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.     

Pharmacological characterization and distribution of muscarinic receptors in human placental syncytiotrophoblast brush-border and basal plasma membranes

Based on the existence of choline acetyltransferase and acetylcholine in human placenta, we have investigated the presence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta. Radioligand binding assay, using [³H]N-methyl-scopolamine as tracer, showed the existence of acetylcholine muscarinic receptors in brush-border (K_d 0.28±0.04 nM; B_max 9.4±1.6 fmol/mg protein) and basal plasma membranes (K_d 0.24±0.05 nM; B_max 34.3±6.3 fmol/mg protein). In order to perform a pharmacological characterization of these receptors, competition binding experiments were carried out using the muscarinic receptor antagonists pirenzepine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6H-pyrido(14)benzodiazepine (AF-DX 116), himbacine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), dicyclomine and hexahydro-sila-difenidol (HHSD). The results obtained showed that the muscarinic receptors in brush-border and basal plasma membranes belong to different subtypes. In brush-border membranes, the receptor found match in terms of affinity for the antagonists with the muscarinic M₁ receptor subtype (K_i pirenzepine, 13.6±8.2 nM; K_i AF-DX 116, 1680±271 nM; K_i himbacine, 212±6.5 nM; K_i 4-DAMP, 1.5±0.4 nM; K_i dicyclomine, 5.1±0.8 nM; K_i HHSD, 34.3±7.3 nM), whereas the receptor in basal plasma membrane seems to be of the muscarinic M₂ receptor subtype (K_i pirenzepine, 202±48 nM; K_i AF-DX 116, 124±60 nM; K_i himbacine, 20.6±4.8 nM; K_i 4-DAMP, 4.5±1.2 nM; K_i dicyclomine, 54.6±22 nM; K_i HHSD, 89.2±15.8 nM). The results obtained show the existence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta with a different distribution pattern in terms of number of receptors and distribution of different subtypes. The functional significance of these findings is as yet unknown, but these receptors probably mediate different functions as they belong to different subtypes and are coupled to different second messengers.     

Muscarinic receptor heterogeneity in rat central nervous system. I. Binding of four selective antagonists to three muscarinic receptor subclasses: a comparison with M2 cardiac muscarinic receptors of the C type

We previously observed that [3H]NMS recognizes three types of muscarinic receptors in rat brain (one M1 subclass with high affinity for pirenzepine, and two M2 subclasses with low affinities for pirenzepine), based on distinct affinity and kinetic constants of [3H]NMS for these three subclasses. In this work, we investigated the binding of four selective antagonists to these three (the M1 and two M2) subclasses. We were able to demonstrate that cardiac-like M2 receptors with low affinity for pirenzepine and low affinity for N-methylscopolamine were present not only in cerebellum (as previously shown; see introduction) but also in cortex, striatum, and hippocampus, and the two M2 receptor subclasses were discriminated by dicyclomine, 4-DAMP, and gallamine, as well as by AF-DX 116 and [3H]NMS. Our findings also suggested that the biphasic association and dissociation kinetics of [3H]NMS observed in various brain regions reflect sequential binding to the different receptors.     

Affinity of muscarinic receptor antagonists for three putative muscarinic receptor binding sites

1. A range of muscarinic receptor antagonists were examined for affinity at the M1 muscarinic binding site, present in rat cerebrocortical membranes and the M2 muscarinic binding sites of rat cardiac and submaxillary gland membranes. 2. The results obtained were consistent with the presence of three classes of muscarinic binding site. 3. Both the M1 binding site, labelled by [3H]-pirenzepine ([3H]-Pir) in rat cerebrocortical membranes, and the M2 gland binding site, labelled by [3H]-N-methyl scopolamine ([3H]-NMS) in rat submaxillary gland membranes, displayed higher affinity for pirenzepine, dicyclomine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and cyclohexylphenyl (2-piperidinoethyl) silanol (CPPS) than did the M2 binding sites of cardiac membranes labelled by [3H]-NMS. 4. The M2 cardiac sites displayed higher affinity for methoctramine, himbacine and AF-DX 116 than did either the M1 binding site of cerebrocortical membranes or the M2 gland binding site present in rat submaxillary gland membranes. 5. The M1 and M2 gland binding sites could only be distinguished by considering the absolute affinity of compounds for these two sites. Thus, all compounds, with the exception of 4-DAMP, displayed between a 2 and 8 fold higher affinity for the M1 than for the M2 gland binding site. There were no antagonists with higher M2 gland than M1 affinity.     

