Bisquaternary pyridinium oximes as allosteric inhibitors of rat brain muscarinic receptors

The mode of interaction of bisquaternary pyridinium oximes with rat brain muscarinic receptors in cerebral cortex and brain stem preparations was studied by the use of the tritium-labeled antagonist N-methyl-4-piperidyl benzilate ( [3H] 4NMPB). Binding of the labeled muscarinic antagonist was inhibited by these drugs, the most potent inhibitors being 1-(2-hydroxyiminoethylpyridinium)-1-(3-cyclohexylcarboxypyridin ium)dimethyl-ether (HGG-42) and its 3-phenylcarboxypyridinium analog (HGG-12) (apparent KI = 1.3-1.7 and 1.8-2.2 microM, respectively). Analysis of the binding properties suggested that binding of the muscarinic antagonist and the bisquaternary pyridinium oximes was nonexclusive. Kinetic binding data provide evidence that the drugs inhibit binding of muscarinic antagonists in an allosteric manner, with a resulting decrease in the rates of both association of [3H]4NMPB to the receptor and its dissociation from it. These effects were observed both in brain stem and in cortical preparations even after pretreatment and washing out of the inhibitors. The selective natures of HGG-12 and HGG-42 were apparent from their irreversible effects on the number of muscarinic binding sites. In brain stem, the presence of these drugs resulted in a loss of about 30% of binding sites, which accounts in part for the apparent decrease in maximal binding capacity observed in the equilibrium binding of [3H]4NMPB. In the cortex, however, only approximately 10% of the muscarinic receptors were lost upon exposure to these drugs. The decrease in the muscarinic receptor population of the brain stem was dependent on both concentration and time and occurred both in vitro and in vivo following injection of HGG-12 into rats. Unlike the in vitro loss of receptor sites, which was irreversible, the in vivo effect was restored 2 hr after the injection. Taken together, the results suggest that the bisquaternary oximes are allosteric inhibitors of the muscarinic acetylcholine receptor and may be capable of distinguishing between receptor states and inducing specific irreversible effects. Because of these properties, the drugs may prove extremely useful as sensitive probes in studies on the nature of the agonist-receptor-effector relationship.     

The oxime HGG-12 as a muscarinic acetylcholine receptor antagonist without intrinsic activity in cardiac membranes

Direct interactions of the bispyridinium oxime HGG-12 with muscarinic acetylcholine receptors were investigated in porcine cardiac atrial membranes. Competition binding experiments using the radiolabeled muscarinic receptor antagonist (³H)QNB revealed specific binding of HGG-12 to muscarinic acetylcholine receptors of porcine atrial membranes with a dissociation constant of 3.8×10^–7 mol/l. Muscarinic acetylcholine receptor-stimulated binding of the radiolabeled GTP analog (³⁵S)GTP[S] to guanine nucleotide binding proteins (G-proteins) was used to study antagonistic and possible agonistic effects of HGG-12 at muscarinic acetylcholine receptors. HGG-12 completely inhibited carbachol- and oxotremorine-stimulated (³⁵S)GTP[S] binding to pertussis toxin sensitive and insensitive G-proteins in a competitive manner. Inhibition constants (K_I) of HGG-12 for blockade of carbachol- and oxotremorine-stimulated GTP[S]-binding (9.7×10^–7 mol/l and 1.7×10^–6 mol/l, respectively) were higher by about a factor of 100 than those of the muscarinic acetylcholine receptor antagonist atropine. In the absence of muscarinic acetylcholine receptor agonists, HGG-12 by itself had no stimulatory effect on (³⁵S)GTP[S] binding in porcine atrial membranes. The results of this study show that the oxime HGG-12 is a competitive antagonist without intrinsic activity at porcine atrial muscarinic acetylcholine receptors. The stimulatory action of HGG-12 on muscarinic acetylcholine receptors which has been described by several authors is, therefore, suggested to be due to partial inhibition of acetylcholinesterase by the oxime rather than to direct agonism at muscarinic acetylcholine receptors.     

