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Stanislav Tucek - One of the best experts on this subject based on the ideXlab platform.
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Clozapine interaction with the M2 and M4 subtypes of muscarinic receptors.
European journal of pharmacology, 1999Co-Authors: P Michal, M Lysíková, E E El-fakahany, Stanislav TucekAbstract:Available evidence indicates that the antipsychotic drug clozapine acts as a partial agonist at the muscarinic M4 and as an antagonist at the M2 receptors. We wondered whether there is indeed a fundamental difference between its action on these two receptor subtypes, and whether it interacts with their classical or allosteric binding sites. In experiments on Chinese hamster ovary cells stably expressing the M2 or M4 receptors, clozapine inhibited the binding of the specific muscarinic ligand [3H]N-Methylscopolamine to either receptor subtype. The affinity of the high-affinity sites for clozapine was diminished by GTP in the way expected for agonists on both the M2 and the M4 receptor subtypes. Arunlakshana-Schild plots of data obtained in saturation binding experiments with [3H]N-Methylscopolamine at different concentrations of clozapine were linear with a slope of unity. Clozapine did not alter the time course of [3H]N-Methylscopolamine dissociation from muscarinic M2 or M4 receptors. It inhibited the synthesis of cyclic AMP in cells expressing the M4 receptor subtype, but did not measurably inhibit the synthesis of cyclic AMP in cells expressing the M2 receptor subtype. We conclude that clozapine has a high affinity for muscarinic M2 and M4 receptor subtypes, that it associates with the classical and not with the allosteric binding site, and that it acts as a partial agonist on both the M2 and the M4 receptor subtype.
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Positive allosteric action of eburnamonine on cardiac muscarinic acetylcholine receptors
European Journal of Pharmacology, 1996Co-Authors: Jan Proska, Stanislav TucekAbstract:Abstract It was discovered recently that alcuronium and strychnine (which is a precursor of alcuronium) allosterically increase the affinity of cardiac muscarinic receptors for the antagonist, N-Methylscopolamine. We have now investigated the effects of l-eburnamonine and vincamine, which are both closely related to strychnine. In experiments on rat heart atria, l-eburnamonine was found to increase the binding of [3H]N-Methylscopolamine with Ehlert's cooperativity coefficient α = 0.35, which indicates that the strength of its allosteric action is close to that of alcuronium and strychnine (α = 0.31 and 0.44, respectively). However, the affinity of l-eburnamonine for the cardiac muscarinic receptors is lower than the affinities of alcuronium and strychnine (KAR = 22.6 μM, 0.15 μM, and 3.4 μM, respectively). In spite of its extremely close similarity to l-eburnamonine, vincamine has a negative allosteric effect on the binding of [3H]N-Methylscopolamine (α = 4.1; KAR = 22.8 μM). It is likely that a systematic investigation of the allosteric effects of the analogues of strychnine will not only yield new allosteric effectors on muscarinic receptors, but also clarify the structural features responsible for the direction (positive or negative) of their allosteric effect.
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Positive allosteric action of eburnamonine on cardiac muscarinic acetylcholine receptors
European Journal of Pharmacology, 1996Co-Authors: Jan Proska, Stanislav TucekAbstract:Abstract It was discovered recently that alcuronium and strychnine (which is a precursor of alcuronium) allosterically increase the affinity of cardiac muscarinic receptors for the antagonist, N-Methylscopolamine. We have now investigated the effects of l-eburnamonine and vincamine, which are both closely related to strychnine. In experiments on rat heart atria, l-eburnamonine was found to increase the binding of [3H]N-Methylscopolamine with Ehlert's cooperativity coefficient α = 0.35, which indicates that the strength of its allosteric action is close to that of alcuronium and strychnine (α = 0.31 and 0.44, respectively). However, the affinity of l-eburnamonine for the cardiac muscarinic receptors is lower than the affinities of alcuronium and strychnine (KAR = 22.6 μM, 0.15 μM, and 3.4 μM, respectively). In spite of its extremely close similarity to l-eburnamonine, vincamine has a negative allosteric effect on the binding of [3H]N-Methylscopolamine (α = 4.1; KAR = 22.8 μM). It is likely that a systematic investigation of the allosteric effects of the analogues of strychnine will not only yield new allosteric effectors on muscarinic receptors, but also clarify the structural features responsible for the direction (positive or negative) of their allosteric effect.
Vladimír Doležal - One of the best experts on this subject based on the ideXlab platform.