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.     

Characterization of the muscarinic receptor subtype that mediates the contractile response of acetylcholine in the swine myometrium

The aim of the present study was to characterize the subtype of muscarinic receptor that mediates acetylcholine-induced contractions in the nonpregnant proestrus swine myometrium by means of mechanical, radioligand ([3H]quinuclidinyl benzilate) binding and biochemical (measurement of cyclic AMP) approaches. Acetylcholine (-logEC50, 6.12), oxotremorine-methiodide (6.47), methacholine (6.35), carbachol (6.18) and muscarine (6.33) caused contractile responses of the uterine circular muscle, with a similar maximum amplitude, but pilocarpine and McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium) were ineffective in causing contraction. The contractile response to acetylcholine was antagonized by the following muscarinic receptor antagonists in a competitive manner (with pA2 values in parentheses): atropine (8.95), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.83), tropicamide (7.07), himbacine (7.01), pirenzepine (6.42) and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyri do[2,3 b][1,4]benzodiazepin-6-one (AF-DX116, 5.96). Electrical field stimulation (10 Hz) caused tetrodotoxin- and atropine-sensitive contractions in the circular muscle. All muscarinic receptor antagonists decreased the electrical field stimulation-induced contraction in a concentration-dependent manner. The order of inhibition (-logIC50) was 4-DAMP (8.35) > tropicamide (6.72) > himbacine (6.54) > pirenzepine (6.31)> AF-DX116 (6.13). Acetylcholine did not affect the cytoplasmic cyclic AMP level, regardless of the presence or absence of forskolin, suggesting the absence of functional muscarinic M2 and/or M4 receptors in the swine myometrium. The receptor binding study indicated that circular muscle layers of the swine myometrium contained a single class of [3H]quinuclidinyl benzilate binding site (Kd = 0.92 nM; Bmax = 126.6 fmol/mg protein). Specific binding was displaced by muscarinic receptor antagonists in the following order (with pKi value and Hill coefficient in parentheses): atropine (8.22 and 0.93) > 4-DAMP (8.18 and 0.94) > tropicamide (6.78 and 0.93) > pirenzepine (5.46 and 0.92) > AF-DX116 (5.12 and 0.94). The present results suggest that in circular muscle layers of the swine myometrium, exogenous and endogenous acetylcholine cause contraction through activation of muscarinic M3 receptors present on smooth muscle cells.     

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.     

Subclassification of atrial and intestinal muscarinic receptors of the rat--direct binding studies with agonists and antagonists

1. Although extensively investigated, the extent of differences between receptors mediating negative inotropic and chronotropic responses is still unclear. In the present study atrial and intestinal muscarinic receptors were identified by [3H]-N-methyl-scopolamine ([3H]-NMS) binding and the affinities of some presumably inotropy- or chronotropy-selective agonists and several antagonists determined. 2. All the agonists tested showed similar affinity for right and left atrial receptors. Accepting an affinity difference of 0.4 log units as experimental error, none of the agonists tested was selective for either atrium. 3. Affinity differences of the cardioselective antagonists himbacine, AF-DX 116 and methoctramine and the M1-selective antagonist dicyclomine for right and left atrial muscarinic receptors were also minimal (less than 2 fold selective). When compared to intestinal receptors, AF-DX 116 was 3 to 4 fold, methoctramine 10 to 13 fold selective and himbacine and dicyclomine non-selective. 4. These data provide evidence for differences between atrial and intestinal but not between right and left atrial muscarinic receptors.     

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)     

Activation of T84 cell chloride channels by carbachol involves a phosphoinositide-coupled muscarinic M3 receptor.

