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Esam E. El-fakahany - One of the best experts on this subject based on the ideXlab platform.
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Role of membrane cholesterol in differential sensitivity of muscarinic receptor subtypes to persistently bound Xanomeline.
Neuropharmacology, 2018Co-Authors: Alena Randáková, Esam E. El-fakahany, Vladimír Doležal, Eva Dolejsi, Vladimír Rudajev, Pavel Zimčík, Jan JakubíkAbstract:Xanomeline (3-(Hexyloxy)-4-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,5-thiadiazole) is a muscarinic agonist that is considered to be functionally selective for the M1/M4 receptor subtypes. Part of Xanomeline binding is resistant to washing. Wash-resistant Xanomeline activates muscarinic receptors persistently, except for the M5 subtype. Mutation of leucine 6.46 to isoleucine at M1 or M4 receptors abolished persistent activation by wash-resistant Xanomeline. Reciprocal mutation of isoleucine 6.46 to leucine at the M5 receptor made it sensitive to activation by wash-resistant Xanomeline. Lowering of membrane cholesterol made M1 and M4 mutants and M5 wild type receptors sensitive to activation by wash-resistant Xanomeline. Molecular docking revealed a cholesterol binding site in the groove between transmembrane helices 6 and 7. Molecular dynamics showed that interaction of cholesterol with this binding site attenuates receptor activation. We hypothesize that differences in cholesterol binding to this site between muscarinic receptor subtypes may constitute the basis for Xanomeline apparent functional selectivity and may have notable therapeutic implications. Differences in receptor-membrane interactions, rather than in agonist-receptor interactions, represent a novel possibility to achieve pharmacological selectivity. Our findings may be applicable to other G protein coupled receptors.
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JPET #90134 1
2016Co-Authors: Marianne K O Grant, Esam E. El-fakahanyAbstract:Persistent binding and functional antagonism by Xanomeline at th
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Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor
2016Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Esam E. El-fakahanyAbstract:Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M1 and M4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of Xanomeline on the binding characteristics and function of the M1 muscarinic receptor. Pretreatment of CHO cells that stably express the M1 receptor for 1 hr with increasing concentrations of Xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed Xanomeline-induced inhibition of [3H]NMS binding from monophasic to biphasic. The high-affinity Xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of Xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with Xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound Xanomeline developed with a time course that parallels the return of receptor activation by prebound Xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of Xanomeline, but not its persistent binding at an allosteric site. Furthermore, th
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Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.
Pharmacological research, 2015Co-Authors: Alena Randáková, Esam E. El-fakahany, Vladimír Doležal, Eva Dolejsi, Vladimír Rudajev, Pavel Zimčík, Jan JakubíkAbstract:We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and Xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist Xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in Xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant Xanomeline.
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Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.
PloS one, 2014Co-Authors: Eva Šantrůčková, Vladimír Doležal, Esam E. El-fakahany, Jan JakubíkAbstract:Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of Xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of Xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to Xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to Xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant Xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation Xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by Xanomeline. 6/NMS prevented formation of Xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of Xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover Xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.
Jan Jakubík - One of the best experts on this subject based on the ideXlab platform.
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Role of membrane cholesterol in differential sensitivity of muscarinic receptor subtypes to persistently bound Xanomeline.
Neuropharmacology, 2018Co-Authors: Alena Randáková, Esam E. El-fakahany, Vladimír Doležal, Eva Dolejsi, Vladimír Rudajev, Pavel Zimčík, Jan JakubíkAbstract:Xanomeline (3-(Hexyloxy)-4-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,5-thiadiazole) is a muscarinic agonist that is considered to be functionally selective for the M1/M4 receptor subtypes. Part of Xanomeline binding is resistant to washing. Wash-resistant Xanomeline activates muscarinic receptors persistently, except for the M5 subtype. Mutation of leucine 6.46 to isoleucine at M1 or M4 receptors abolished persistent activation by wash-resistant Xanomeline. Reciprocal mutation of isoleucine 6.46 to leucine at the M5 receptor made it sensitive to activation by wash-resistant Xanomeline. Lowering of membrane cholesterol made M1 and M4 mutants and M5 wild type receptors sensitive to activation by wash-resistant Xanomeline. Molecular docking revealed a cholesterol binding site in the groove between transmembrane helices 6 and 7. Molecular dynamics showed that interaction of cholesterol with this binding site attenuates receptor activation. We hypothesize that differences in cholesterol binding to this site between muscarinic receptor subtypes may constitute the basis for Xanomeline apparent functional selectivity and may have notable therapeutic implications. Differences in receptor-membrane interactions, rather than in agonist-receptor interactions, represent a novel possibility to achieve pharmacological selectivity. Our findings may be applicable to other G protein coupled receptors.