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Binding of N-Methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors.
Scientific reports, 2017Co-Authors: Jan Jakubík, Esam E. El-fakahany, Alena Randáková, Pavel Zimčík, Vladimír DoležalAbstract:Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-Methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.
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Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy
British Journal of Pharmacology, 2011Co-Authors: Jan Jakubík, Helena Janickova, Esam E. El-fakahany, Vladimír DoležalAbstract:BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-γ−thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [35S]GTPγS and [3H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M2 muscarinic acetylcholine receptor. KEY RESULTS Agonists displayed biphasic competition curves with the antagonist [3H]-N-Methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [3H]GDP from membranes. The inverse agonist N-Methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from Gi/o G-proteins but only its dissociation from Gs/olf G-proteins. CONCLUSIONS AND IMPLICATIONS These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of Gi/o versus Gs/olf G-proteins that are not identified by conventional GTPγS binding.
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Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of presynaptic M2 and M4 muscarinic receptors in rat brain
The Journal of pharmacology and experimental therapeutics, 2007Co-Authors: Eva Machová, Jan Jakubík, Esam E. El-fakahany, Vladimír DoležalAbstract:We studied the effects of 3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) wash-resistant binding on presynaptic muscarinic regulation of electrically evoked [ 3 H]acetylcholine (ACh) release from rat brain slices. In both cortical and striatal tissues that possess M 2 and M 4 autoreceptors, respectively, immediate application of 10 μM xanomeline had no effect on evoked [ 3 H]ACh release or its inhibition by 10 μM carbachol. In contrast, preincubation with 1, 10, or 100 μM xanomeline for 15 min decreased evoked release of ACh measured after 53 min of washing in xanomeline-free medium in a concentration-dependent manner. The maximal inhibitory effect equaled the immediate effect of the muscarinic full agonist carbachol, and it was completely (at 1 and 10 μM xanomeline) or partially (at 100 μM xanomeline) blocked by 1 μM N -Methylscopolamine. Neither presence of N -Methylscopolamine during 100 μM xanomeline treatment nor previous irreversible inactivation of the classical receptor binding site using propylbenzylcholine mustard in cortical slices prevented the inhibitory effect of wash-resistantly bound xanomeline. Treatment of cortical slices with xanomeline slightly decreased the number of muscarinic binding sites, and it markedly decreased affinity for N -Methylscopolamine. When applied as in acetylcholine release experiments, xanomeline did not impair presynaptic α 2 -adrenoceptor-mediated regulation of noradrenaline release. The functional studies in brain tissue reported in this work demonstrate that xanomeline can function as a wash-resistant agonist of native presynaptic muscarinic M 2 and M 4 receptors with both competitive and allosteric components of action.
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Determinants of positive cooperativity between strychnine-like allosteric modulators and N-Methylscopolamine at muscarinic receptors.
Journal of Molecular Neuroscience, 2006Co-Authors: Jan Jakubík, Vladimír DoležalAbstract:It has been shown previously that the third extracellular loop (o3) and its vicinity play a critical role in allosteric modulation at muscarinic acetylcholine receptors (mAChRs) (Ellis et al., 1993; Krejci and Tucek, 2001; Buller et al., 2002). In this study interaction of four chemically related substances (strychnine, its dimethoxy derivate brucine, precursor for synthesis of strychnine Wieland-Gumlich aldehyde (WGA), and precursor for synthesis of alcuronium propargyl-WGA) with orthosteric antagonist N-Methylscopolamine (NMS) was investigated on the M3 subtype of mAChRs mutated at the o3 loop.
Klaus Mohr - One of the best experts on this subject based on the ideXlab platform.
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bisquaternary dimers of strychnine and brucine a new class of potent enhancers of antagonist binding to muscarinic m2 receptors
Bioorganic & Medicinal Chemistry, 2003Co-Authors: Darius P Zlotos, Stefan Buller, Ulrike Holzgrabe, Klaus MohrAbstract:Abstract Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M 2 receptors was examined. The compounds retarded the dissociation of the antagonist [ 3 H] N -Methylscopolamine ([ 3 H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [ 3 H]NMS-occupied M 2 receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.
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Contribution of lateral substituents in symmetrical and non-symmetrical heptane-bisammonio compounds to the allosteric stabilization of N-Methylscopolamine binding to muscarinic M2 receptors.
