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.     

Different muscarine receptors mediate the prejunctional inhibition of [3H]-noradrenaline release in rat or guinea-pig iris and the contraction of the rabbit iris sphincter muscle

Summary To investigate the muscarine receptor type mediating inhibition of [³H]-noradrenaline release from the isolated rat and guinea-pig iris we have determined the potency of antimuscarinic drugs to antagonize the methacholine-induced inhibition of [³H]-noradrenaline overflow evoked by field stimulation (3 Hz, 2 min). The prejunctional apparent affinities were compared with those obtained for postjunctional muscarine receptors mediating the methacholine-induced contraction of the isolated rabbit iris sphincter muscle.Prejunctional apparent affinity constants of pirenzepine (6.67), himbacine (8.51), methoctramine (7.92), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.00), hexahydro-difenidol enantiomers (6.92, (R); 5.77, (S)) in the rat iris and methoctramine (7.58) in the guinea-pig iris indicate the presence of M₂ receptors. Although the post-junctional affinity constants in the rabbit iris sphincter of methoctramine (5.93), gallamine (3.92), and 4-DAMP (9.07) confirm our previous suggestions of the presence of M₃-like receptors, the results obtained with the hexahydro-difenidol enantiomers do not agree with that concept. The post-junctional affinity constants of the hexahydro-difenidol enantiomers were not different from the prejunctional values (6.86, (R); 5.55, (S)), indicating a similar and low degree of stereoselectivity for these stereoisomers at both receptor sites (14 and 17, (R)/(S)-ratios, respectively). Hence, the postjunctional muscarine receptor in the rabbit iris sphincter fails to exhibit the high degree of stereo selectivity observed for hexahydro-difenidol enantiomers at M₃ receptors on other smooth muscles.This study was supported by the Deusche Forschungsgemeinschaft (Fu 163/2) Send offprint requests to H. Fuder at the above address     

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     

Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors

Summary The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [³H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [³H]QNB was saturable, reversible and of high affinity (K _D = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M₁), AF-DX 116 (M₂) and 4-DAMP (M₃). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M₃ receptors and a much smaller population (12%) exhibiting characteristics of M₂ receptors. The binding curve for the displacement of [³H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (K _D = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (K _D = 4.4 M). When oxotremorine displacement of [³H]QNB binding was determined in the presence GTPS, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [³²P]NAD caused the [³²P]-labelling of a 40 kD protein that may represent the subunit(s) of G proteins that are known to be NAD-ribosylated by the toxin. We conclude that both M₃ and M₂ receptors may be coupled to G proteins in a pertussis-sensitive manner. Send offprint requests to T. W. Honeyman at the above address     

Muscarinic Receptor Binding Sites of the M4 Subtype in Porcine Lung Parenchyma

Abstract: Muscarinic acetylcholine receptors regulate distal airway resistance and secretion. The subtype expressed in the lung in different species remains uncertain. It has recently become possible to identify the M₄ subtype by careful comparison of antagonist affinities. We characterized the binding of [³H]quinuclidinyl benzilate ([³H]QNB) to muscarinic receptors in cell membranes from lung parenchyma of 2–8 week old pigs in comparison to cloned human M₃ and M₄ receptors expressed in COS cells, to M₂ in rat atria and to M₄ in bovine adrenal medulla. In porcine lung, [³H]QNB bound with high affinity (K_d=95±9pM) to a single homogeneous population of muscarinic receptor sites (B_max=340±10 fmol/mg protein). Competition studies showed that the affinity (expressed as pK_i) of 3 selective blockers was in close agreement between pig lung and cloned human m₄ (r=0.996). A series of 10 blockers showed affinities closely matching reported values for M₄ receptors of the adrenal medulla (r=0.965). Conversely, affinity values in porcine lung differed significantly (P< 0.05, t-test) from those determined in parallel with either human cloned M₃ or with rat atria expressing the M₂ subtype. We conclude that pig lung muscarinic receptor binding sites most closely resemble the M₄ subtype, in contrast to the M₃ subtype typical of large airways in this species.     