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Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.
Pharmacological research, 2015Co-Authors: Alena Randáková, Esam E. El-fakahany, Vladimír Doležal, Eva Dolejsi, Vladimír Rudajev, Pavel Zimčík, Jan JakubíkAbstract:We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and Xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist Xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in Xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant Xanomeline.
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Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.
PloS one, 2014Co-Authors: Eva Šantrůčková, Vladimír Doležal, Esam E. El-fakahany, Jan JakubíkAbstract:Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of Xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of Xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to Xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to Xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant Xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation Xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by Xanomeline. 6/NMS prevented formation of Xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of Xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover Xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.
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Equilibrium dissociation constants (KD) of [3H]NMS binding to the membranes of the cells treated with Xanomeline, carbachol, oxotremorine or pilocarpine is expressed in nM.
2014Co-Authors: Eva Šantrůčková, Vladimír Doležal, Esam E. El-fakahany, Jan JakubíkAbstract:Intact cells were exposed to 10 µM Xanomeline for 1, 3 or 10 min or for 10 min to 1 µM carbachol, 1 µM oxotremorine or 3 µM pilocarpine or sham-treated (control) and washed with KHB for 10 min or 1 hour and then membranes were prepared as described in Methods. *, different from control, a, different from shorter treatment with Xanomeline, b, different from 10-min washing, P
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Effects of NMS on delayed elevation of intracellular calcium levels induced by short-term application of Xanomeline at hM1 through hM4 receptors.
2014Co-Authors: Eva Šantrůčková, Vladimír Doležal, Esam E. El-fakahany, Jan JakubíkAbstract:Changes in the concentration of intracellular calcium (ordinate) are expressed as changes in fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. First (control) stimulation: 300 nM carbachol (CBC) for 5 s was applied. Second stimulation: At 300 s stimulation with 10 µM Xanomeline (Xano) was applied for 20 s. After 2-min washing with KHB cells were superfused with 10 µM NMS for 2 min and then washed with KHB for additional 4 min. Traces are averages of 8 to 12 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.015 at the base line to ±0.037 at peaks. Results were confirmed in 5 additional independent experiments. Parameters of Xanomeline effects are summarized in Table S6 in File S1.
Marianne K O Grant - One of the best experts on this subject based on the ideXlab platform.
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JPET #90134 1
2016Co-Authors: Marianne K O Grant, Esam E. El-fakahanyAbstract:Persistent binding and functional antagonism by Xanomeline at th
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Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor
2016Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Esam E. El-fakahanyAbstract:Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M1 and M4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of Xanomeline on the binding characteristics and function of the M1 muscarinic receptor. Pretreatment of CHO cells that stably express the M1 receptor for 1 hr with increasing concentrations of Xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed Xanomeline-induced inhibition of [3H]NMS binding from monophasic to biphasic. The high-affinity Xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of Xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with Xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound Xanomeline developed with a time course that parallels the return of receptor activation by prebound Xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of Xanomeline, but not its persistent binding at an allosteric site. Furthermore, th
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Effects of Xanomeline pretreatment, followed by washout, on [3H]NMS binding in CHO cells stably expressing rat M1 wild-type or rat M1 mutant123 muscarinic acetylcholine receptors.
2013Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Jan Jakubík, Vladimír Doležal, Esam E. El-fakahanyAbstract:For all figures, radioligand binding assays were performed for 1 hour at 37°C using naïve cells (closed squares), or after pretreating with Xanomeline for 1 h (open circles) or 24 h (open diamonds) followed by washing and immediate use in the binding assay, or after pretreating for 1 h followed by washing and incubation in agonist-free media for an additional 23 h before use in the binding assay (closed circles). Top row: Inhibition of binding of 0.2 nM [3H]NMS, was measured in (A) rat wild-type or (B) rat mutant123 cells in the presence of increasing concentrations of Xanomeline in naïve cells or after pretreating with increasing concentrations of Xanomeline. Bottom row: Saturation binding of [3H]NMS in (C) rat wild-type or (D) rat mutant123 cells. Cells were pretreated with 300 nM Xanomeline as described above and were subsequently incubated with increasing concentrations of [3H]NMS. Nonspecific binding was defined by 10 µM atropine. Values represent the means ± standard error of four to seven experiments conducted in triplicate.
