Different muscarinic receptor subtypes mediating the phasic activity and basal tone of pig isolated intravesical ureter.

1. We have studied the effects of muscarinic cholinoceptor agonists and specific antagonists on both phasic activity and basal tone of the isolated intravesical ureter of the pig by means of isometric techniques in vitro. 2. Acetylcholine in the presence and absence of physostigmine increased both phasic activity and basal tone of ureteral strips in a concentration-dependent manner. Moreover carbachol, methacholine and oxotremorine-M increased both contractile parameters while bethanechol and McN-A-343 evoked only increases in tone without affecting the frequency of the phasic contractions. 3. The nicotinic receptor blocker, hexamethonium (10(-6)-10(-4) M), failed to modify the contractions evoked by a single dose of carbachol (10(-5) M), whilst the muscarinic antagonist, atropine inhibited both phasic and tonic responses. 4. The muscarinic M1 (pirenzepine), M2 (AF-DX 116 and methoctramine), M3 (4-DAMP, HHSiD and p-F-HHSiD), and putative M4 receptor (tropicamide) antagonists significantly reversed increases in both frequency of phasic activity and baseline tone induced by a submaximal dose of carbachol (10(-5) M). The pIC50 values for inhibition of the induced phasic activity were: atropine (10.16) > 4-DAMP (9.12) > HHSiD (8.22) = methoctramine (7.98) = p-F-HHSiD (7.88 > tropicamide (7.62) = pirenzepine (7.53) = AF-DX 116 (7.45) and for inhibition of basal tone were: atropine (10.73) > 4-DAMP (9.32) > HHSiD (8.65) = pirenzepine (8.43) = p-F-HHSiD (8.38) > methoctramine (7.79) > tropicamide (7.53) > AF-DX 116 (7.04).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of muscarinic receptors of bovine coronary artery by functional and radioligand binding studies

The nature of the muscarinic receptor subtype mediating contraction of the endothelium-denuded bovine coronary artery was investigated in vitro by functional measurements and radioligand binding studies. The acetylcholine (ACh)-induced isotonic contraction of circularly cut muscle strips was recorded and expressed as a percentage of the maximum contraction obtained with 80 mM K+. In order to distinguish between M1, M2 and M3 receptors, the potency of the five subtype-selective antagonists, 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), parafluor-hexahydro-siladifenidol (pFHHSiD), pirenzepine, AF-DX 116 and methoctramine, to block the ACh-induced contraction was estimated. All the antagonists competitively inhibited the responses induced by ACh, with one exception, namely, 4-DAMP, whose Schild plot had a slope greater than one. The low affinity of pirenzepine (pA2 7.14 +/- 0.14) excluded an action at the M1 subtype. The low affinity of AF-DX 116 (pA2 6.49 +/- 0.18) and methoctramine (pA2 5.88 +/- 0.07) suggest that the bovine coronary artery smooth muscle receptor is not of the M2 (cardiac) subtype. In contrast, 4-DAMP (pA2 9.04 +/- 0.03) and pFHHSiD (pA2 7.64 +/- 0.04) potently inhibited the ACh-induced contraction with affinities similar to those reported for the M3 (glandular) receptor. In addition, the muscarinic receptors mediating coronary artery contraction were characterized in antagonist/[3H]N-methyl-scopolamine ([3H]NMS) competition binding studies. With the exception of AF-DX 116, all antagonists bound to a homogeneous population of receptors with pseudo-Hill slopes not different from unity. The pKi values, albeit somewhat lower, essentially substantiated the functional affinity estimates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of muscarinic receptors in lamb isolated coronary resistance arteries.

