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.     

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)     

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)     

M3-muscarinic receptor mediates prejunctional inhibition of noradrenaline release and the relaxation in cat femoral artery.

The aim of the present study was to analyse the muscarinic receptors involved in the vasodilation elicited by acetylcholine (ACh) and the carbachol inhibition of electrically-evoked [3H]noradrenaline (NA) release in cat femoral artery. For this purpose, the following receptor antagonists were used, atropine, pirenzepine (M1-antagonist), AF-DX 116 (M2-antagonist) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3-antagonist). The order of potency (pA2 values) of these drugs at postjunctional level was: atropine (9.7) greater than or equal to 4-DAMP (9.6) greater than pirenzepine (7.2) greater than AF-DX 116 (6.0), and at prejunctional level (pIC50 values) was: 4-DAMP (9.3) greater than atropine (8.5) greater than AF-DX 116 (7.1) greater than pirenzepine (5.9). These findings indicate that the muscarinic receptors mediating the vasodilation induced by ACh and the carbachol inhibition of NA release are of the M3-subtype.     

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)     

Comparison of the muscarinic receptors in the coronary artery,cerebral artery and atrium of the pig

Summary The affinity of various muscarinic antagonists for the muscarinic receptors mediating contraction (induced by acetyl-\-methylcholine) of the isolated pig coronary and basilar artery was determined in order to compare the muscarinic receptor subtype involved in the contractile response of these arteries. In order to identify the muscarinic receptor subtype(s) involved, the affinity of the antagonists for the M₂ receptor present in the pig atria was also investigated. The following muscarinic antagonists were used: atropine, pirenzepine, AF-DX 116 (11-2{{2-{(diethyl-amino)methyl} -1- piperidinyl}acetyl} - 5, 11- dihydro - 6H - pyrido {2, 3 - b} {, 4}benzodiazepin - 6 - one), 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide), HHSiD (hexahydrosiladifenidol), methoctramine (N, N- bis{6 - {(2 - methoxybenzyl)amino} hexyl} -1, 8 - octane - diamine tetrahydrochloride) and ipratropium.The order of affinity of the antagonists with respect to the muscarinic receptor in the coronary artery was clearly different from that for the muscarinic receptor in the basilar artery. The order of affinity established on the basilar artery closely resembled that for the M2 receptor in the atria.It is concluded that the muscarinic receptors on smooth muscle of the coronary and basilar arteries are not identical. The muscarinic receptor involved in the contraction of the basilar artery adheres to the M2 receptor subtype. A comparison of the selectivity of the antagonists suggests that the muscarinic receptor involved in the contraction of the coronary artery belongs to the M₃ (like in exocrine glands) or M₄ (as found in ileal smooth muscle) receptor subtype.     

Pre- and postjunctional muscarinic receptor subtypes in dog airways.

To examine muscarinic receptor subtypes involved in cholinergically mediated contractions of the airway, we studied the effects of the M1-selective antagonist, pirenzepine, the M2-selective antagonist, AF-DX 116, the M3-selective antagonist, 4-diphenyl-acetoxy-N-methylpiperidine (4-DAMP) methiodide, and the non-selective antagonist, atropine, on acetylcholine (ACh)- and electrically induced contractions in dog bronchi and bronchioles. The relative potencies of the antagonists based on IC50 values of each antagonist for contractions induced by the two concentrations of ACh that produced 50% of the maximum (ED50) and the maximum (EDmax) contractions and the pA2 values were atropine greater than or equal to 4-DAMP methiodide greater than pirenzepine = AF-DX 116 in both the bronchi and bronchioles. The IC50 and pA2 values of each antagonist did not differ significantly between the bronchi and bronchioles. 4-DAMP methiodide significantly inhibited the contractile response to electrical field stimulation (EFS) at 5 Hz at concentrations that did not alter the contractile responses to exogenous ACh in both the bronchi and bronchioles, whereas pirenzepine, AF-DX 116 and atropine inhibited the EFS-induced contraction only at the concentrations that reduced the contraction induced by exogenous ACh. The present results suggest that the cholinergic contraction is mediated via the postsynaptic receptor M3, based on functional potencies of muscarinic antagonists and presynaptic receptor auto-facilitatory M3, based on the suppression of the contractile response to EFS by 4-DAMP methiodide in central and peripheral airways.     