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Effects of atropine on the long-term effects of Xanomeline pretreatments on [3H]NMS saturation binding in CHO cells stably expressing human M1 muscarinic acetylcholine receptors.
2013Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Jan Jakubík, Vladimír Doležal, Esam E. El-fakahanyAbstract:(A) Presence of atropine during 1 h pretreatment followed by 23 h wait in ligand-free media. Cells were pretreated with 3 µM Xanomeline in the presence of 10 µM atropine (open circles), or with atropine alone (open squares) for 1 h followed by washing and incubation for 23 h in ligand-free media. (B) Alternatively, atropine was added during the 23 h wait following pretreatment with Xanomeline and washing. Cells were pretreated with 3 µM Xanomeline (open circles) or sham treated (open squares) for 1 h followed by washing and incubation for 23 h in the presence of 10 µM atropine. For all experiments, cells were subsequently incubated for 1 h at 37°C with increasing concentrations of [3H]NMS. Nonspecific binding was defined by 10 µM atropine. Values represent the means ± standard error of four experiments conducted in triplicate.
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Effects of Xanomeline pretreatment on [3H]NMS saturation binding parameters in CHO hM1, rM1, and mutant123 cells.
2013Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Jan Jakubík, Vladimír Doležal, Esam E. El-fakahanyAbstract:Cells were pretreated with 300 nM or 3 µM Xanomeline for 1 h or 24 h at 37°C followed by washing and immediate use in the binding assay or for 1 h followed by washing and further incubation in the absence of free Xanomeline for 23 h. Cells were then incubated with increasing concentrations of [3H]NMS at 37°C for 1 h. Parameters derived from computer-assisted non-linear regression analysis as described in Methods are presented as mean ± S.E.M. of three to ten experiments conducted in triplicate.aEquilibrium dissociation constant for [3H]NMS binding.bMaximal cell-surface receptor density (dpm/100,000 cells).cControl, naïve cells were incubated with radioligand.dNot measured.*ANOVA followed by Dunnett’s post-test detected a significant difference (p
Vladimír Doležal - One of the best experts on this subject based on the ideXlab platform.
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Role of membrane cholesterol in differential sensitivity of muscarinic receptor subtypes to persistently bound Xanomeline.
Neuropharmacology, 2018Co-Authors: Alena Randáková, Esam E. El-fakahany, Vladimír Doležal, Eva Dolejsi, Vladimír Rudajev, Pavel Zimčík, Jan JakubíkAbstract:Xanomeline (3-(Hexyloxy)-4-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,5-thiadiazole) is a muscarinic agonist that is considered to be functionally selective for the M1/M4 receptor subtypes. Part of Xanomeline binding is resistant to washing. Wash-resistant Xanomeline activates muscarinic receptors persistently, except for the M5 subtype. Mutation of leucine 6.46 to isoleucine at M1 or M4 receptors abolished persistent activation by wash-resistant Xanomeline. Reciprocal mutation of isoleucine 6.46 to leucine at the M5 receptor made it sensitive to activation by wash-resistant Xanomeline. Lowering of membrane cholesterol made M1 and M4 mutants and M5 wild type receptors sensitive to activation by wash-resistant Xanomeline. Molecular docking revealed a cholesterol binding site in the groove between transmembrane helices 6 and 7. Molecular dynamics showed that interaction of cholesterol with this binding site attenuates receptor activation. We hypothesize that differences in cholesterol binding to this site between muscarinic receptor subtypes may constitute the basis for Xanomeline apparent functional selectivity and may have notable therapeutic implications. Differences in receptor-membrane interactions, rather than in agonist-receptor interactions, represent a novel possibility to achieve pharmacological selectivity. Our findings may be applicable to other G protein coupled receptors.
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Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.
Pharmacological research, 2015Co-Authors: Alena Randáková, Esam E. El-fakahany, Vladimír Doležal, Eva Dolejsi, Vladimír Rudajev, Pavel Zimčík, Jan JakubíkAbstract:We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and Xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist Xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in Xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant Xanomeline.
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Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.
PloS one, 2014Co-Authors: Eva Šantrůčková, Vladimír Doležal, Esam E. El-fakahany, Jan JakubíkAbstract:Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of Xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of Xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to Xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to Xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant Xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation Xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by Xanomeline. 6/NMS prevented formation of Xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of Xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover Xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.