Characterization of the leukotriene D4 receptor in dimethylsulphoxide-differentiated U937 cells: comparison with the leukotriene D4 receptor in human lung and guinea-pig lung

The leukotriene D₄ receptor has been fully characterized by radioligand binding in membrane preparations from dimethyl sulphoxide-differentiated U937 cells, a human monocyte leukemia cell line, and, in parallel experiments, compared with leukotriene D₄ receptor found in human lung and guinea-pig lung preparations. [³H]Leukotriene D₄ specific binding in differentiated U937 cell membranes is of high affinity (K_D = 0.35 nM), saturable (B_max = 287 fmol/mg protein), with differentiation resulting in a 3–5-fold increase in the number of detectable binding sites. [³H]Leukotriene D₄-specific binding in differentiated U937 cell membranes displays several features of G-protein-cupled receptors, being inhibited by GTP analogues and sodium ions, but increased by divalent cations. These characteristics are shared with [³H]leukotriene D₄-specific binding in human and guinea-pig lung preparations. However, differences between these leukotriene D₄ receptor types were observed. [³H]Leukotriene D₄ equilibrium binding to differentiated U937 cell membranes could be dissociated to non-specific binding levels by 1000-fold excess of competing ligand, whereas binding to guinea-pig lung membranes was only partially dissociated under these conditions. In addition, differences in potency were demonstrated in competition studies with leukotriene E₄ and leukotriene C₄, although leukotriene D₄ and the leukotriene D₄-receptor antagonists MK-571 and ICI 204,219 were equipotent in competing for [³H]leukotriene D₄-specific binding in all three membranes preparations. In conclusion, the leukotriene D₄ receptor in differentiated U937 cell membranes resembles that in human lung, validating the use of this cell line as a suitable source of receptor in the development of potent specific antagonists.     

Radioligand binding to muscarinic receptors of bovine aortic endothelial cells

1. Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (-)-[3H]-N-methyl quinuclidinyl benzilate ([3H]-NMeQNB) was used as radioligand. 2. Binding of [3H]-NMeQNB to crude membranes of freshly isolated endothelial cells was atropine-displaceable and of high affinity (KD = 0.48 nM) to a single class of sites (maximum binding capacity: 14 +/- 3 fmol mg-1 protein). Stereospecificity of the binding sites was demonstrated in experiments in which [3H]-NMeQNB binding was inhibited by dexetimide in the nanomolar range (KI = 0.63 nM) and by levetimide, its stereoisomer in the micromolar range (KI = 3.2 microM) (selectivity factor: approximately 5000). 3. Drug competition curves indicated a single class of binding sites for antagonists and the following apparent affinities (KI, nM): methyl atropine: 1.1: 4-diphenylacetoxy N-methyl piperidine methyl bromide (4-DAMP): 3.4; pirenzepine: 16; 11-[2-diethylamino-methyl)-1-piperidinyl- acetyl]-5,11-dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one (AF-DX 116); 2.500. Competition of acetylcholine with [3H]-NMeQNB was best described by two affinity sites (or states) (KH = 0.82 microM, KL = 1.6 microM). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 microM), acetylcholine affinity (IC50) was slightly, but significantly reduced (factor approximately 4). 4. Binding of [3H]-NMeQNB to freshly harvested intact cells was also atropine-displaceable, stereospecific (selectivity factor: approximately 3500) and of high affinity (KD = 0.35 nM). The maximum binding capacity (9 +/- 2 fmol mg-1 total cell protein) was comparable to that of membranes and corresponded to approximately 900 binding sites per endothelial cell.(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.     