1. In vitro experiments in a microvascular myograph were designed to characterize postjunctional muscarinic receptors producing contraction both in the presence and absence of the endothelium in coronary resistance arteries (normalized diameter of 150-450 microns), isolated from the left ventricle of hearts from 3-6 month old lambs. Preferential muscarinic receptor antagonists were used to determine the receptor subtype: pirenzepine (M1 receptor), AFDX 116 (M2 receptor), 4-DAMP and pFHHSiD (M3 receptor). 2. The rank order of potency for muscarinic agonist-induced increases in tension in endothelium-intact preparations was oxotremorine-M = methacholine = acetylcholine (ACh) > carbachol. Removal of the endothelium increased the potency of ACh, but this procedure did not change either the sensitivity or maximal response to carbachol. 3. The contractile response to ACh was reproducible. Incubation with 3 x 10(-7)-3 x 10(-6) M pirenzepine induced non-parallel rightward shifts and depressed the maximum of the concentration-response curve to ACh in endothelium-intact arteries. The slope by Schild analysis was 2.9 +/- 0.8 (P < 0.05, n = 7). Atropine, AFDX 116, 4-DAMP and pFHHSiD produced parallel rightward shifts of the curves to ACh and the slopes of the Schild plots were not significantly different from unity. The pKB values for the antagonists from plots constrained to unity in endothelium-intact segments were: atropine (9.4), 4-DAMP (9.0), pFHHSiD (7.9) and AFDX 116 (6.2). 4. In endothelium-denuded arteries, pirenzepine, AFDX 116 and pFHHSiD caused concentration-dependent, parallel rightward displacements of the concentration-response curves to ACh and the slopes of the Schild plots were not significantly different from unity. The plots constrained to a slope of unity gave the following pKB values: pFHHSiD (8.7), pirenzepine (7.5) and AFDX 116 (6.2). 5. In the presence of the endothelium, low concentrations of pirenzepine (10(-9)-10(-7) M) produced leftward shifts of the ACh concentration-response curves. This potentiating effect of pirenzepine was reversed by endothelial cell removal. In preparations precontracted with the thromboxane-mimetic, U46619, the putative M1-selective agonist, McN-A-343, induced a biphasic relaxation with log IC50 of 8.53 +/- 0.14 and 5.02 +/- 0.08 for the first and second phase of the relaxation, respectively, and maximal relaxations of 22.8 +/- 4.3% and 41.1 +/- 5.4% (n = 16). McN-A-343 relaxed the vessels in the presence of 10(-7) M pFHHSiD and 3 x 10(-7) M AFDX 116, but not after incubation with 10(-9) M pirenzepine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mediation by the same muscarinic receptor subtype of phasic and tonic contractile activities in the rat isolated portal vein.

1. The effects of several agonists on the phasic and tonic contractile responses to muscarinic receptor stimulation have been investigated in the rat portal vein in vitro. 2. Neither chemical denervation with 6-hydroxydopamine nor the presence of the alpha 1-adrenoceptor antagonist, prazosin, influenced the spontaneous or the stimulated myogenic activity of the portal vein. 3. Indomethacin and NG-nitro-L-arginine were used to investigate the influence of vasoactive factors in this preparation. They slightly increased the frequency and the amplitude of the spontaneous myogenic activity of the portal vein, respectively. NG-nitro-L-arginine but not indomethacin enhanced the maximal phasic response to carbachol. Both indomethacin and NG-nitro-L-arginine failed to influence the tonic response to carbachol. 4. Muscarinic agonists increased phasic activity according to the rank order of potency: acetylcholine > muscarine > methacholine > carbachol > aceclidine > bethanechol. These effects were superimposed on a sustained contracture at higher concentrations. Oxotremorine was more potent than arecoline in increasing the mechanical phasic activity, without inducing a sustained contracture. Pilocarpine and McN A343 were weak agonists, producing submaximal effects only on phasic activity. 5. The muscarinic antagonists AF-DX116, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), P-fluorohexahydrosiladiphenidol (pFHHSiD) and pirenzepine antagonized the phasic and tonic mechanical responses to carbachol. Although the tonic contracture was slightly more sensitive to all antagonists studied, the rank order of potency: 4-DAMP > pFHHSiD > pirenzepine > AF-DX 116 was the same for both types of responses, which is indicative of a M3-receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the functional muscarinic receptors in the rat urinary bladder.