Muscarinic agonists stimulate Cl- secretion across monolayers of the colon tumor epithelial cell line, T84. The muscarinic receptor has been characterized in T84 cell homogenates by radioligand binding using [3H]N-methylscopolamine ([3H]NMS). [3H]NMS bound to a single population of sites at 25 degrees C in 100 mM NaCl, 20 mM HEPES, 10 mM MgCl2, pH 7.4 buffer, with calculated Kd = 278 (+/- 44) pM and Bmax = 40 (+/- 6) fmol/mg protein (n = 4). Binding was reversible (diss. t1/2 = 18 +/- 3 min) and stereoselective (dexetimide Ki = 0.3 nM) much greater than levetimide (Ki = 8300 nM). Antagonists exhibited the following rank order of potencies and Ki values (nM): atropine (0.54) greater than 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) (0.84) greater than dicyclomine (14) = hexahydrosiladifenidol (18) greater than pirenzepine (136) greater than AF-DX 116 (3610). The same sequence was observed for inhibition of carbachol-induced 125I efflux from T84 monolayers. This is indicative of an M3 'glandular' muscarinic receptor. Coupling to second messenger systems was examined by labelling monolayers with [14C]arachidonic acid (AA) or [3H]inositol. Carbachol (0.3 mM) did not release [14C]AA from labelled lipids, but ionomycin produced a dose-dependent increase in media [14C]AA. Carbachol (0.3 mM) elevated inositol monophosphate 14-fold. The results suggest that muscarinic agonists stimulate Cl- secretion by interacting with an M3 receptor coupled to inositide lipid hydrolysis.     

Pharmacological differences between muscarinic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase inhibition

Pharmacological differences between muscarinic cholinergic receptors coupled to phosphoinositide turnover and those coupled to adenylate cyclase were studied. Stimulation of muscarinic receptors from SK-N-SH human neuroblastoma cells resulted in phosphoinositide hydrolysis, but not in inhibition of cAMP formation. As has been shown previously, stimulation of muscarinic receptors from NG108-15 neuroblastoma x glioma cells, on the other hand, resulted in inhibition of cAMP formation without any observable phosphoinositide hydrolysis. These two cell lines provide a useful model system in which to study differential coupling of muscarinic cholinergic receptors. Inhibition of [3H]N-methyl scopolamine [( 3H]NMS) binding and inhibition of carbachol-stimulated function by the antagonists pirenzepine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were studied in this system. Pirenzepine inhibited [3H]NMS binding in both cell lines with low affinity (Ki of 130 and 160 nM in NG108-15 and SK-N-SH cells respectively), indicating that both cell lines express M2 receptors. None of the three antagonists studied exhibited any clear selectivity for the receptors in one cell line over those of the other. In contrast, several agonists including acetylcholine, bethanechol and carbachol exhibited pronounced selectivity. These agonists inhibited [3H]NMS binding to membranes from SK-N-SH cells with IC50 values that were 17-, 3- and 38-fold higher, respectively, than those of NG108-15 cells. This selectivity was still observed when whole cells rather than membranes were studied. These findings indicate that pharmacological differences between receptors coupled to phosphoinositide turnover and those coupled to cAMP inhibition can be detected with certain agonists, but not with the antagonists pirenzepine, AF-DX 116 or 4-DAMP.     

Muscarinic receptor heterogeneity in rat central nervous system. II. Brain receptors labeled by [3H]oxotremorine-M correspond to heterogeneous M2 receptors with very high affinity for agonists

We compared the binding characteristics of muscarinic receptors labeled by [3H]oxotremorine-M ([3H]oxo-M) in homogenates of brain cortex and heart from rat. In both tissues [3H]oxo-M bound, with the same KD (6.5 nM), to a fraction of the receptors labeled by [3H]-N-methylscopolamine ([3H]NMS). This [3H]oxo-M receptor population represented, respectively, 15-20% and 35-40% of the total number of [3H]NMS receptors in cortex and heart. The three unlabeled agonists oxotremorine, carbamylcholine, and pilocarpine, when tested in competition with [3H]oxo-M, displayed a homogeneous super high affinity toward [3H]oxo-M-labeled receptors, and were unable to discriminate between brain and heart receptors labeled by [3H]oxo-M. By contrast, selective muscarinic antagonists showed some selectivity for either brain or heart [3H]oxo-M-labeled receptors. We analyzed competition curves between [3H]oxo-M and secoverine, pirenzepine, AF-DX 116, dicyclomine, or gallamine, assuming the existence of one or two receptor subclasses. Heart muscarinic receptors labeled by [3H]oxo-M were homogeneous M2 receptors of the C type with very low affinity for pirenzepine (Ki = 400 nM). Brain [3H]oxo-M-labeled receptors were heterogeneous receptors, with 30% (the B type) having a higher affinity for dicyclomine and a lower affinity for AF-DX 116 and gallamine than cardiac receptors, whereas the remaining 70% (the C type) showed "cardiac-like" binding properties. Both [3H]oxo-M-labeled subtypes in cortex homogenates had a low affinity for pirenzepine, indicating that [3H]oxo-M labeled only B and C (M2) receptors in this tissue. GTP inhibited completely [3H]oxo-M binding in heart homogenates with an IC50 at 300 nM. In cortex homogenates, GTP showed the same potency, but its efficacy was much lower (with only 30% maximal inhibition). [3H]oxo-M dissociation kinetics were monophasic in heart homogenates and biphasic in cortex homogenates. [3H]oxo-M dissociation from both tissues was slowed by gallamine and d-tubocurarine and accelerated by GTP. We found no correlation between B versus C [3H]oxo-M receptors, GTP-sensitive versus GTP-insensitive receptors, and rapidly versus slowly dissociating receptors, suggesting that [3H] oxo-M labeled a large variety of muscarinic receptor-regulatory protein complexes, all having an SH affinity for agonists.     

