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.     

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.     

Muscarinic cholinoceptor subtypes mediating tracheal smooth muscle contraction and inositol phosphate generation in guinea pig and rat.

The effects of the muscarinic cholinoceptor antagonists atropine (non-selective), pirenzepine (M1-selective), methoctramine (M2-selective) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; M3-selective) were examined on the responsiveness of guinea pig and rat tracheal tissue to acetylcholine and carbachol. Results indicate that smooth muscle contraction in isolated tracheal tissue from both species was mediated primarily by muscarinic M3 cholinoceptors. The effects of atropine, pirenzepine and 4-DAMP were similar against the contractile actions of acetylcholine and carbachol in both species and in epithelium-intact and epithelium-denuded tissue. In contrast, differences in the effects of methoctramine in antagonising contractile responses to acetylcholine and carbachol were observed between the two species and following epithelium removal in the guinea pig. Thus, whilst this study has found that tracheal smooth muscle contraction in the guinea pig and rat is mediated primarily by muscarinic M3 cholinoceptors, anomalies in the functional inositol phosphate generation results obtained with the muscarinic cholinoceptor antagonists highlight species differences in the actions of acetylcholine and carbachol in eliciting smooth muscle contraction suggesting the possible existence of functional non-M3 muscarinic cholinoceptors.     

Muscarinic receptor agonist-antagonist interaction in the rat rectum: are there ways of activating the same receptors?

The interaction between the muscarinic receptor agonists, carbachol, acetylcholine (ACh) and methacholine, and antagonists, atropine, gallamine, 4-DAMP and pirenzepine, was studied on the rat isolated rectum preparation. ACh (1.93 X 10(-8)-1.95 X 10(-6) M), methacholine (8.7 X 10(-8)-1.1 X 10(-6) M) and carbachol (1.1 X 10(-7)-3.5 X 10(-6) M) induced contractions that were reversibly antagonized by atropine (1.9 X 10(-9)-4.8 X 10(-8) M), 4-DAMP (1.5 X 10(-8)-2.86 X 10(-7) M) gallamine (1.12 X 10(-6)-1.12 X 10(-4) M) and pirenzepine (2.8 X 10(-7)-7.0 X 10(-6) M). The pA2 values were atropine: 8.99 +/- 0.28, 9.29 +/- 0.14 and 8.86 +/- 0.05; 4-DAMP: 8.39 +/- 0.10, 8.66 +/- 0.15 and 8.26 +/- 0.30, gallamine: 5.85 +/- 0.23, 5.73 +/- 0.25 and 5.96 +/- 0.10 and pirenzepine: 6.85 +/- 0.44, 7.17 +/- 0.13 and 7.21 +/- 0.03 against ACh, methacholine and carbachol, respectively. The experimental dose-ratio (atropine + gallamine) was greater than the expected dose-ratio (as predicted by the Paton & Rang rule) for ACh and methacholine while the experimental dose-ratio closely approximates the expected dose-ratio for carbachol. It is suggested that atropine, 4-DAMP pirenzepine and gallamine act on the same receptors but gallamine allosterically altered the binding of the agonists and antagonists to varying extents.     

Evidence for prejunctional M2 muscarinic receptors in pulmonary cholinergic nerves in the rat.

1. The effects of muscarinic antagonists considered to be selective for M1 receptors (pirenzepine) and for M2 receptors (gallamine and methoctramine) were used to investigate the existence of prejunctional muscarinic receptors on cholinergic nerves in the rat lung. The tracheal tube preparation was used in vitro, and contraction of the trachealis muscle was induced by electrical field stimulation (EFS) and by application of an exogenous muscarinic agonist (pilocarpine), and measured as an increase in intraluminal pressure in the tube. 2. The muscarinic antagonists, gallamine and methoctramine, enhanced the contractions induced by nerve stimulation, while contractions elicited by exogenous application of pilocarpine were inhibited by the antagonists. 3. In contrast, pirenzepine blocked contractions induced by both EFS and pilocarpine in a dose-dependent manner (EC50 0.1 microM) due to blockade of the postjunctional muscarinic receptors on airway smooth muscle. Potentiation of the response to EFS was never seen with this antagonist. 4. The muscarinic agonist, pilocarpine, caused a slow maintained increase in tone of the tracheal tube and at the same time reduced the contractions induced by EFS. This inhibitory effect was blocked by gallamine and methoctramine. 5. The results suggest that prejunctional inhibitory muscarinic receptors may be localised on the parasympathetic cholinergic nerve terminals innervating tracheal smooth muscle in the rat. This confirms previous findings obtained by measuring transmitter release in this species. The present results suggest that these receptors are of the M2 subtype. Blockade of these autoreceptors with gallamine or methoctramine would increase the output of acetylcholine (ACh) and thereby enhance the nerve-induced contraction of tracheal smooth muscle.     