1. Muscarinic receptors mediating contraction of the rat urinary bladder were characterized functionally in vitro by use of atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP methiodide), 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard), hexahydro-sila-diphenidol hydrochloride (HHSiD), the p-fluoro analogue of hexahydro-sila-diphenidol hydrochloride (p-F-HHSiD), methoctramine, and pirenzepine. 2. (+)-cis-Dioxolane contracted bladder strips in a concentration-dependent manner with an EC50 of 0.169 +/- 0.018 microM and an Emax of 7.84 +/- 0.67 g. 3. Concentration-effect curves to (+)-cis-dioxolane were shifted to the right in the presence of the antagonists in a concentration-dependent manner. The rank order of antagonist affinities against the (+)-cis-dioxolane response was (pA2 values in the parentheses) atropine (9.28) > or = 4-DAMP methiodide (9.04) > HHSiD (8.01) > p-F-HHSiD (7.28) = pirenzepine (7.12) > or = methoctramine (6.77, 7.25). The profile resembles that associated with the M3 receptor subtype. 4. Atropine, 4-DAMP methiodide, pirenzepine, and methoctramine had no effects on the contractile response to 120 mM KCl. However, HHSiD and p-F-HHSiD decreased the response to KCl, and 4-DAMP mustard increased it. 5. Contractile responses to electrical field stimulation (1-32 Hz, 0.05 ms pulse duration) were biphasic in nature. The tonic response was suppressed more than the phasic response by all antagonists except methoctramine. The suppression was not always concentration-dependent, and did not seem to be related to antagonism of any one receptor subtype. 6. Our findings are consistent with the minority M3 receptors mediating the contractile response to muscarinic stimulation by (+)-cis-dioxolane in the rat bladder.     

Muscarinic receptor subtype mediating vasodilation feline middle cerebral artery exhibits M3 pharmacology

The nature of the muscarinic receptor subtype mediating the endothelium-dependent relaxation of the cat middle cerebral artery was investigated in vitro by recording the smooth muscle isometric tension of precontracted arterial segments. Relaxation induced by several agonists (acetylcholine (ACh), acetyl-beta-methylcholine, oxotremorine, carbachol and McN-A-343) was recorded. The ability of selective (pirenzepine, dicyclomine, adiphenine, AF-DX 116, methoctramine, gallamine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and hexahydro-sila-difenidol (HHSiD] and non-selective antagonists (atropine, scopolamine and quinuclidinyl benzilate (QNB] to block the relaxation induced by ACh was also estimated. The weak activity of the poorly selective M1 muscarinic receptor as together with the intermediate affinity of pirenzepine and adiphenine tend to exclude the M1 muscarinic receptor as the primary mediator of the cholinergic relaxation. The low affinity of AF-DX 116 and methoctramine further suggested that the cerebrovascular muscarinic receptor does not correspond to the M2 cardiac subtype. In contrast, 4-DAMP and HHSiD potently inhibited the ACh-induced relaxation with affinities similar to those reported at the M3 glandular receptor. We conclude that a similar to the pharmacological M3 muscarinic receptor subtype is responsible for the cholinergic relaxation of the cat middle cerebral artery.     

Functional and binding studies with muscarinic M2-subtype selective antagonists.