Reactivating and protective effects of pyridinium compounds in human erythrocyte acetylcholinesterase inhibition by organophosphates in vitro

The reactivation of human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by O-ethyl-S-2-di-isopropylaminoethyl methylphosphonothioate (VX) and the protection against AChE inhibition by O-1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) was studied with sixteen quaternized pyridinium compounds. TMB-4 which is known as a good reactivator of AChE inhibited by organophosphates proved to be the most effective reactivator. Of the tested newly synthetised compounds 3 were fairly good reactivators of methylethoxyphosphonylated AChE. These compounds have 2 pyridinium rings connected by a dimethylether link and a hydroxiiminomethyl group in position 2 of one pyridinium ring, while the radicals of the other pyridinium ring are benzoylcarbonyl, cyclohexylcarbonyl or amidocarbonyl residue.The rate of reactivation with these compounds followed a two-phase pattern, being fast at the beginning and then slowing down to an equilibrium. Kinetic treatment of the first-phase reaction course yielded the second-order rate constants of reactivation. All 3 compounds had similar reactivating efficiency (k _r values range from 0.8×10³ to 3.6×10³ M^–1 min^–1) and in effective concentrations (1 to 100 M) they also inhibited AChE (K _i(app) values range from 0.11 to 0.19 mM). Their reactivating properties were not better than those revealed by TMB-4 (k _r= 19.4×10³ M^–1 min^–1) which was tested as a reference compound.HGG-12, HGG-42 and HI-6 were also found to exert a good protective effect against AChE inhibition by Soman; no protection was obtained with TMB-4.     

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     

Prevention of guanine nucleotide-induced reductions in muscarinic agonist binding to rabbit ileal submucosal membranes by lidamidine and tetracaine

Summary The effects of low concentrations (< = 1 mM) of the anti-diarrhoeal drug lidamidine and sub-anaesthetic concentrations (< = 1 M) of tetracaine on the binding of the muscarinic antagonist [³H] N-methylscopolamine ([³H]NMS) and the high affinity muscarinic agonist, [³H] oxotremorine-M ([³H]oxo-M) to broken cell preparations of rabbit ileal submucosal membranes were investigated. High affinity [3H]NMS binding (K _D = 0.79 ± 0.05 nM, B _max = 1.75 ± 0.13 pmoles · mg^–1) was unaffected by either lidamidine or tetracaine. Inhibition of NMS binding by the muscarinic agonist carbachol was reduced by the nonhydrolyzable analogue of GTP, GppNHp. This effect of GppNHp was partially prevented by lidamidine. Analysis of equilibrium binding of the muscarinic agonist [³H] oxotremorine-M revealed that the binding of oxotremorine-M could be best described by the presence of the least two populations of sites with affinities of 0.9 ± 0.2 and 27.7 ± 0.9 nM respectively. High affinity [³H] oxotremorine-M binding was markedly reduced by GppNHp, GDPS and fluoride (5 mM). Neither lidamidine, nor tetracaine had any effect on the binding of oxotremorine-M when added alone. However, lidamidine and tetracaine prevented GppNHp, GDPS and fluoride-induced reductions in oxotremorine-M binding. The present findings are consistent with an allosteric interaction between lidamidine or tetracaine and GppNHp-induced reductions in oxotremorine-M binding to submucosal muscarinic receptors. These effects are discussed in relation to the observed action of lidamidine in potentiating presynaptic inhibition of acetylcholine release by muscarinic agonists. Send offprint requests to D. Bleakman     

The interaction of bis-pyridinium oximes with mouse brain muscarinic receptor

The bispyridinium oximes toxogonin [N, N′-oxydimethylene bis (pyridinium 4-aldoxim) dichloride] and its structural analogs HS-3, HS-6, HI-6 and MMB-4, and the bispyridinium salt SAD-128, which serve as antidotes to certain types of organophosphorus poisoning, bind competitively to mouse brain muscarinic receptors. This was determined in vitro employing the potent and specific muscarinic antagonist ³H-4NMPB (³H-4-N-methyl piperidyl benzilate). All the bispyridinium compounds also exerted a mild anti-acetylcholine activity (K_d = 10^?4?10^?5M) measured physiologically in the guinea pig ileum, which correlated well with the dissociation constants obtained from binding studies with mouse brain homogenate. The most potent muscarinic blocker was SAD-128 (K′_d = (7.1 ± 1.2) × 10^?6M for whole mouse brain), whose remarkable therapeutic action against soman intoxication may be partly attributed to this antimuscarinic activity.The binding data are best fitted by a competitive model, and the deviation from the law of mass action observed here may be related either to the heterogeneity of muscarinic receptors in the mouse brain or to nonequivalency of the number of binding sites for bisquaternary pyridines and 4-NMPB.     