No evidence for a role of muscarinic M2 receptors in functional antagonism in bovine trachea.

1. The functional antagonism between methacholine- or histamine-induced contraction and beta-adrenoceptor-mediated relaxation was evaluated in bovine tracheal smooth muscle in vitro. In addition, the putative contribution of muscarinic M2 receptors mediating inhibition of beta-adrenoceptor-induced biochemical responses to this functional antagonism was investigated with the selective muscarinic antagonists, pirenzepine (M1 over M2), AF-DX 116 and gallamine (M2 over M3), and hexahydrosiladiphenidol (M3 over M2). 2. By use of isotonic tension measurement, contractions were induced with various concentrations of methacholine or histamine, and isoprenaline concentration-relaxation curves were obtained in the absence or presence of the muscarinic antagonists. Antagonist concentrations were chosen so as to produce selective blockade of M2 receptors (AF-DX 116 0.1 microM, gallamine 30 microM), or half-maximal blockade of M3 receptors (pirenzepine 0.1 microM, AF-DX 116 0.5 microM, hexahydrosiladiphenidol 0.03 microM). Since these latter antagonist concentrations mimicked KB values towards bovine tracheal smooth muscle M3 receptors, antagonist-induced decreases in contractile tone were compensated for by doubling the agonist concentration. 3. It was found that isoprenaline-induced relaxation of bovine tracheal smooth muscle preparations was dependent on the nature and the concentration of the contractile agonist used. Thus, isoprenaline pD2 (-log EC50) values were decreased 3.7 log units as a result of increasing cholinergic tone from 22 to 106%, and 2.4 log units by increasing histamine tone over a similar range. Furthermore, maximal relaxability of cholinergic tone decreased gradually from 100% at low to only 1.3% at supramaximal contraction levels, whereas with histamine almost complete relaxation was maintained at all concentrations applied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pirenzepine and gallamine on cardiac and pulmonary muscarinic receptors in the rabbit.

1. The effect of muscarinic antagonists considered to be selective for M1 receptors (pirenzepine) and for M2 receptors (gallamine) were studied on bronchoconstriction and bradycardia elicited by stimulation of the vagal nerves and by i.v. acetylcholine (ACh) in anaesthetized rabbits. 2. Pirenzepine was equipotent as an antagonist of ACh-induced responses at postjunctional muscarinic receptors in the heart, lung and blood vessels, whereas gallamine was at least ten times less potent at pulmonary and vascular muscarinic receptors. Thus, gallamine never caused complete inhibition of bronchoconstrictor or hypotensive responses to i.v. ACh, whereas doses of pirenzepine in excess of 1 mumol kg-1 abolished all muscarinic responses. 3. In the lung, both antagonists inhibited bronchoconstriction caused by vagal stimulation and ACh-induced bronchoconstriction to the same extent (pirenzepine, mean ED50 65 +/- 22 and, 130 +/- 28 nmol kg-1 respectively; gallamine, ED50 greater than 10,000 nmol kg-1 for both responses). Enhancement of vagally-induced bronchoconstriction was never observed. 4. In the heart, however, both pirenzepine and gallamine were ten times less potent as antagonists of vagally-induced bradycardia than of ACh-induced bradycardia. This differential blockade was unaltered by propranolol (1 mg kg-1) pretreatment. 5. It is concluded that there is no evidence for M1 or M2 muscarinic receptors in the pulmonary innervation of the rabbit and the potency of the antagonists in abolishing in abolishing vagally-induced bronchoconstriction was consistent with blockade of M3 muscarinic receptors on airway smooth muscle. 6. The results suggest that M2 muscarinic receptors may exert an inhibitory effect on transmission in the parasympathetic nerves innervating the heart in the rabbit.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle

The muscarinic receptor subtype involved in human airway smooth muscle contraction was characterised for the first time, using subtype-selective muscarinic antagonists. It was demonstrated that methacholine-induced contraction of central (trachea) and peripheral (small bronchi) airway smooth muscle preparations was antagonised by pirenzepine, AF-DX 116, 4-DAMP methobromide, hexahydrosiladifenidol, and methoctramine with pA₂-values characteristic of M₃ (smooth muscle/glandular) muscarinic receptors. Since these pA₂-values demonstrate significant correlations with those found in bovine and guinea-pig tracheal smooth muscle contraction, it is concluded that these animal tissues provide a good model for the study of M₃ subtype-selective muscarinic antagonists to be used as bronchodilators.     