1. The potency of a series of selective muscarinic antagonists has been measured on two functional isolated tissue preparations (rat ileum and atria) and these compared with their potency on a range of binding preparations in order to determine whether the subtypes of M2 receptor measured functionally are the same as those measured in binding studies. 2. On the functional preparations pirenzepine, hexahydrosiladiphenidol (HSD) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were more potent on the ileum than on the atrium (3 fold, 29 fold and 5 fold respectively), whereas himbacine, AF-DX 116 and methoctramine showed the opposite selectivity (5 fold, 3 fold and 56 fold respectively). Atropine had a similar potency on the ileum and atrium. 3. [3H]-N-methyl scopolamine was used to study M2 binding sites on membranes from rat heart and rat submandibular gland. Each preparation appeared to contain a homogeneous binding site population. The potencies of the five M2 selective antagonists (and pirenzepine) in binding studies to heart membranes were very similar to those observed in functional studies of rat atria (correlation coefficient = 0.98). Similarly the binding to submandibular gland membranes was very similar to that observed in functional studies on rat ileum (correlation coefficient = 0.97). 4. [3H]-pirenzepine was used to examine the binding of these antagonists to M1 binding sites on membranes from rat cerebral cortex. The affinities of 4-DAMP, HSD, AF-DX116 and himbacine at M1 sites were similar to their affinities on the gland. Only pirenzepine and methoctramine had higher affinity on M1 sites than on the gland.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of guinea pig ileal electrolyte transport by M3-muscarinic acetylcholine receptors in vitro

To determine the muscarinic receptor subtype mediating guinea pig ileal mucosal electrolyte secretion, we compared the potencies (Kb) of selective M1 (pirenzepine) (PZ), M2 (AF-DX 116, methoctramine), and M3 [4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladifenidol (HHSiD)] antagonists as inhibitors of carbachol-induced reductions in guinea pig atrial heart rate and ileal longitudinal muscle contractions, responses mediated by M2 and M3 receptors, respectively. Pretreatment with all five muscarinic antagonists shifted the carbachol concentration-response curve to the right, in a manner suggesting competitive antagonism. The following affinity profiles (Kb, nM) were obtained for: 1) ileal mucosa: 4-DAMP (2.7) greater than HHSiD (23.0) greater than PZ (110) greater than or equal to methoctramine (395) greater than AF-DX 116 (784); 2) atrial heart rate: 4-DAMP (9.5) congruent to methoctramine (11) greater than AF-DX 116 (63) greater than HHSiD (222) greater than PZ (256); and 3) ileal longitudinal muscle: 4-DAMP (3.1) greater than HHSiD (21) greater than PZ (143) greater than methoctramine (388) greater than or equal to AF-DX 116 (482). The selectivity profiles of these antagonists suggest that muscarinic receptors in the ileal mucosa more closely resemble those in the ileal muscle (M3) than those in atrial muscle (M2). Moreover, M1-muscarinic receptors appear to be relatively unimportant in mediating the effects of carbachol on short circuit current (ISC). Carbachol-induced increases in ISC were also unaffected by pretreatment with 0.5 microM tetrodotoxin, suggesting that electrolyte transport in the guinea pig ileal mucosa may be mediated, in part, by postsynaptic M3-muscarinic receptors on the enterocytes.     

Chimeric m2/m3 muscarinic receptors: role of carboxyl terminal receptor domains in selectivity of ligand binding and coupling to phosphoinositide hydrolysis

The cloning and expression of five mammalian muscarinic receptor genes (m1-m5) have shown that the individual receptor subtypes differ in their functional and ligand-binding properties. To study the role of the carboxyl terminal receptor domains in this pharmacological diversity, we constructed chimeric m2/m3 receptors in which a region comprising part of transmembrane domain VI, the third extracellular loop, transmembrane region VII, and the cytoplasmic tail (collectively referred to as C-terminal domains) was exchanged between the human m2 and the rat m3 receptor. The ability of the cloned receptors to mediate stimulation of phosphoinositide hydrolysis and to bind subtype-selective muscarinic ligands was studied after their transient expression in COS-7 cells. Whereas wild-type m3 strongly stimulated phosphoinositide breakdown, wild-type m2 gave only a poor response. Exchange of the C-terminal domains between m2 and m3 had no significant effect on the magnitude of these responses. In N-[3H]methylscopolamine competition binding studies, the muscarinic antagonists AF-DX 116 and methoctramine showed 11- and 23-fold higher affinities, respectively, for m2 than for m3, whereas hexahydro-silad-ifenidol (HHSiD) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) displayed the reverse selectivity profile, having approximately 10-fold higher affinities for m3. In comparison with wild-type m3, the mutant m3 receptor containing the C-terminal domains of m2 displayed 2.5- and 8-fold higher affinities for AF-DX 116 and methoctramine but 7- and 3-fold lower affinities for HHSiD and 4-DAMP, respectively. The mutant m2 receptor with the C-terminal domains of m3 showed 2-3-fold lower affinities for AF-DX 116 and methoctramine but 2-3-fold higher affinities for HHSiD and 4-DAMP, as compared with wild-type m2. These data suggest that the C-terminal domains of the muscarinic receptors are not involved in conferring selectivity of coupling to phosphoinositide hydrolysis but contain major structural determinants of antagonist binding selectivity.     