Characterization of the muscarinic receptor in human tracheal smooth muscle

Summary Muscarinic receptors in human tracheal smooth muscle were characterized by radioligand binding and functional studies. Specific [³H]-(–)-quinuclidinylbenzilate ([³H]-(–)-QNB) binding to tracheal smooth muscle membranes was reversible, stereoselective and of high affinity (K _d=47±4 pmol/l;R _T=920±120 fmol/g tissue). Inhibition of specific [³H]-(–)-QNB binding by the M-1 selective antagonist pirenzepine was found to occur at relative high concentrations classifying the muscarinic receptor population as belonging to the M-2 subclass.Inhibition of specific [³H]-(–)-QNB binding by muscarinic agonists revealed the presence of high and low affinity sites in nearly equal proportions. 5-Guanylylimidodiphosphate converted high affinity sites into low affinity sites although its effect was minimal. Log dose-contraction curves of methacholine had Hill coefficients of 1.10±0.04 with pD₂-values of 6.75±0.02.Inhibition of specific [³H]-(–)-QNB binding by methacholine, however, was best described by a two binding site model with pK _i-values considerably lower. The difference between these affinity values points to the presence of substantial receptor reserve.     

Investigating the interaction of McN-A-343 with the M1 muscarinic receptor using its nitrogen mustard derivative and ACh mustard

Background and purpose:

We investigated how McN-A-343 inhibited the alkylation of the M₁ muscarinic receptor by its nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically.

Experimental approach:

We incubated the M₁ muscarinic receptor expressed in Chinese hamster ovary cells with ACh mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping the reaction and removing unreacted ligands, unalkylated receptors were measured using [³H]N-methylscopolamine. Analogous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model.

Key results:

The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants estimated from alkylation kinetics agreed with those measured by displacement of [³H]N-methylscopolamine binding.

Conclusions and implications:

Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively with ACh and N-methylscopolamine at the orthosteric site on the M₁ muscarinic receptor. Measuring how drugs modulate the kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric modulators and competitive inhibitors.     

The influence of guanyl-5′-yl imidodiphosphate and sodium chloride on the binding of the muscarinic agonist, [3H] cis methyldioxolane

[³H]Quinuclydinyl benzylate([³H]QNB) binding was carried out on crude synaptosomal membranes isolated from cat cerebral cortex. The specific binding showed a single type of site with K_D 0.25 nM, Hill number 0.89 and B_max 114 pmol/g protein. The local anesthetics procaine, tetracaine, and the adrenergic antagonists phentolamine and propranolol, in concentrations between 1 nM and 500 μM, inhibited [³H]QNB binding with K_i varying between 9 μM for procaine and 80 μM for propranolol. The Hill coefficients obtained from logit/log plots suggested that there was no cooperativity between the binding sites for local anesthetics. At various concentrations the inhibition by procaine and propranolol may appear as competitive or non-competitive. The Hill numbers obtained from the saturation curves suggest that there was no cooperativity between anesthetics and [³H]QNB binding sites. Neither 1 mM Ca²⁺ nor Mg²⁺ affected [³H]QNB binding or the action of the drugs. The effect of local anesthetics and adrenergic antagonists was reversible and these drugs did not protect the muscarinic receptor from the deleterious effect of Triton X-100 as was the case with muscarinic agents. Our findings suggest that local anesthetics inhibit [³H]QNB binding to the muscarinic receptor by acting at some accessory site but not on the true receptor site. The possible mechanism of action of local anesthetics on synaptic transmission is discussed.     

Synthesis and investigation of anti-inflammatory activity of novel nitric oxide donating hybrid drugs

A small library of nitric oxide donating groups, 4-acetamidophenyl-2-[{2-(nitrooxy)ethyl}(phenyl) amino]benzoate (5a–e) possessing a variety of substituents (–H, –NO₂, –CH₃, –acetamidophenyl, –SO₂NH₂) attached to the fourth position of phenyl ring were synthesized and evaluated for anti-inflammatory, analgesic and ulcerogenic potential. Structure–activity relationship data showed that the 2-phenylaminobenzoic acid skeleton is required for selective COX-2 inhibition. Among all compounds 4-acetamidophenyl-2-[{2-(nitrooxy)ethyl}(phenyl)amino]benzoate (5a) has shown potent anti-inflammatory activity while 4-acetamidophenyl-2-[{4-{(4-acetamidophenoxy)carbonyl} phenyl}{2-(nitrooxy)ethyl}amino]benzoate (5d) has shown potent analgesic activity compared to standard drug diclofenac.     