Characterization of the muscarinic receptor subtype involved in phosphoinositide metabolism in bovine tracheal smooth muscle

1. The muscarinic receptor subtype involved in the methacholine-induced enhancement of phosphoinositide metabolism in bovine tracheal smooth muscle was identified by using the M2-selective antagonist AF-DX 116 and the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide, in addition to the M1-selective antagonist pirenzepine, in a classical Schild analysis. 2. All the antagonists shifted the methacholine dose-response curve to the right in a parallel and concentration-dependent fashion, yielding Schild plots with slopes not significantly different from unity. The pA2 values (6.94, 6.32 and 8.54 for pirenzepine, AF-DX 116 and 4-DAMP methobromide respectively) indicate that it is the M3 (smooth muscle/glandular), but not the M2 (cardiac) muscarinic receptor subtype, present in this tissue, that mediates phosphoinositide turnover, in accordance with our previous contractile studies. 3. The results provide additional evidence for the involvement of phosphoinositide turnover in the pharmacomechanical coupling between muscarinic receptor stimulation and contraction in (bovine tracheal) smooth muscle.     

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)     

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)     

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.     

Characterisation of the prejunctional inhibitory muscarinic receptor on cholinergic nerves in the rat urinary bladder

The nature of the prejunctional inhibitory muscarinic receptor on cholinergic nerve endings in the rat urinary bladder was investigated by measuring stimulated endogenous acetylcholine release via high pressure liquid chromatography (HPLC), in the presence of various selective muscarinic antagonists. The rank order of potencies for the antagonists used was: atropine (-log concentration = 7.8) > 4-DAMP (4-diphenylacetoxy-N-methylpiperidine) (7.6) > tripitramine (7.3) = HHD (hexahydrodifenidol) (7.3) > pFHHSiD (p-fluoro-hexahydrosiladifenidol hydrochloride) (7.0) > himbacine (6.5) > methoctramine (5.9) > or = pirenzepine (5.8) > gallamine (4.3). A comparison of the antagonist potencies obtained, with affinity constants at muscarinic M(1) to M(5) receptors, suggests that the prejunctional inhibitory muscarinic receptor is of the M(4) receptor subtype.     

The affinity of some selective muscarinic receptor antagonists for the muscarinic receptor mediating endothelial-dependent relaxation of the rabbit and rat thoracic aorta

The pKB values determined for pirenzepine, 4-DAMP, secoverine and gallamine against acetylcholine-mediated relaxant effects in rabbit aorta indicate that this muscarinic receptor closely resembles that which mediates contraction of non-vascular smooth muscle. The results of the present study argue against the presence of a novel type of muscarinic receptor mediating endothelium-dependent relaxation.     

M3 muscarinic receptors but not M2 mediate contraction of the porcine detrusor muscle in vitro

1. The objective of the study was to determine the role of muscarinic receptor subtypes in mediating contraction of the porcine detrusor smooth muscle in vitro. 2. Strips of pig detrusor muscle were set up in physiological salt solution and the tensions developed by the tissues were recorded. Responses to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (4-DAMP, methoctramine, darifenacin, oxybutynin, tolterodine and pirenzepine). Antagonist affinity values (pKB values) were calculated and compared with those quoted in the literature for these antagonists at each of the muscarinic receptor subtypes. 3. The M3-selective antagonists, 4-DAMP and darifenacin had high affinities (pKB values of 9.4 and 8.6, respectively). Oxybutynin, tolterodine and pirenzepine had affinities of 8.2, 8.1 and 6.8, respectively, whilst the M2-selective agent methoctramine had a relatively low affinity (pKB = 6.1). The rank order of affinities was, therefore, 4-DAMP > darifenacin > oxybutynin > tolterodine > pirenzepine > methoctramine for the pig detrusor. Correlation of the antagonist affinities obtained on the bladder with those published for these antagonists at the five muscarinic receptor subtypes identified the M3(m3)-receptor as the muscarinic subtype mediating detrusor contractile responses in vitro. 4. These data suggest that a small population of M3-muscarinic receptors must mediate direct contractile responses of the pig detrusor muscle to muscarinic receptor stimulation in vitro.     

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.     

Antimuscarinic action of liriodenine, isolated from Fissistigma glaucescens, in canine tracheal smooth muscle.