Functional characterization of muscarinic receptors in murine airways.

1. The effects of muscarinic receptor antagonists considered to be selective for M1 receptors (pirenzepine; PZ), M2 receptors (AFDX-116), and for M3 receptors (4-diphenyl acetoxy N-methyl-piperidine (4-DAMP)) were used to investigate the existence of muscarinic receptors subtypes in murine airways. Atropine was used as a nonselective antagonist. The effects of these antagonists were studied upon tracheal contractions induced either by EFS (electric field stimulation) or by application of an exogenous cholinoceptor agonist (arecoline). 2. The muscarinic receptor antagonists tested inhibited arecoline-induced tracheal contractions with the following rank order of potency: 4-DAMP = atropine > pirenzepine = AFDX-116. The rank order of potency of the muscarinic antagonists used in inhibiting EFS-induced tracheal contractions was: 4-DAMP = atropine > PZ > AFDX-116. The pA2 values for these antagonists were similar when compared to the pA2 values determined in guinea-pig and bovine airway smooth muscle. 3. In addition to in vitro studies, the effects of inhalation of the different muscarinic antagonists on lung function parameters in vivo were investigated. Inhalation of 4-DAMP induced a decrease in airway resistance and an increase in lung compliance. In contrast, inhalation of AFDX-116 induced an increase in airway resistance and almost no change in lung compliance. Apart from some minor effects of atropine on airway resistance, atropine, PZ, and pilocarpine failed to induce changes in lung mechanics as determined by in vivo lung function measurements. 4. The results provide evidence for the existence of M3 receptors on murine tracheae that are involved in the contraction of tracheal smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

A pharmacological study of the responses induced by muscarinic agonists on the isolated superior cervical ganglion of the guinea-pig

We have studied the muscarinic agonist induced responses on the guinea-pig superior cervical ganglion in vitro, as recorded from the internal carotid nerve using a grease-gap. The principal response was a depolarization, but a small hyperpolarizing response could be revealed under certain conditions. We determined the pA2 of a number of muscarinic antagonists against the muscarine induced depolarization. Four selective antagonists and atropine appeared to act competitively. The rank order of their pA2s was 4-DAMP (8.5), atropine (8.4), pirenzepine (8.0), methoctramine (7.2) and AF-DX 116 (6.3). In addition to muscarine, we assessed the potency and relative maximum response of nine other muscarinic compounds to depolarize this preparation: carbachol, 5-methylfurmethide, oxotremorine, oxotremorine-M, pilocarpine, RS 86, AF102B and two novel compounds L-670548 and L-679512. L-670548 was the most potent and AF102B was the least potent agonist tested. Only AF102B evoked a maximum depolarization that was significantly smaller than muscarine. A hyperpolarizing response to carbachol (1 microM) could be recorded when the superfusing medium contained 0.3 microM pirenzepine and only 0.1 mM CaCl2 (cf. usual 2.5 mM). This response was relatively small compared to that evoked on the superior cervical ganglion of the rat. It was blocked by the cardioselective antagonists methoctramine (0.1-0.3 microM) and AF-DX 116 (0.3-1.0 microM). Of the 10 agonists tested, only carbachol, oxotremorine and oxotremorine-M reproducibly evoked a hyperpolarizing response. It was concluded that muscarinic agonists can induce a depolarization of the guinea-pig superior cervical ganglion mediated by M1 receptors. The activation of cardiac-like M2 receptors resulted in a hyperpolarizing response that was relatively small.     

Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function

Previous work showing that AF-DX 116, a cardioselective muscarinic antagonist in functional experiments, does not discriminate between muscarinic receptors in bovine cardiac and tracheal membranes has been extended. In addition to AF-DX 116 we used the muscarinic antagonists, atropine, pirenzepine, 4-DAMP methobromide, gallamine, hexahydrosiladifenidol and methoctramine, in radioligand binding experiments on bovine cardiac left ventricular and tracheal smooth muscle membranes. The functional antagonism of the methacholine-induced contraction of bovine tracheal smooth muscle strips was also evaluated. An excellent correlation was found for all compounds between the binding affinities for muscarinic receptors in cardiac and tracheal smooth muscle membranes; moreover, the affinities found in cardiac membranes correspond with the pA2 values reported for atrial preparations of rat and guinea pig. However, significant and occasionally marked discrepancies were found between binding and functional affinities of these muscarinic antagonists on bovine tracheal smooth muscle.     

Presynaptic and postjunctional muscarinic receptors in dog ileum: binding studies

[3H]N-Methylscopolamine identified two distinct populations of muscarinic receptors in membranes derived from the longitudinal smooth muscle/myenteric plexus of dog ileum. In isolated axonal varicosities, the half-maximal saturation of binding sites occurred at 2.38 +/- 0.39 nM [3H]N-methylscopolamine, with maximal binding capacity 140 +/- 35 fmol/mg protein (mean +/- S.D., n = 8). In purified smooth muscle plasma membranes, the Kd value was 16 +/- 3 nM with Bmax 1960 +/- 494 fmol/mg. The displacement potencies of subtype-selective muscarinic antagonists in the fraction of axonal varicosities followed the order 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methiodide much greater than pirenzepine = methoctramine greater than AF-DX 116 with pKi values 7.38, 5.67, 5.70 and 5.13, respectively. Both 4-DAMP methiodide and pirenzepine were approximately 4-fold less potent in displacing the ligand from the receptors in smooth muscle plasma membranes as compared to varicose receptors. The potency ratios of cardioselective antagonists methoctramine and AF-DX 116 on varicose and smooth muscle receptors were 1 and 1.7. It is concluded that presynaptic receptors located on isolated axonal varicosities have pharmacological properties similar to glandular (M3) subtype of muscarinic receptors. The binding properties of receptors present in smooth muscle plasma membranes were found incompatible with those of any of the M1, M2 or M3 subtypes.     

Characterization of muscarinic receptors mediating endothelium-dependent relaxation of bovine coronary artery.

In order to identify the receptor subtype responsible for acetylcholine (ACh)-induced relaxation of bovine coronary artery, we determined the affinity of six subtype-selective muscarinic antagonists and compared them with affinity estimates obtained for bovine left atria. At low concentrations, ACh potently relaxed circular strips of coronary artery with endothelium (EC50 0.15 microM), but contracted them at higher agonist concentrations with potencies that depended on the presence or absence of endothelium: EC50 1.8 microM (without endothelium); 4.6 microM (with endothelium). The pA2 values obtained for antagonism of relaxant responses to ACh were: pirenzepine (M1-selective) 7.38 +/- 0.12; AF-DX 116 (11-[2-(diethylamino-methyl)-1-piperidinyl-acetyl]-5,11- dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one; M2-selective) 5.79 +/- 0.09; and 4-diphenylacetoxy-N-methyl-piperidine-methobromide (4-DAMP; M3/M1-selective) 9.07 +/- 0.12. The corresponding Schild slopes were 0.98 +/- 0.07 for pirenzepine, 1.17 +/- 0.09 for AF-DX 116 and 1.01 +/- 0.04 for 4-DAMP. For the following three antagonists, pKB values were determined at two different antagonist concentrations: dicyclomine (M1-selective) 7.49 +/- 0.10, cyclohexylphenyl-(2-piperidinoethyl)-silanol (CPPS; M3-selective) 8.0 +/- 0.10, and parafluoro-hexahydrosila-difenidol (pFHHSiD; M3-selective) 7.87 +/- 0.10. For comparison, the antagonism of methacholine-induced negative inotropy in left atria was determined for three antagonists, yielding the following pA2 values: pirenzepine 5.98 +/- 0.14; AF-DX 116 6.81 +/- 0.14 and 4-DAMP 7.99 +/- 0.14. The slopes of the corresponding Schild plots were 1.05 +/- 0.10, 1.14 +/- 0.12 and 1.08 +/- 0.08, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation.