Allosteric Effects of the Alkane-bis-ammonium Compound W84 and of Tacrine on [3H]Pirenzepine Binding at M1-Receptors in Rat Cerebral Cortex

Abstract: The bis-quaternary W84, hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)-ammonium bromide], is a potent allosteric modulator of M₂-cholinoceptors. In this study we aimed at quantifying its allosteric effect on the dissociation of [³H]pirenzepine from M₁-cholinoceptors in rat cerebral cortex and to measure the effects on association and equilibrium binding of [³H]pirenzepine. For sake of comparison tacrine was included which is known to be a potent allosteric modulator of [³H]pirenzepine binding to M₁-receptors. Under control conditions (3 mM MgHPO₄, 50 mM Tris-HCl, pH 7.4, 23°) [³H]pirenzepine binding was characterized by K_D=5 nM and B_max=965 fmol/mg membrane protein, the rate constants amounting to k₊₁ = 5.0 μM^?1xmin^?1 and k_-1=0.031 min^?1. W84 and tacrine reduced [³H]pirenzepine binding concentration-dependently with IC₅₀-values of 1.9 μM and 2.6 μM, respectively. [³H]pirenzepine association was inhibited by the compounds with EC_50,ass⁼1.8 μM for W84 and EC_50,ass=2.4 μM for tacrine. The concentrations reducing the dissociation rate by 50% amounted to EC₅₀,_diss=21 μM for W84 and to EC_50,diss⁼54 μM for tacrine. Compared with W84, the dose-response curves of tacrine for the investigated effects were significantly steeper. In conclusion, W84 affected [³H]pirenzepine binding to M₁-receptors allosterically with a higher potency than tacrine but probably by a different mechanism.     

Characterization of porcine coronary muscarinic receptors

Summary To determine the muscarinic receptor subtype involved in the contractile response of coronary smooth muscle, we investigated the profiles of various muscarinic receptor antagonists competing for [³H]N-methyl-scopolamine ([³H]NMS) binding to membrane preparations from porcine coronary arteries. [³H]NMS binds to a single population of muscarinic binding sites with a K_D of 135 pM and a B_max of 57 fmol/mg. The affinity profiles of AF-DX 116 [11-2((–((diethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one], atropine, 4-DAMP [4-diphenylacetoxy-N-methylpiperidine methiodide], methoctramine [N,N-bis (6-((2-methoxybenzyl) amino)hexyl)-1,8-octane-diamine tetrahydrochloride], HHSiD [hexahydrosiladi-fenidol] and pirenzepine are consistent with binding to a mixed population of muscarinic binding sites, namely of the M₂ and M₃ subtype.Binding curves for AF-DX 116 and methoctramine are shallow with Hill-coefficients significantly less than unity. Comparison of data from binding studies with results obtained in functional experiments, i.e. antagonism of methacholine induced contraction of porcine coronary artery rings, it was found that only the low-affinity pK_i values of AF-DX 116 (6.26) and methoctramine (6.51) correlated well with functional pA₂ values.It is concluded that a mixed population of the M₂ and M₃ muscarinic receptor subtypes is present in porcine coronary arteries. Functional experiments do not support the contribution of the M₂ subtype to the contractile response. Cholinergic induced contractions of porcine coronary arteries appear to be evoked via stimulation of the muscarinic M₃ receptor subtype. However, since the compounds investigated here do not markedly discriminate between cloned m₃, m₄ and m₅ receptors the involvement of muscarinic receptors different from M₁, M₂ and M₃ cannot be excluded. Send offprint requests to M. Entzeroth at the above address     

Cholinergic muscarinic receptors of bovine adrenal medulla

Muscarinic receptor binding sites were identified on bovine adrenal medulla membranes, using [³H]-quinuclidinyl benzylate. A Scatchard analysis revealed a k_d of 0.07 nM and a Bmax of 3 fmol/mg prot. Muscarinic receptors of the medulla may, in part, be responsible for the release of catecholamines and the modulation of the cyclic nucleotide content of chromaffin cells.     