1. The antimuscarinic properties of liriodenine, isolated from Fissistigma glaucescens, were compared with methoctramine (cardioselective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, smooth muscle selective M3 antagonist) by radioligand binding tests, functional tests and measurements of second messenger generation in canine cultured tracheal smooth muscle cells. 2. Liriodenine, pirenzepine, methoctramine and 4-DAMP displaced [3H]-N-methyl scopolamine ([3H]-NMS) binding in a concentration-dependent manner with Ki values of 2.2 +/- 0.4 x 10(-6), 3.3 +/- 0.7 x 10(-7), 8.9 +/- 2.3 x 10(-8) and 2.3 +/- 0.6 x 10(-9) M, respectively. The curves for competitive inhibition of [3H]-NMS with liriodenine, methoctramine and 4-DAMP were best fitted according to a two site model of binding, but pirenzepine was best fitted according to a model with one site. 3. Liriodenine and 4-DAMP displayed a high affinity for blocking tracheal contraction (pKB = 5.9 and 9.1, respectively) and inositol phosphate formation (pKB = 6.0 and 8.9, respectively), but a low affinity for antagonism of cyclic AMP inhibition (pKB = 4.7 and 7.8, respectively). 4. Methoctramine blocked cyclic AMP inhibition with a high affinity (pKB = 7.4), but it antagonized tracheal contraction and inositol phosphate formation with a low affinity (pKB = 6.1 and 6.0, respectively). 5. In conclusion, both M2 and M3 muscarinic receptor subtypes coexist in canine tracheal smooth muscle and are coupled to the inhibition of cyclic AMP formation and phosphoinositide breakdown, respectively. The antimuscarinic characteristics of liriodenine are similar to those of 4-DAMP. It may act as a selective M3 receptor antagonist in canine tracheal smooth muscle.     

Mediation by M3-muscarinic receptors of both endothelium-dependent contraction and relaxation to acetylcholine in the aorta of the spontaneously hypertensive rat.

1. Experiments were designed to characterize the subtype(s) of endothelial muscarinic receptor that mediate(s) endothelium-dependent relaxation and contraction in the aorta of spontaneously hypertensive rats (SHR). 2. Rings of SHR aorta with endothelium were suspended in organ baths for the measurement of isometric force. Ecothiopate (an inhibitor of acetylcholinesterase) was present throughout the experiments. Endothelium-dependent contraction to acetylcholine was studied in quiescent aortic rings in the presence of NG-nitro-L-arginine (to prevent the formation of nitric oxide). Endothelium-dependent relaxation to acetylcholine was obtained during contraction to phenylephrine and in the presence of indomethacin (to inhibit cyclo-oxygenase activity). Responses to acetylcholine were assessed against the non-preferential muscarinic receptor antagonist, atropine, and the preferential antagonists pirenzepine (M1), methoctramine (M2) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3). 3. The potency of acetylcholine in inducing endothelium-dependent contraction was 6.54 +/- 0.07 (EC50). Atropine, pirenzepine, methoctramine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent contraction to acetylcholine. The pA2 values for these muscarinic receptor antagonists were estimated from Arunlakshana-Schild plots to be (-log M) 9.48 +/- 0.07, 6.74 +/- 0.22, 6.30 +/- 0.20 and 9.39 +/- 0.22 respectively. The potency of acetylcholine in inducing endothelium-dependent relaxation was 7.82 +/- 0.09 (IC50). Atropine, pirenzepine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent relaxation to acetylcholine but methoctramine had no effect. The pA2 values for atropine and 4-DAMP for the relaxation to acetylcholine were estimated from Arunlakshana-Schild plots to be (-log M) 9.15 +/- 0.23 and 9.63 +/- 0.28, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of selective compounds with muscarinic receptors at dispersed intestinal smooth muscle cells.

1. The characterization of muscarinic receptors on single cells of the guinea-pig ileum longitudinal smooth muscle, devoid of neuronal elements, was functionally studied by estimating the affinities of muscarinic antagonists on acetylcholine-induced contractions. 2. Atropine (5 x 10(-11) to 5 x 10(-6) M), 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP, 5 x 10(-8) to 5 x 10(-6) M), cyclohexyl(4-fluoro-phenyl) (3-piperidinopropyl) silanol (pFHHSiD, 5 x 10(-7) to 5 x 10(-5) M) as well as pirenzepine (5 x 10(-7) to 5 x 10(-5) M) competitively antagonized the acetylcholine-dependent contractions with different affinities (atropine > 4-DAMP > pFHHSiD > pirenzepine). 3. Methoctramine (5 x 10(-7) to 5 x 10(-5) M), and AF-DX 116 (5 x 10(-6) and 5 x 10(-5) M) also showed antagonist properties but these deviated from simple competition. These compounds, which discriminate between M2 and M3 receptors, showed a potency lower than that of pirenzepine, the rank order of potencies being pirenzepine > methoctramine > AF-DX 116. When concentrations of AF-DX 116, methoctramine and pirenzepine were increased an unspecific contractile effect occurred. 4. McN-A-343, a partial agonist on intact guinea-pig longitudinal smooth muscle strips, on this preparation induced a weak contraction (about 7% in comparison to control) that was not reversed by antimuscarinic agents. 5. These data indicate that M3 rather than M2 receptor sites are present on this tissue.