1. By measuring the binding of N-[3H-methyl]-scopolamine ([3H]-NMS) and of unlabelled subtype-specific muscarinic antagonists, two populations of muscarinic binding sites can be distinguished in the membranes of cardiac ventricles taken from 1-day-old chicks. One of them, corresponding to approximately 80% of [3H]-NMS binding sites, has higher affinities for AF-DX116 (pKi = 6.42) and methoctramine (pKi = 7.33); the rate of [3H]NMS dissociation from these sites is fast. The other population, corresponding to approximately 20% of [3H]-NMS binding sites, has lower affinities for AF-DX116 (pKi = 5.00) and methoctramine (pKi = 6.19); the rate of [3H]-NMS dissociation from these sites is slow. Both populations have high affinities for pirenzepine, but the affinity of the former (major) population is lower (pKi = 7.99) than that of the latter (minor) population (pKi = 10.14). 2. Since it has been shown earlier that two mRNAs for muscarinic receptors are expressed in the chick heart, one of them close to the genetically defined m2 and the other to the m4 subtype, we propose that the major population of binding sites with high affinities for AF-DX116 and methoctramine and the lower affinity for pirenzepine represents the M2-like receptors, while the minor population represents the M4-like receptors. 3. It proved possible to obtain isolated samples of either population by selectively protecting the M2-like sites with AF-DX116 and the M4-like sites with pirenzepine, and by inactivating the unprotected sites with benzilylcholine mustard.(ABSTRACT TRUNCATED AT 250 WORDS)     

An M3-like muscarinic autoreceptor regulates the in vivo release of acetylcholine in rat striatum

Selective muscarinic antagonists were used in an attempt to characterize the muscarinic autoreceptor modulating the release of acetylcholine in the striatum of the rat. In vivo microdialysis was applied to infuse atropine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), pirenzepine or AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro[2,3-b][1,4]benzodiazepine-6-one), leading to a dose-dependent increase in the overflow of acetylcholine, the order of potency being: atropine greater than 4-DAMP greater than pirenzepine greater than AF-DX 116. We conclude from these data that the muscarinic receptor modulating release in the striatum is of the M3 type.     

Pharmacological characterization of muscarinic receptors in the uterus of oestrogen-primed and pregnant rats

1. Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen-treated and late pregnant rats.
2. Competition binding studies with uterine membrane preparations and [³H]-QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pK_i) for each antagonist as follows; oestrogen-treated – atropine (7.98)⩾himbacine (7.83)>methoctramine (7.52)⩾hexahydrosiladiphenidol (HHSiD; 7.32)⩾5,11-dihydro-11-[[[2-[2 - [(dipropylamino)methyl] - 1piperidinyl]ethyl]amino] - carbonyl] - 6H-pyrido- [2,3 - b][1,4] - benzodiazepin - 6-one (AF - DX 384; 7.10)>11 - [[2 - [(diethylamino)methyl]-1-piperidinyl]- acetyl]5,11-dihydro-6H-pyridol]2,3,-b][1,4]benzodiazepin-6-one (AF-DX 116, 6.77)>pirenzepine (6.17); late pregnant – atropine (8.05)⩾methoctramine (7.95)⩾himbacine (7.71)⩾HHSiD (7.52)⩾AF-DX 384 (7.34)>AF-DX 116 (6.72)>pirenzepine (6.18).
3. The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non-pregnant animals (pD₂=5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration-response curves. The pA₂ estimates were: oestrogen-treated – atropine (9.42)>himbacine (8.73)⩾HHSiD (8.68)⩾methoctramine (8.49)⩾AF-DX 384 (7.91)⩾AF-DX 116 (7.36)⩾pirenzepine (7.26); late pregnant – atropine (9.48)>himbacine (8.37)⩾HHSiD (8.22)⩾methoctramine (8.01)⩾AF-DX 116 (7.73)⩾AF-DX 384 (7.44)⩾pirenzepine (6.92).
4. The relative pK_i estimates for antagonists obtained in membrane preparations from oestrogen-treated rats suggest the presence of muscarinic M₂ subtypes. In functional studies pA₂ values indicated the additional presence of muscarinic M₃ receptor or, possibly an atypical receptor subtype. The similarity between pK_i and pA₂ estimates obtained in uteri from oestrogen-treated and pregnant animals, respectively, indicates that pregnancy does not affect myometrial muscarinic receptors in the rat.

     

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

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 microM) or pFHHSiD (3 microM), 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 microM 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 microM) in atria or atropine (30 nM) and pFHHSiD (3 microM) in ileum. Similar results in ileum were obtained when pilocarpine was used as the agonist. 4. These data were consistent with muscarinic M2 receptors mediating responses in atria and M3 receptors mediating responses in ileum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholinesterase histochemistry and functional characterization of the muscarinic receptor mediating the contraction of the bovine oesophageal groove

1 By using acetylcholinesterase (AChE) histochemistry and invitro isometric techniques, we have studied the presence and distribution of AChE-positive nerves, as well as the effects of muscarinic cholinoceptor agonists and selective antagonists, in the bovine oesophageal groove. 2 AChE-positive nerves and cells were distributed widely on the oesophageal groove floor. These fibres originated from adventitial ganglia containing bodies with high AChE activity and were shown grouped as large adventitial nerve bundles. 3 Both in the presence and absence of physostigmine, acetylcholine (ACh) induced concentration dependent contractions of bovine oesophageal groove strips. The rank order of the pD₂ values for muscarinic agonists was: oxotremorine-M (7.37) = carbachol (7.14) > acetylcholine plus physostigmine (6.46) > bethanechol (5.42) > McN-A-343 (4.45) > acetylcholine (4.06). 4 Hexamethonium (10^?6–10^?4M ), a nicotinic receptor blocker, did not affect the carbachol concentration–response curve, which was significantly inhibited by the muscarinic antagonist, atropine (10^?9–10^?8M ). 5 The preferential muscarinic antagonists pirenzepine (M₁), 11-(2(-(diethyl-amino)methyl)-1-piperidinylacetyl)-5,-11-dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepine-6-one (AF-DX 116) and methoctramine (M₂), 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) and p-fluoro-hexahydrosiladiphenidol (p-F-HHSiD) (M₃) and tropicamide (M₄) evoked rightwards displacements in a parallel manner of the carbachol control curve, and there was no decrease of the maximum response with the highest concentration of antagonist utilized. The muscarinic antagonist affinities, expressed in terms of pA₂ values, were: atropine (9.51) = 4-DAMP (9.32) > p-F-HHSiD (7.78) > tropicamide (7.40) > pirenzepine (6.91) = AF-DX 116 (6.88) = methoctramine (6.71). This muscarinic antagonist profile suggests that an M₃ receptor is involved in the carbachol induced contraction. 6 The present results suggest that a rich network of AChE- positive fibres is present in the oesophageal groove floor, where they form a nerve trunk and thinner branches accompanying blood vessels and sometimes around ganglia. The muscarinic cholinergic contraction of the bovine oesohageal groove seems to be mediated via activation of an M₃ postsynaptic muscarinic receptor.