Properties of the muscarinic cholinergic receptors in rat atrium

Summary 1. Properties of the muscarinic cholinergic receptor sites in the rat atrial homogenate and microsomal fraction were studied by the use of tritium labelled 3-quinuclidinyl benzilate ([³H]-QNB), a potent muscarinic antagonist. 2. The specific [³H]-QNB binding to the receptor sites displayed saturability, stereospecificity as well as reversibility. 3. The competition studies showed that muscarinic antagonists were more potent than muscarinic agonists. 4. Certain neuromuscular blocking agents, antipsychotics, antiarrhythmics and antihistamines also were capable of interacting with the [³H]-QNB binding sites. However, - and -adrenergic agents, calcium antagonist (D-600) and ionophore (A-23187) failed to show any effect. 5. Analyses by the double reciprocal plot, Hill plot and Scatchard plot of the dependence of the specific [³H]-QNB binding on the concentration of QNB suggested that binding was occurring to a single population of receptor sites in the atrial homogenate or microsomal fraction. Further, there was no evidence of any detectable site to site interactions (positive or negative cooperative type). From the Scatchard plot, the equilibrium dissociation constant (K_D) of 1.1 nM was calculated and the Hill coefficient was close to 1.0 6. Interaction of the muscarinic antagonists with the [³H]-QNB sites showed the Hill coefficients close to 1.0 whereas for the agonists, the coefficients were much less than 1.0 indicating that agonist-receptor site interactions have some different characteristics from those following antagonist-receptor site interaction. 7. The rate and the maximal level of QNB binding to the receptor sites was markedly influenced by the temperature; various cations, on the other hand, displayed no effect either on the association or dissociation of QNB binding. The specific QNB binding exhibited a broad pH optimum from pH 6.0–8.5. 8. Treatment of the membrane fraction (or homogenate) with either phospholipase A or C and with p-chloromercuribenzoate caused significant inhibition of [³H]-QNB binding. 9. The QNB binding site was resistant to tryptic digestion. Even when about 40% of the membrane proteins were removed by the tryptic proteolysis, the [³H]-QNB binding ability of the membrane remained unaffected; in fact, the removal of tryptic proteolytic products by centrifugation markedly increased the specific QNB binding to the membrane.     

Effect of bay K 8644, a calcium channel agonist,on dog cardiac muscarinic receptors

To investigate further whether the effects of the dihydropyridine (DHP) drugs on calcium channels are related to those of these drugs on muscarinic receptors, the binding characteristics of the DHP calcium channel agonist, Bay K 8644, on muscarinic receptors and calcium channels were compared to those of the DHP calcium channel antagonists, nicardipine and nimodipine in the dog cardiac sarcolemma. Bay K 8644, nicardipine and nimodipine inhibited the specific [³H]QNB binding with K _i values of 16.7μM, 3.5μM and 15.5μM respectively. Saturation data of [³H]QNB binding in the presence of these DHP drugs showed this inhibition to be competitive. Bay K 8644, like nicardipine and nimodipine, blocked the binding of [³H]nitrendipine to the high affinity DHP binding sites, but atropine did not, indicating that the muscarinic receptors and the DHP binding sites on calcium channels are distinct. The K _i value of Bay K 8644 for the DHP binding sites was 4 nM. Nicardipine and nimodipine (K _i :0.1–0.2 nM) were at least 20 times more potent than Bay K 8644 in inhibiting [³H]nitrendipine binding. Thus, the muscarinic receptors were about 4000 times less sensitive than these high affinity DHP binding sites to Bay K 8644. These results suggest that the DHP calcium agonist Bay K 8644 binds directly to the muscarinic receptors but its interaction with the muscarinic receptors is not related to its binding to the DHP binding sites on calcium channels.     