Involvement of M3 muscarinic receptors in ACh-induced increase in membrane-associated RhoA of rat bronchial smooth muscle

It is known that RhoA is translocated from cytoplasm to cell membrane in bronchial smooth muscle when activated by acetylcholine (ACh) stimulation. In the present study, the effects of selective muscarinic receptor antagonist methoctramine, AF-DX116 (for M(2)) and 4-diphenylacetoxy N-methylpiperidine (4-DAMP; for M(3)) on the ACh-induced rat bronchial smooth muscle contraction and increase in membrane-associated RhoA were investigated to elucidate the muscarinic receptor subtype participating in these responses. To evaluate ACh-induced contraction of bronchial smooth muscle, bronchial ring of rat was prepared, suspended in an organ bath and the tension was measured isometrically. To quantify the ACh-induced increase in membrane-associated RhoA protein, western blot analysis was performed by using homogenates of membrane and cytosolic fractions of the rat bronchi. The muscarinic M(2) and M(3) receptors were detected by using RT-PCR in rat bronchial smooth muscle. Both the ACh-induced smooth muscle contraction and increase in membrane-associated RhoA were markedly inhibited by 4-DAMP, but not by methoctramine or AF-DX116. In conclusion, these results indicated contraction for the first time that the activation of RhoA occurs via M(3) receptor in rat bronchial smooth muscle.     

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)     

Binding of selective antagonists to four muscarinic receptors (M1 to M4) in rat forebrain

To compare the proportions of four muscarinic receptors in different rat brain regions, we used competition curves with four selective antagonists, at 1-[N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) binding equilibrium and after allowing [3H]NMS dissociation for 35 min. Himbacine and methoctramine were shown to discriminate two muscarinic receptor subtypes having a high affinity for 4-diphenylacetoxy-N-methylpiperidine methiodide and hexahydrosiladifenidol, intermediate affinity for pirenzepine, and low affinity for AF-DX 116. One M4 subtype had a high affinity for himbacine and methoctramine; it was found predominantly in homogenates from rat striatum (46% of total [3H]NMS receptors) and in lower proportion in cortex (33% of [3H]NMS receptors) and hippocampus (16% of [3H]NMS receptors). Its binding properties were identical to those of muscarinic receptors in the neuroblastoma x glioma NG 108-15 hybrid, suggesting that it was encoded by m4 mRNA. The M3 subtype (typically found in rat pancreas, a tissue expressing the m3 mRNA) had a low affinity for himbacine and methoctramine and represented about 10% of all [3H]NMS receptors in rat brain cortex, hippocampus, striatum, and cerebellum. M1 and M2 receptors were identified in rat brain by their high affinity for pirenzepine and AF-DX 116, respectively.     

M3 muscarinic cholinoceptors are linked to phosphoinositide metabolism in rat cerebellar granule cells.

Primary cultures of rat cerebellar granule cells are shown to possess a high density (283 +/- 48 fmol/mg of protein) of muscarinic receptor sites, defined using N-[3H]methylscopolamine [( 3H]NMS), with a KD of 0.18 +/- 0.01 nM measured after culture in vitro for 7 days. Displacement of specific [3H]NMS binding demonstrated a muscarinic receptor with low affinity for pirenzepine (Ki: 240 nM); further investigation using antagonists, AF-DX 116 and 4-DAMP to discriminate between M2 and M3 receptors respectively, revealed low M2 affinity (Ki: 600 nM) and high M3 affinity (Ki: 2.4 nM), indicative of the M3 receptor subtype. The robust muscarinic receptor stimulation of [3H]inositol phosphate formation, previously observed in these cells, was confirmed. Inhibition of this response followed a similar profile to the binding data, exhibiting weak inhibitory effects for pirenzepine (Ki: 710 nM) and AF-DX 116 (Ki: 5000 nM), but a potent action for 4-DAMP (Ki: 2.4 nM). The opposite profile seen for AF-DX 116 and 4-DAMP is indicative of a M3 receptor subtype expressed on these cells and linked to phosphoinositide hydrolysis. Further studies demonstrated that M3 receptor stimulation caused a rapid, transient increase in the second messenger inositol 1,4,5-trisphosphate, suggesting that potential Ca(2+)-homeostatic and neuromodulatory effects may be mediated by this response.     

Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.     

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.     

The human astrocytoma cell line 1321 N1 contains M2-glandular type muscarinic receptors linked to phosphoinositide turnover.

1. Muscarinic receptors present in the human astrocytoma cell line 1321 N1 were characterized in radioligand binding studies and in functional studies of carbachol-stimulated phosphatidylinositol (PI) turnover. 2. In radioligand binding studies the muscarinic receptor in intact cells could be labelled using [3H]-N-methylscopolamine ([3H]-NMS) but not by [3H]-pirenzepine. In the intact cells these receptors displayed low pirenzepine affinity (pKi = 6.83) indicating that they were not of the M1 subtype. Furthermore, the 1321 N1 muscarinic receptors displayed low affinity for the two M2-cardiac selective ligands methoctramine (pKi = 5.82) and AF-DX 116 (pKi = 6.29). This pharmacology was consistent with the 1321 N1 cells containing a single population of muscarinic receptors that displayed a similar pharmacology to the M2-receptor present in exocrine gland tissue. 3. The M2-gland nature of the receptors was further indicated in the functional studies where antagonist affinities were determined from their ability to antagonize carbachol-stimulated PI turnover in 1321 N1 cells. pA2 values for pirenzepine (7.31), methoctramine (6.10) and AF-DX 116 (6.52) were similar to those determined in the binding studies. 4. From these studies we conclude that 1321 N1 astrocytoma cells contain an M2-gland muscarinic receptor which mediates muscarinic receptor-mediated stimulation of PI turnover in these cells.     