Archiv der Pharmazie, 2003Co-Authors: Markus Staudt, Klaus Mohr, Christian Tränkle, Ulrike HolzgrabeAbstract:Allosteric modulators are able to enhance or decrease the equilibrium binding of orthosteric agonists or antagonists. The treatment of Alzheimer's disease and the organophosphorus poisoning can take advantage of the enhancement of the ligand binding. Prerequisite is the formation of ternary complexes consisting of the receptor protein, the orthosteric ligand, e. g. N-Methylscopolamine (NMS), and the alloster optimized for the corresponding orthoster. In this study, heptane-bisammonio compounds were optimized with regard to the orthosteric antagonist NMS. Comparing pairs of compounds characterized by phthalimides, cyclohexanedicarbonic acid imide and succinimides at both ends or a phthalimide at one end and either of the three imides at the other end stressed the importance of an aromatic moiety at both ends of the heptane-bisammonio chain.
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opposite effects of alcuronium on agonist and on antagonist binding to muscarinic receptors
European Journal of Pharmacology, 1996Co-Authors: Andrea Maas, Klaus MohrAbstract:Abstract Alcuronium is known to retard allosterically the dissociation of [3H]N-Methylscopolamine from muscarinic M2 receptors, thereby augmenting the binding of this antagonist. Functionally, alcuronium behaves as a weak antimuscarinic agent and induces in combination with N-Methylscopolamine an overadditive antimuscarinic action with oxotremorine-M as the agonist. The effect of alcuronium on the binding of [3H]oxotremorine-M was studied in porcine heart homogenates. Agonist binding was concentration dependently inhibited with a Ki = 0.48 ± 0.03 μM (means ± S.D., n = 3). Under identical conditions [3H]N-Methylscopolamine binding was elevated. Alcuronium, 100 μM, which nearly prevented the dissociation of [3H]N-Methylscopolamine, retarded the rate of dissociation of [3H]oxotremorine-M only by a factor of two. These findings support the notion that the overadditive antimuscarinic action of alcuronium in conjunction with N-Methylscopolamine is based on a shift by alcuronium of the interplay between agonist and antagonist in favour of the antagonist.
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potentiation by alcuronium of the antimuscarinic effect of n Methylscopolamine in guinea pig left atria
European Journal of Pharmacology, 1995Co-Authors: Andrea Maas, Evi Kostenis, Klaus MohrAbstract:Abstract Alcuronium is known to stabilize allosterically the binding of the muscarinic antagonist N-Methylscopolamine to muscarinic M2 receptors and thus to elevate the equilibrium binding of N-Methylscopolamine in homogenized cardiac tissue. In order to check for a functional consequence of this effect, the action of alcuronium alone and in combination with N-Methylscopolamine was determined in contracting guinea pig left auricles with oxotremorine-M as the negative inotropic agonist. For sake of comparison, the allosteric modulator W84 = hexane-1,6-bis(dimethyl-3′-phthalimidopropyl-ammonium bromide) was included. Alcuronium displayed a weak antimuscarinic action (pA2 = 5.7). In conjunction with 10−7 M N-Methylscopolamine, alcuronium (≥ 10−6 M) induced a more pronounced antimuscarinic effect than expected for a combination of competitive antagonists. The extent of overadditivity with combinations of W84 and 10−7 M N-Methylscopolamine was smaller. In conclusion, alcuronium potentiates the antimuscarinic effect of N-Methylscopolamine in contracting cardiac preparations with high effectivity.
Alice Schade Powers - One of the best experts on this subject based on the ideXlab platform.
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Role of acetylcholine in negative patterning in turtles (Chrysemys picta).
Behavioral neuroscience, 2009Co-Authors: Alice Schade Powers, Phillip Hogue, Christian Lynch, Brian W. Gattuso, Shmuel Lissek, Christine NayalAbstract:Turtles were run on a negative patterning task involving 2 positive elements, a key with white stripes on a black background, and a solid red key, and a compound stimulus combining the 2 elements, white stripes on a red background. Injections of scopolamine, Methylscopolamine, or saline were started at the same time that the compound stimulus was introduced, after the animals had been autoshaped to press the key for each of the elements. Scopolamine disrupted the learning of negative patterning, but Methylscopolamine had no effect. In contrast, learning of a simple discrimination between the elements was not affected by scopolamine. These results show that muscarinic cholinergic receptors are involved in the learning of negative patterning in turtles.