EFFECT OF THYROID STATUS ON β-ADRENORECEPTORS AND MUSCARINIC RECEPTORS IN THE RAT LUNG

• 1 The effects of thyroid status on the specific binding of the muscarinic ligand (–)-[³H] quinuclidinyl benzilate (QNB) and of the β-adrenoreceptor ligand (–)-[³H] dihydroalprenolol (DHA) in the adult rat lung were investigated.
• 2 The specific binding of (–)-[³H] quinuclidinyl benzilate (QNB) to lung membranes was saturable and the equilibrium dissociation constant (K_D) determined from Scatchard analysis was 54 pM. Kinetic analysis of the binding of [³H] QNB yielded a K_D of 42 pM. [³H] QNB binding was inhibited by muscarinic agonists and antagonists, the order of their potency was l-hyoscyamine>atropine>scopolamine>oxotremorine>carbachol. These data were consistent with [³H] QNB binding to the muscarinic receptor.
• 3 Adult male rats treated for 2 weeks with the antithyroid agent 3-amino-1,2,4-triazole (ATZ) showed a 52% and 80% reduction in the serum concentration of triiodothyronine (T₃) and thyroxine (T₄) respectively. These hypothyroid rats also had a 39% decrease in the concentration of lung β-adrenoreceptors and a 37% decrease in the concentration of lung muscarinic receptors as compared to euthyroid controls. Concurrent treatment of rats with ATZ and T₄ for 2 weeks resulted in a reduction of 15% and 20% in the concentration of lung β-adrenoreceptors and muscarinic receptors respectively. The K_D values for [³H] DHA and [³H] QNB binding did not change with the ATZ or ATZ + T₄ treated groups.
• 4 Administration of T₄ (500 μg/kg/day) to male rats for 12 days did not result in any significant change in the concentration of either β-adrenoreceptors or muscarinic receptors compared to euthyroid controls. No change in the K_K values for [³H] DHA or [³H] QNB binding were detected.
• 5 The results show that hypothyroid rats have a reduced lung concentration of both β-adrenoreceptors and muscarinic receptors whereas in hyperthyroid rats these receptors do not significantly change from euthyroid controls.

     

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     

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     

An acute effect of triazolam on muscarinic cholinergic receptor binding in the human brain measured by positron emission tomography

An acute effect of triazolam, a potent benzodiazepine agonist, on cholinergic receptor binding in the human brain was measured by PET (positron emission tomography) using [¹¹C]N-methyl-4-piperidylbenzilate ([¹¹C]NMPB), a potent muscarinic cholinergic receptor antagonist. Two PET scans were performed in each subject: (1) control scan; (2) after oral administration of 0.5 mg triazolam or placebo. The previously discussed amnestic effect of triazolam was measured by immediate and delayed recall of meaningful and meaningless syllables. A compartment model employing the radioactivity in the cerebellum as an input function was used for the quantification of receptor binding. The binding parameter,k ₃, was decreased after triazolam administration in all measured regions, whereas no change was observed after placebo treatment. The reduction compared to the control study varied from 8.6±3.7% in the temporal cortex to 16.3±6.3% in the thalamus. Triazolam administration impaired both immediate and delayed recall of syllables, whereas placebo administration had no effects. Benzodiazepine agonists are reported to decrease the cortical acetylcholine release. The decrease of acetylcholine release in the synaptic cleft might be the explanation for the decreased binding of [¹¹C]NMPB.     

Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors

Background

Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity.

Results

At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 μM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [³H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [³⁵S]GTPγS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [³⁵S]GTPγS binding at M₂ and M₄ receptors. In contrast, 0.1 μM rapacuronium significantly increased ACh potency at M₁, M₃, and M₅ receptors. Kinetic measurements at M₃ receptors showed acceleration of the rate of ACh-induced [³⁵S]GTPγS binding by rapacuronium.

Conclusions

Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 μM) but not at high concentrations (10 μM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M₃ receptor-mediated bronchoconstriction.     