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)     

Muscarinic receptor binding characteristics of a human neuroblastoma SK-N-SH and its clones SH-SY5Y and SH-EP1

The present study examines the muscarinic receptor binding characteristics of parent human neuroblastoma (SK-N-SH) and its neuroblast (SH-SY5Y) and epithelial-like (SH-EP1) clones using [3H]methylscopolamine [( 3H]NMS). Specific [3H]NMS binding to intact SK-N-SH and SH-SY5Y cells was saturable with a Kd of 0.2 nM and Bmax of 100-150 fmol/mg protein. Specific [3H]NMS binding to whole cell preparations of SH-EP 1 could not be detected. Pharmacological analysis of the binding site both in whole cells and membranes of SK-N-SH are indicative of an homogeneous receptor population possessing low affinity for the M1-selective antagonist pirenzepine. The muscarinic receptors expressed by the neuroblast clone, SH-SY5Y were further characterized and shown to have the properties of an homogeneous M3 subtype with low affinity for the M1-selective antagonist pirenzepine and the M2-cardioselective AFDX-116 but high affinity for 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP). In conclusion the SH-SY5Y neuroblastoma should provide an important human neuronal cell model with which to define the regulation of post-receptor events driven by a single receptor population.     

The allosteric binding profile of himbacine: a comparison with other cardioselective muscarinic antagonists

The possibility of an allosteric interaction by himbacine, a cardioselective antagonist, with rat cardiac muscarinic receptors was studied. Himbacine allosterically decelerated the dissociation of bound [3H]N-methylscopolamine [( 3H]NMS) in a concentration-dependent manner with an IC50 value of 103.7 microM. When compared to the IC50 values of other cardioselective antagonists, the rank order of potencies was: methoctramine greater than gallamine greater than himbacine greater than AF-DX 116. In contrast, the potencies of these compounds to displace [3H]NMS binding were: himbacine greater than methoctramine greater than AF-DX 116 greater than gallamine. The allosteric potencies were found not to be correlated with binding potencies (correlation coefficient = -0.15). A striking common feature of the cardioselective antagonists is their ability to bind to an allosteric site on cardiac muscarinic receptors.     

Characterization of the muscarinic receptor subtypes in the rat urinary bladder

We investigated the nature of the muscarinic receptors present in the rat urinary bladder by performing binding studies with various selective (pirenzepine, AF-DX 116, hexahydrosiladifenidol, benzhexol, 4-diphenyl-acetoxy-N-methyl piperidine methiodide, dicyclomine, secoverine) and classical (N-methylscopolamine, atropine) antagonists. Competition experiments were carried out against [3H]N-methyl scopolamine at 30 degrees C in Na+/Mg2+ HEPES buffer; non-specific binding was determined in the presence of 1 microM 3-quinuclidinyl benzilate. Of all the antagonists examined, only AF-DX 116 exhibited a heterogeneous binding profile (nH less than 1). Computer-assisted analysis showed that the data fitted best to a two-binding site model, revealing the existence of high and low affinity receptors. The affinity values of AF-DX 116, determined in binding experiments carried out in heart and gland homogenates, allowed us to classify the rat urinary bladder receptors into cardiac and glandular subtypes. We suggest that the glandular receptor subtype is involved in smooth muscle contraction, since AF-DX 116 was equally potent in inhibiting smooth muscle contraction and the secretion of saliva.     