Muscarinic receptor subtypes mediating vasodilation in the pulmonary artery

Binding studies in several species have demonstrated a high proportion of M1 muscarinic receptors in the lung but their localization is uncertain. Using [3H]quinuclidinyl benzylate we have confirmed that binding sites with high affinity for pirenzepine account for 50% of muscarinic receptors in the rat lung. Our functional studies using the muscarinic antagonists 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), methoctramine and pirenzepine have demonstrated that the muscarinic receptor on the rat pulmonary artery endothelium which mediates vasodilation is of the M3 subtype and cannot account for the high proportion of M1 receptors identified in lung homogenates.     

Binding studies with [3H]cis-methyldioxolane in different tissues

Summary Special conditions - tricine buffer containing Ca²⁺ and Mg²⁺, 22°C (TCM) — allow to label a much higher proportion of muscarinic receptors by [³H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984 a). Taking the maximum number of binding sites, B _max, of [³H]QNB as 100%, B _max of [³H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [³H]CD and [³H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [³H]CD system, affinities of antagonists are slightly higher in the [³H]QNB system. In both assay systems receptors of heart and pons/ medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [³H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [³H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in B _max and not in the dissociation constant K _D. Competition of unlabelled pirenzepine with [³H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M₂-receptors) are labelled by [³H]CD. In regions rich in M₁ receptors like hippocampus (80% M₁ receptors) or cortex (65–70% M₁ receptors) the proportion of M₁ receptors labelled by [³H]CD is smaller than expected considering the concentration of M₁ receptors present in these tissues. Thus [³H]CD, under the conditions described in this paper, seems to label preferentially but not exclusively M₂ receptors in their agonist high affinity form. Send offprint requests to A. Closse at the above address     

[3H]sumatriptan labels both 5-HT1D and 5-HT1F receptor binding sites in the guinea pig brain: an autoradiographic study

We have used in vitro autoradiography to visualize [³H]sumatriptan binding sites in sections of guinea-pig and rat brain. In saturation studies, this ligand recognized a single saturable population of high affinity binding sites in all regions examined (pK_D = 8.3–9.3). While 5-HT and the sumatriptan derivative CP-122,288 (5-methyl-aminosulfonylmethyl-3-(N-methylpyrrolidin-2R-yl-methyl)-1H-indole) competed for [³H]sumatriptan binding sites with a high affinity and monophasic profile, displacement experiments with 5-carboxamidotryptamine revealed the existence of 2 classes of binding sites. The high affinity component (pKD = 9.2–9.9) probably corresponded to 5-HT_1B (rat) or 5-HT_1D (guinea-pig) receptors. The intermediate affinity (pK_D = 5.7–7.3) of the other component, taken together with their high affinity for [³H]sumatriptan, was similar to that of the cloned 5-HT_1F receptor. The regional distribution of the 5-HT_1B/1D [³H]sumatriptan binding sites was in agreement with previously published studies (striatonigral system, hypothalamus, central gray, superficial layer of the superior colliculus) and corresponded to the pattern of serotonin-5-O-carboxymethyl-glycyl [¹²⁵I]tyrosinamide labeling in consecutive sections. [³H]sumatriptan binding sites with a low affinity for 5-CT predominated in the intermediate neocortical layers, the claustrum (in the guinea-pig only), the mammillary nuclei, most of the thalamic nuclei and the principal oculomotor nucleus (in the guinea-pig only). This distribution is very similar to that of 5-HT_1F mRNA, indicating further the identity of these sites with 5-HT_1F receptors. Very high densities of 5-HT_1F sites were also found in the rat parafascicular nucleus.Some regions, such as the caudate/nucleus, the lateral geniculate nuclei and the spinal trigeminal nucleus appeared to contain both 5-HT_1B/1D and 5-HT_1F binding sites. Ketanserin had a low affinity for [³H]sumatriptan binding sites in all guinea-pig brain regions, compatible with the presence of the 5-HT_1D\ subtype. An exception was the substantia nigra, where a significant proportion of sites displayed an intermediate affinity for this compound, suggesting the presence of 5-HT_1D receptors. [³H]5-HT labeled 5-HT_1F sites in the claustrum and intermediate cortical layers in the guinea-pig. However these data show that [³H]sumatriptan, in the presence of 10 nM 5-carboxamidotryptamine, is a more suitable radioligand to study the distribution of 5-HT_1F binding sites.     