Interaction of benzilylcholine mustard,benzilylcholine and lachesine with the histamine receptor in the longitudinal muscle of guinea-pig ileum

1. The histamine receptors of longitudinal muscle strips from guinea-pig ileum were inactivated on extended exposure to concentrations of benzilylcholine mustard (BCM) above 10^-6M. These concentrations are 3 orders of magnitude greater than those required for inactivation of the muscarinic receptors. Mepyramine (3 × 10^-8M) afforded complete protection for the histamine receptor against the effects of BCM.2. Recovery from block at 30° C was very slow with a first-order rate constant of approximately 10^-7 s^-1.3. The rate constant, k₂, for the alkylation reaction in which the reversible BCM-receptor complex is converted into an irreversible complex is much greater than the rate constant, k_-1, for dissociation of the reversible complex. The value of the rate constant, k₁, for formation of the reversible complex was 1·1 ± 0·2 × 10³ M^-1 s^-1.4. These observations suggested that k₁ was probably not much greater than for BCM acting on the muscarinic receptor, even though the value of k₁ was reduced by more than 2 orders of magnitude. This effect has been established for benzilylcholine and lachesine, reversible analogues of BCM. For both drugs k₁ for the histamine receptor was approximately 3 orders of magnitude less than for the muscarinic receptor, whereas k₁ was greater by a factor of only 6.     

Mixed Dopaminergic/Serotonergic Properties of Several 2-Substituted 4-[2-(5-Benzimidazole)ethyl]-1-arylpiperazines

A series of substituted 4-[2-(5-benzimidazole)ethyl]-arylpiperazines was synthesized by introducing different substituents into position 2 of benzimidazole ring of 4-[2-(N,N-di-n-propyl-amino)ethyl]-1,2-diaminobenzenes. They were evaluated for in vitro binding affinity at the D₁ and D₂ dopamine and 5-HT_1A serotonin receptors using synaptosomal membranes of the bovine caudate nuclei and hippocampi, respectively. Tritiated SCH 23390 (D₁ receptor-selective), spiperone (D₂ receptor selective) and 8-OH-DPAT (5-HAT_1A receptor selective) were employed as the radioligands. Only compound 6 expressed a moderate binding affinity at the dopamine D₁ receptor, while the remaining ligands were inefficient or weak competitors of [³H]SCH 23390. Compound 12 was an absolutely inactive competitor of all three radioligands. Also, compound 7 was an inefficient displacer of [³H]-8-OH-DPAT. Compound 19 with a K_i value of 3.5 nM was the most potent competitor of [³H]spiperone and compound 13 (K_i = 3.3 nM) was the most efficient in displacing [³H]-8-OH-DPAT from the 5-HAT_1A serotonin receptor. Ligands 5, 6, 8–11 , and 13–20 expressed mixed dopaminergic/serotonergic activity in nanomolar range of concentrations with varying affinity ratios which strongly depended on the properties of the substituents introduced into position 2 of benzimidazole ring of the parent compounds.     

Structure-activity studies of N,N-dialkyl and cycloalkyl carbamate esters of dimethylethanolamine and choline with nicotinic and muscarinic cholinergic properties

Structure-activity studies were performed on a series of newly synthesized N-sub-stituted carbamate esters of choline and dimethylethanolamine which exhibited either or both nicotinic and muscarinic cholinergic properties. Substitutions on the carbamyl N consisted of varying lengths of dialkyls, diphenyl, and various heterocycles. The compounds were evaluated for their nicotinic and muscarinic receptor binding properties in rat brain membranes, their effect on muscarinic receptor-activated phosphoinositide (PI) turnover in rat cortical slices, and for muscarinic receptor-mediated intracelluar Ca release in a mouse adrenal carcinoma cell line transfected with an m1 muscarinic cholinergic receptor gene. N,N-diethylcarbamylcholine had the highest affinity for nicotinic cholinergic receptors; the K_i value of 1 × 10^?9 M approached that of nicotine and being 1/5 that for the dimethyl analogue. With increasing chain length of the dialkyl substituents the nicotinic affinity progressively decreased, while muscarinic affinity increased. N,N-diphenylcarbamylcholine had a K_i value of 1 × 10^?7 M for nicotinic receptors and exhibited nicotinic antagonist activity. Contrary to the results with [³H]-N-methylcarbamylcholine binding, the tertiary derivatives exhibited higher K_i values for [³H]-3-quinuclidinyl benzilate binding than the quarternaries; the tertiary dibutyl and diphenyl had the highest affinities with K_i values of 4 × 10^?7 M. The inhibition of muscarinic receptor-mediated PI turnover and release of intracellular calcium were related to the muscarinic antagonist potency of the compounds. © 1994 Wiley-Liss, Inc.