Muscarinic receptor subtypes in human and rat colon smooth muscle.

Muscarinic receptor subtypes in human and rat colon smooth muscle homogenates were characterized with [3H]N-methylscopolamine ([3H]NMS) by ligand binding studies. [3H]NMS saturation experiments show the existence of a homogeneous population of non-interacting binding sites with similar affinity (KD values of 1.38 +/- 0.20 nM in human colon smooth muscle and 1.48 +/- 0.47 nM in rat colon smooth muscle) and with Hill slopes close to unity in both samples of tissue. However, a significant (P less than 0.01) increase in muscarinic receptor density (Bmax) is found in human colon (29.9 +/- 2.9 fmol/mg protein) compared with rat colon (17.2 +/- 1.5 fmol/mg protein). Inhibition of [3H]NMS binding by non-labelled compounds shows the following order in human colon: atropine greater than AF-DX 116 greater than pirenzepine. Whereas in rat colon the rank order obtained is atropine greater than pirenzepine greater than AF-DX 116. Atropine and pirenzepine bind to a homogeneous population of binding sites, although pirenzepine shows higher affinity to bind to the sites present in rat colon (Ki = 1.08 +/- 0.08 microM) than those in human colon (Ki = 1.74 +/- 0.02 microM) (P less than 0.05). Similarly, IC50 values obtained in AF-DX 116 competition experiments were significantly different (P less than 0.01) in human colon (IC50 = 1.69 +/- 0.37 microM) than in rat colon (IC50 = 3.78 +/- 0.75 microM). Unlike atropine and pirenzepine, the inhibition of [3H]NMS binding by AF-DX 116 did not yield a simple mass-action binding curve (nH less than 1, P less than 0.01) suggesting the presence of more than one subtype of muscarinic receptor in both species. Computer analysis of these curves with a two binding site model suggests the presence of two populations of receptor. The apparent Ki1 value for the high affinity binding site is 0.49 +/- 0.07 microM for human colon smooth muscle and 0.33 +/- 0.05 microM for rat colon smooth muscle. The apparent Ki2 for the low affinity binding site is 8.01 +/- 1.0 microM for human samples and 6.07 +/- 1.1 microM for rat samples. These values are close enough to suggest that the first subtype of muscarinic receptor may be considered cardiac (M2) and the second subtype glandular (M3). The relative densities of the receptor subtypes are significantly different for both species. Human colon samples show the major densities of subtype M2, 22.62 +/- 1.11 fmol/mg protein, this represents 75.66 +/- 3.73% of the total receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding characteristics of the muscarinic receptor subtype of the NG108-15 cell line

Summary Kinetic, saturation and competition binding studies were conducted on the muscarinic receptor binding sites labeled by [³H]N-methylscopolamine ([³H]NMS) in membranes prepared from NG108-15 cells. The pharmacology of the NG108-15 cell muscarinic receptors was compared to that of the M₁ receptors of rat cortex labeled using [³H]pirenzepine, the M₂ and M₃ receptors of rat heart and submaxillary gland, respectively, labeled using [³H]NMS and the muscarinic receptors of the PC12 cell line also labeled using [³H]NMS.The rate of dissociation of [³H]NMS from the NG10815 cell muscarinic receptor was similar to that obtained at the M₃ receptor and at the muscarinic receptor of the P12 cells but was slower that the dissociation rate obtained at the M₂ cardiac muscarinic receptor. The Kd of [³H]NMS in the NG108-15 cells was significantly lower than that obtained at the M₂ and M₃ receptor but was similar to the Kd obtained in PC12 cells. In competition studies the affinity estimates for AF-DX 116, 4-DAMP, methoctramine and pirenzepine were not consistent with the presence of either an M₁, M₂ Or M₃ receptor but were identical to the affinity estimates obtained at the muscarinic receptor of the PC12 cell line.On the basis of these data we conclude that the muscarinic receptor present in the NG108-15 cells is different to the M₁, M₂ or M₃ subtypes already described but is similar to the muscarinic receptor present in the PC12 cell line. Since NG108-15 cells expresses mRNA for the m4 muscarinic receptor gene described by Bonner et al. (1987) we propose that the muscarinic receptors present in this cell line be denoted as M4 receptors.Send offprint requests to A. Michel at the above address     

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)