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.     

Binding characteristics and functional G protein coupling of muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes

Summary The non-selective labelled antagonist [³H]N-methyl-scopolamine ([³H]NMS) was used to identify muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes. Saturation and kinetic experiments revealed a binding site with a K_D-value of 0.2–0.3 nmol/l and a receptor concentration (B_max) of 100 fmol/mg protein. The affinities of eight selective muscarinic antagonists were determined and compared with those at M₁ (rat cerebral cortex), M₂ (rat heart), M₃ (rat submandibular gland) and M₄ (data from Dörje et al. 1991) receptors. The M₂-selective agent AF-DX 116, the group of M₂/M₄-selective compounds himbacine, AF-DX 384, AQ-RA 741 and methoctramine but also the M3-selective HHSiD showed affinities corresponding to M₂ and/or M₄ sites. The intermediate affinity of 4-DAMP favours a mixed M₂/M₄ receptor population mainly containing M₂ receptors. Two compounds, pirenzepine and AQ-RA 741, displayed biphasic displacement curves indicating the presence of a small population of putative M1 receptors. The rat duodenum antagonist binding profile, however, is not consistent with the presence of M3 receptors. We further demonstrate a concentration-dependent stimulation of [³⁵S]GTP[S] binding to duodenal G proteins by the muscarinic agonist oxotremorine. Estimation of the binding parameters of GTP[S] in absence and presence of oxotremorine provided evidence for a catalytic activation of G proteins by agonist-activated muscarinic receptors in rat duodenal membranes and a strong signal amplification on the G protein level. Send offprint requests to C. Liebmann at the above address     

Effects of NIK-247 and Tacrine on Muscarinic Receptor Subtypes in Rats

• 1.The purpose of this study was to compare the effect of NIK-247 on muscarinic receptor subtypes with that of tacrine (THA) in rats.
• 2.NIK-247 and tacrine dose dependently inhibited the binding of [³H]pirenzepine (M₁), [³H]AF-DX 384 (M₂), and [³H]4-DAMP (M₃). The IC₅₀ values for NIK-247 were 4.4×10⁻⁶ M, 1.1×10⁻⁵ M, and 1.5×10⁻⁵ M, respectively, whereas those for tacrine were 5.8×10⁻⁷ M, 2.0×10⁻⁶ M, and 5.8×10⁻⁶ M, respectively.
• 3.Gpp[NH]p, a GTP analogue, slightly shifted the curve of displacement of [³H]AF-DX 384 binding for NIK-247 to the right. However, Gpp[NH]p did not shift the curve of displacement of [³H]pirenzepine and [³H]4-DAMP binding to the right.
• 4.NIK-247 moderately decreased the rate of beating in right atrial preparations, but did not decrease it below 50% of control level.
• 5.These findings indicate that NIK-247 is an M₁ antagonist, M₂ partial agonist, and M₃ antagonist.

     

Presynaptic muscarinic receptor subtypes involved in the inhibition of acetylcholine and noradrenaline release in bovine cerebral arteries