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A role for acetylcholine in spatial memory in turtles
Physiology & behavior, 1994Co-Authors: Martin Petrillo, Carol A. Ritter, Alice Schade PowersAbstract:The present research was undertaken to determine whether acetylcholine plays a role in memory for a maze in turtles. Cholinergic cells have been observed in the basal forebrain of turtles, and the basal forebrain of turtles projects to the dorsal cortex, a region that has been implicated in associative function. In Experiment 1, turtles were trained on an X-maze for water reward and then given lesions of the dorsal cortex or basal forebrain or sham lesions and retested postoperatively on the maze. Both dorsal cortex and basal forebrain lesions impaired performance on the maze. In Experiment 2, turtles were trained on the maze and then given saline, scopolamine, or Methylscopolamine on a 1-day retention test. Scopolamine in the higher doses impaired maze performance on the test day, but Methylscopolamine did not. The highest dose of scopolamine had no effect on measures of general activity, showing that the effects of the drug were specific to the learned task.
Jan Jakubík - One of the best experts on this subject based on the ideXlab platform.
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Binding of N-Methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors.
Scientific reports, 2017Co-Authors: Jan Jakubík, Esam E. El-fakahany, Alena Randáková, Pavel Zimčík, Vladimír DoležalAbstract:Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-Methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.
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Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy
British Journal of Pharmacology, 2011Co-Authors: Jan Jakubík, Helena Janickova, Esam E. El-fakahany, Vladimír DoležalAbstract:BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-γ−thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [35S]GTPγS and [3H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M2 muscarinic acetylcholine receptor. KEY RESULTS Agonists displayed biphasic competition curves with the antagonist [3H]-N-Methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [3H]GDP from membranes. The inverse agonist N-Methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from Gi/o G-proteins but only its dissociation from Gs/olf G-proteins. CONCLUSIONS AND IMPLICATIONS These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of Gi/o versus Gs/olf G-proteins that are not identified by conventional GTPγS binding.
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Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of presynaptic M2 and M4 muscarinic receptors in rat brain
The Journal of pharmacology and experimental therapeutics, 2007Co-Authors: Eva Machová, Jan Jakubík, Esam E. El-fakahany, Vladimír DoležalAbstract:We studied the effects of 3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) wash-resistant binding on presynaptic muscarinic regulation of electrically evoked [ 3 H]acetylcholine (ACh) release from rat brain slices. In both cortical and striatal tissues that possess M 2 and M 4 autoreceptors, respectively, immediate application of 10 μM xanomeline had no effect on evoked [ 3 H]ACh release or its inhibition by 10 μM carbachol. In contrast, preincubation with 1, 10, or 100 μM xanomeline for 15 min decreased evoked release of ACh measured after 53 min of washing in xanomeline-free medium in a concentration-dependent manner. The maximal inhibitory effect equaled the immediate effect of the muscarinic full agonist carbachol, and it was completely (at 1 and 10 μM xanomeline) or partially (at 100 μM xanomeline) blocked by 1 μM N -Methylscopolamine. Neither presence of N -Methylscopolamine during 100 μM xanomeline treatment nor previous irreversible inactivation of the classical receptor binding site using propylbenzylcholine mustard in cortical slices prevented the inhibitory effect of wash-resistantly bound xanomeline. Treatment of cortical slices with xanomeline slightly decreased the number of muscarinic binding sites, and it markedly decreased affinity for N -Methylscopolamine. When applied as in acetylcholine release experiments, xanomeline did not impair presynaptic α 2 -adrenoceptor-mediated regulation of noradrenaline release. The functional studies in brain tissue reported in this work demonstrate that xanomeline can function as a wash-resistant agonist of native presynaptic muscarinic M 2 and M 4 receptors with both competitive and allosteric components of action.
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Determinants of positive cooperativity between strychnine-like allosteric modulators and N-Methylscopolamine at muscarinic receptors.
Journal of Molecular Neuroscience, 2006Co-Authors: Jan Jakubík, Vladimír DoležalAbstract:It has been shown previously that the third extracellular loop (o3) and its vicinity play a critical role in allosteric modulation at muscarinic acetylcholine receptors (mAChRs) (Ellis et al., 1993; Krejci and Tucek, 2001; Buller et al., 2002). In this study interaction of four chemically related substances (strychnine, its dimethoxy derivate brucine, precursor for synthesis of strychnine Wieland-Gumlich aldehyde (WGA), and precursor for synthesis of alcuronium propargyl-WGA) with orthosteric antagonist N-Methylscopolamine (NMS) was investigated on the M3 subtype of mAChRs mutated at the o3 loop.