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.     

Direct binding studies on ileal and cardiac muscarinic receptors

1 Functional studies have indicated that muscarinic receptors in cardiac tissue differ from those in the ileum. In the present study ileal and cardiac muscarinic receptors identified by [3H]-N-methyl scopolamine ([3H]-NMS) were characterized and the selectivity of currently available ileal and atrial selective antagonists determined. 2 In terms of the current functional classification of muscarinic receptors both ileal and cardiac muscarinic receptors were of the M2 subtype based upon their low affinity for pirenzepine. 3 Cyclohexylphenyl(2-piperidinoethyl)silanol (CPPS), a highly ileal selective antagonist in functional studies, was unable to distinguish between ileal and atrial muscarinic receptors identified in binding studies. Furthermore, although AF-DX 116 and dicyclomine were able to differentiate atrial and ileal muscarinic receptors, neither compound was more than 2 fold selective. These data indicate that it is not possible to subclassify ileal and atrial muscarinic receptors using direct ligand binding studies with these antagonists. 4 In circular ileal smooth muscle, apparent heterogeneity of the M2 muscarinic receptor population was observed. Thus AF-DX 116 identified two populations of sites with affinities differing by 30 fold. These two populations of M2 muscarinic receptors may represent the typical M2 muscarinic receptors identified in cardiac tissue and the more recently discovered 'gland type' M2 muscarinic receptors. 5 The circular ileal smooth muscle tissue homogenate was able to decrease dramatically the apparent affinity of adiphenine. This activity, which appeared to result from a phenylmethylsulphonylfluoride (PMSF) sensitive protease effect, should be considered when conducting studies using this tissue preparation and compounds of similar structure to adiphenine.     

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.     

Analysis of muscarinic cholinoceptors mediating phosphoinositide hydrolysis in guinea pig cardiac muscle.

The muscarinic receptor mediating stimulation of PI hydrolysis in guinea pig atria and ventricles has been studied. The non-selective muscarinic agonist (+)-cis-dioxolane elicited this response, concentration-dependently, with a potency indicative of a low receptor reserve. The potency of a novel, M2-selective agonist, L-660,863 (-log EC50 = 6.3, atria; 6.0, ventricles) was observed to be lower than its apparent affinity (-log KA = 7.6) for M2 receptors, indicating an action probably mediated by a population distinct from that producing negative inotropy in the same tissue. The inhibition of the response to (+)-cis-dioxolane by several muscarinic antagonists (atropine, pirenzepine, AF-DX 116, methoctramine, HHSiD and pFHHSiD) generated an affinity profile for this receptor also dissimilar to that described for the receptor mediating the classical cardiac 'M2' response. Although no other muscarinic receptor mRNA has been detected in this tissue, these data suggest the presence of a second population of muscarinic sites, which may signify an M2 receptor diversity.     

Characterization of muscarinic receptors that mediate contraction of guinea-pig isolated trachea to choline esters: effect of removing epithelium.

1. The muscarinic receptor subtype that mediates contraction of guinea-pig trachea, in the presence and absence of epithelium, to acetic and carbamic acid choline esters was determined by use of preferential muscarinic receptor antagonists: pirenzepine (M1 receptor), methoctramine (M2 receptor) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3 receptor). 2. Acetylcholine (ACh), methacholine (MeCh), carbachol (CCh), bethanechol (BeCh) and oxotremorine induced concentration-dependent contraction of guinea-pig isolated tracheal strips in the presence and absence of epithelium. Contraction to acetic choline esters (ACh and MeCh) was augmented by removal of the epithelium, whereas contraction to carbamic acid choline esters (CCh and BeCh) and oxotremorine was not influenced by removal of the epithelium. 3. Pirenzepine, methoctramine and 4-DAMP caused parallel rightward displacements of the concentration-contraction curves to the muscarinic agonists. The pA2 values (determined from Arunlakshana-Schild graphs) for pirenzepine and 4-DAMP in guinea-pig trachea in the presence of epithelium were: ACh as the agonist, 7.6 and 9.0, respectively; CCh as the agonist, 7.6 and 9.1, respectively. The apparent pKB values for methoctramine with the same system were: ACh as the agonist, 5.6; CCh as the agonist, 5.6. Similar values were obtained with MeCh, BeCh and oxotremorine as the agonists. These values were agonist- and epithelium-independent. 4. It is concluded from the pA2 and apparent pKB values obtained for the muscarinic receptor antagonists used in this study that contraction of guinea-pig isolated trachea, with and without epithelium, to both acetic and carbamic acid choline esters is mediated via the muscarinic M3 receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)