Summary Experiments were performed in bovine cerebral arteries preincubated with [³H]-choline or [³H]-noradrenaline to analyze the presynaptic muscarinic receptors involved in inhibition of acetylcholine and noradrenaline release induced by electrical stimulation (4 Hz, 200 mA, 0.3 ms, 1 min). For this purpose, the actions of several muscarinic receptor antagonists on the ³H overflow and on the carbacol-induced inhibition of this overflow were assessed. The evoked [³H]-acetylcholine release and [³H]-noradrenaline release were markedly reduced by the presence of tetrodotoxin, Ca²⁺-free medium, and the inhibitor of both choline transport and choline acetyltransferase, AF64A. Chemical sympathetic denervation with 6-hydroxydopamine (6-OHDA) decreased the uptake of[³H]-noradrenaline, and AF64A reduced mainly the uptake of [³H]-choline, but also of [³H]-noradrenaline. Carbachol reduced the evoked [³H]-noradrenaline and [³H]-acetylcholine release; the IC₅₀ values were 0.37 and 0.43 mol/l, respectively.Atropine and 4-DAMP, but not AF DX 116, methoctramine or pirenzepine, increased the evoked [³H]-acetylcholine release. However, these muscarinic antagonists failed to modify the evoked [³H]-noradrenaline release. Carbachol inhibited the release of both acetylcholine and noradrenaline. The inhibition was blocked by the antagonists. The rank orders of potency (based on plC₅₀ values) were, in the case of [³H]-acetylcholine release, atropine > 4-DAMP >AF-DX 116 >- pirenzepine >- methoctramine, and, in the case of [³H]-noradrenaline release, atropine > 4-DAMP > AF-DX 116 >- methoctramine >-pirenzepine. These results suggest (1) that the prosynaptic receptors that modulate endogenous acetylcholine release are likely of the M₃ subtype, whilst those involved on the effect of the exogenous agonist Carbachol are of M₂ subtype, and (2) that those which inhibit noradrenaline release are probably a mixture of M₂ and M₃ subtypes as well. The autoinhibition of the acetylcholine release was funtionally active under our experimental conditions, while noradrenaline release does not appear to be modulated by muscarinic receptors in physiological conditions.Send offprint requests to G. Balfagón at the above address     

Muscarinic cholinergic receptors in the human right coronary artery: a receptor binding and autoradiographic study

Summary We used a combination of radioreceptor binding and autoradiographic techniques to study the pharmacological characteristics and anatomical localization of [³H]-quinuclidinyl benzilate (QNB) binding sites in the human right coronary artery. The ligand was bound to sections of the human right coronary artery in a manner consistent with the labelling of muscarinic receptors. The addition of pirenzepine or of carbachol to the incubation medium to generate displacement curves was indicative of the presence of M₁ and M₂ receptors in the right coronary artery. Autoradiography showed the localization of M₁ sites primarily in the medial layer of the right coronary artery. M₂ sites were located primarily in the adventitia. No [³H]-QNB binding sites were observed in the endothelium. A possible role of muscarinic receptors in the pathogenesis of coronary vasospasm is discussed.     

Displacement of [3H]oxotremorine-M binding by muscarinic antagonists in guinea-pig atrial and ileal membrane homogenates

Displacement of [3H]oxotremorine-M [( 3H]oxo-M) binding by muscarinic antagonists that are functionally non-selective (atropine), ileoselective (4-DAMP) or cardioselective (gallamine, pancuronium, vecuronium, himbacine) was investigated in guinea-pig atrial and ileal longitudinal muscle membranes. [3H]Oxo-M bound to a single population of high affinity sites in atrial (KD = 11.40 nM) and ileal (KD = 6.15 nM) membranes. Atropine displaced [3H]oxo-M binding sites in a competitive manner, showing similar affinities in the two tissues. 4-DAMP showed two binding sites in ileum but not in atria. The dissociation constant at the high affinity site in ileum was ca 5-fold lower than the value observed in atria, indicating ileoselectivity. Vecuronium also displaced [3H]oxo-M binding in a competitive manner and exhibited similar affinities in both tissues. Gallamine, pancuronium and himbacine displayed two binding sites in each of the two tissues with the majority of sites (ca. 60-80%) showing high affinity. Overall the cardioselective antagonists do not exhibit any consistent correlation between the affinities found in functional experiments and those determined in binding experiments.