al, 1994 for a review). Radioligand binding studies have or by treatment with pertussis toxin, which inactivates the demonstrated that the majority of guinea-pig ileal muscarinic guanine nucleotide regulatory protein Gi (Eglen et al, 1988), receptors are of the M2 subtype (-70%), while a minority has minimal effects on guinea-pig ileal contractile responses are of the M3 subtype (-30%), with no measurable quanto muscarinic agonists.…”

In vitro effects of bethanechol on specimens of intestinal smooth muscle obtained from the duodenum and jejunum of healthy dairy cows

“…Studies 11,44,[53][54][55] have revealed that > 1 mAChR subtype may be responsible for mediation of cholinergic contraction in tissues. Analysis of results of the study reported here indicated that the effect of bethanechol in the intestinal segments of interest was mediated by both M 2 and M 3 mAChRs, with activation of M 3 mAChRs causing stronger increases in contractility traits than for activation of M 2 mAChRs, as indicated by use of both sets of receptor antagonists.…”

“…[41][42][43] The fact that this concentration of atropine almost completely abolished the effects of bethanechol confirmed that the mAChRs were blocked and corroborated that the effect of bethanechol is mediated via mAChRs. Concentrations of specific mAChR antagonists used in the study were determined from antagonist affinity values reported in other studies, 8,15,[44][45][46] which allowed maximal occupancy of the receptor subtype of interest with minimal occupation of other subtypes. The mAChR antagonists used in the study reported here possess differing selectivity for the various mAChR subtypes, depending on their affinity patterns.…”

“…Thus, the affinity pattern for pirenzepine 47 15,45,50 Affinity data for tropicamide are sparse, but this receptor antagonist appears to bind primarily to M 4 . 51,52 We are not aware of any receptor antagonists with high affinity for M 5 receptors.Studies 11,44,[53][54][55] have revealed that > 1 mAChR subtype may be responsible for mediation of cholinergic contraction in tissues. Analysis of results of the study reported here indicated that the effect of bethanechol in the intestinal segments of interest was mediated by both M 2 and M 3 mAChRs, with activation of M 3 mAChRs causing stronger increases in contractility traits than for activation of M 2 mAChRs, as indicated by use of both sets of receptor antagonists.…”