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In order to analyse the subtype of muscarinic receptors involved in the methacholine-induced contraction of the rabbit iris sphincter we have determined equilibrium dissociation constants (KB) of various antagonists in the sphincter muscle. The values were compared with those observed at M1 (rabbit vas deferens), M2 (heteroreceptors in rat iris) and M3 receptors (guinea-pig ileum), or at the muscarinic receptors in the guinea-pig uterus. The methacholine-induced contraction of the uterus from immature guinea-pigs was competitively antagonized by pirenzepine (6.64, -log KB), 4-DAMP (8.39), hexahydrodifenidol (HHD; 7.00 for the (R)- and 5.40 for the (S)-enantiomer), p-fluoro-hexahydrosiladifenidol (p-F-HHSiD; 6.25) and valethamate bromide (8.04). The affinity of the antagonists is consistent with the presence of an M2 receptor. The -log KB values of the antagonists in the rabbit iris sphincter (6.43, p-F-HHSiD; 6.22, AQ-RA 741; 7.23 and 5.34, (R)- and (S)-trihexyphenidyl) were lower than, or within the lowest range of, estimates in the other experimental models, irrespective of the subtype selectivity of the antagonist. This excludes the presence of an M1, M2, M3 or M4 receptor in this smooth muscle. The affinity of UH-AH 37 in the iris was intermediate between that for M1 or M3, and M2 receptors. The low affinity of AQ-RA 741 and the low enantiomeric ratio of trihexyphenidyl (THP) in the iris (77.6) would be compatible with a presumed M5 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

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Experiments were carried out on rat isolated perfused hearts with both vagus nerves attached. The acetylcholine stores were labelled with [14C]-choline. The effects of muscarinic receptor antagonists on the [14C]-overflow and increase in perfusion pressure evoked by vagus nerve stimulation (10 Hz, 4-10 mA) were studied in order to determine the muscarinic receptor type involved in autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat heart. Stimulation of the vagus nerves (1200 pulses) caused an increase in [14C]-overflow and in perfusion pressure which was significantly reduced by hexamethonium 500 mumol/l and abolished by tetrodotoxin 0.3 mumol/l or perfusion with Ca2(+)-free solution. The fractional rate of evoked [14C]-overflow per pulse upon stimulation at 10 Hz (720 pulses) was doubled in the presence of the non-selective antagonist atropine (0.01-1 mumol/l) as well as in that of the M2-selective compounds methoctramine (0.1 mumol/l) and AF-DX 116 (0.1-1 mumol/l), but remained unaffected by the M3-selective hexahydrosiladifenidol (0.1 mumol/l). The increase in perfusion pressure upon nerve stimulation was reduced by atropine (0.01 mumol/l) or hexahydrosiladifenidol (0.1 mumol/l) to approximately 50% and increased by about 50% in the presence of AF-DX 116 (0.1 mumol/l). The results show that the autoinhibition of acetylcholine release in the rat heart is mediated by M2 receptors. On the other hand, the increase in perfusion pressure upon vagus nerve stimulation is caused by a different muscarinic receptor, more sensitive to hexahydrosiladifenidol than to M2-selective antagonists.

The potencies of several muscarine receptor antagonists in blocking either the autoinhibition of acetylcholine release or the muscarinic contraction of the sphincter muscle upon acetylcholine release were investigated in the guinea-pig iris. The agonist at pre- or postjunctional muscarine receptors was acetylcholine released upon field stimulation (5.5 Hz, 2 min) of the irides preloaded with 14C-choline. The stimulation-evoked 14C-overflow was doubled in the presence of atropine 0.1 mumol/l but unaffected by the agonist (+/-)-methacholine (50 mumol/l). Thus, under the present stimulation conditions, the autoinhibition of acetylcholine release on the guinea-pig iris cholinergic nerves was nearly maximally activated. Isotonic contractions of the irides upon field stimulation consisted of a rapid, atropine (0.1 mumol/l)-sensitive peak phase followed by a sustained contraction which involved a cholinergic and a non-cholinergic stimulation of the sphincter muscle. The M2-selective antagonists methoctramine (10 mumol/l) and gallamine (100 mumol/l) increased both the 14C-overflow and the peak contractions evoked by field stimulation. In contrast, the M3-selective antagonist hexahydrosiladifenidol (0.1-10 mumol/l) failed to affect the evoked 14C-release but concentration-dependently (1-10 mumol/l) reduced the iris contractions. Pirenzepine (10 mumol/l) enhanced the evoked 14C-overflow and inhibited the peak contractions (0.1-10 mumol/l; maximal effect at 10 mumol/l). The low potency of the antagonist at both receptor sites indicates that an M1 muscarine receptor is not involved. The results are consistent with the idea of M2 muscarine receptors mediating autoinhibition of acetylcholine release in the guinea-pig iris and M3-like receptors inducing the contraction of the sphincter muscle.