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Effects of NMS on delayed elevation of intracellular calcium levels induced by short-term application of Xanomeline at hM1 through hM4 receptors.
2014Co-Authors: Eva Šantrůčková, Vladimír Doležal, Esam E. El-fakahany, Jan JakubíkAbstract:Changes in the concentration of intracellular calcium (ordinate) are expressed as changes in fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. First (control) stimulation: 300 nM carbachol (CBC) for 5 s was applied. Second stimulation: At 300 s stimulation with 10 µM Xanomeline (Xano) was applied for 20 s. After 2-min washing with KHB cells were superfused with 10 µM NMS for 2 min and then washed with KHB for additional 4 min. Traces are averages of 8 to 12 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.015 at the base line to ±0.037 at peaks. Results were confirmed in 5 additional independent experiments. Parameters of Xanomeline effects are summarized in Table S6 in File S1.
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Concentration response to acute treatment with Xanomeline.
2014Co-Authors: Eva Šantrůčková, Vladimír Doležal, Esam E. El-fakahany, Jan JakubíkAbstract:Cells were seeded, handled and loaded with Fura-2 as described in Methods. After an initial 10-s period cells were stimulated with 300 nM carbachol (CBC) for 5 s, washed with KHB for 5 min, then stimulated with 0.1 (black), 1 (red) or 10 µM (green) Xanomeline (Xano) for 20 s and washed with KBH for 7 min. Traces are averages from 10 to 12 cells from representative experiment confirmed by 3 independent experiments. Signal variation (SD) among cells ranges from ±0.019 at the base line to ±0.035 at peaks. Parameters of calcium response are summarized in Table S2 in File S1. Calculated pEC50 and EMAX of response to Xanomeline are in Table 1.
Kayla C De Lorme - One of the best experts on this subject based on the ideXlab platform.
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Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor
2016Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Esam E. El-fakahanyAbstract:Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M1 and M4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of Xanomeline on the binding characteristics and function of the M1 muscarinic receptor. Pretreatment of CHO cells that stably express the M1 receptor for 1 hr with increasing concentrations of Xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed Xanomeline-induced inhibition of [3H]NMS binding from monophasic to biphasic. The high-affinity Xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of Xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with Xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound Xanomeline developed with a time course that parallels the return of receptor activation by prebound Xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of Xanomeline, but not its persistent binding at an allosteric site. Furthermore, th
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Effects of Xanomeline pretreatment on [3H]NMS saturation binding parameters in CHO hM1, rM1, and mutant123 cells.
2013Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Jan Jakubík, Vladimír Doležal, Esam E. El-fakahanyAbstract:Cells were pretreated with 300 nM or 3 µM Xanomeline for 1 h or 24 h at 37°C followed by washing and immediate use in the binding assay or for 1 h followed by washing and further incubation in the absence of free Xanomeline for 23 h. Cells were then incubated with increasing concentrations of [3H]NMS at 37°C for 1 h. Parameters derived from computer-assisted non-linear regression analysis as described in Methods are presented as mean ± S.E.M. of three to ten experiments conducted in triplicate.aEquilibrium dissociation constant for [3H]NMS binding.bMaximal cell-surface receptor density (dpm/100,000 cells).cControl, naïve cells were incubated with radioligand.dNot measured.*ANOVA followed by Dunnett’s post-test detected a significant difference (p
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Effects of atropine on the long-term effects of Xanomeline pretreatments on [3H]NMS saturation binding in CHO cells stably expressing human M1 muscarinic acetylcholine receptors.
2013Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Jan Jakubík, Vladimír Doležal, Esam E. El-fakahanyAbstract:(A) Presence of atropine during 1 h pretreatment followed by 23 h wait in ligand-free media. Cells were pretreated with 3 µM Xanomeline in the presence of 10 µM atropine (open circles), or with atropine alone (open squares) for 1 h followed by washing and incubation for 23 h in ligand-free media. (B) Alternatively, atropine was added during the 23 h wait following pretreatment with Xanomeline and washing. Cells were pretreated with 3 µM Xanomeline (open circles) or sham treated (open squares) for 1 h followed by washing and incubation for 23 h in the presence of 10 µM atropine. For all experiments, cells were subsequently incubated for 1 h at 37°C with increasing concentrations of [3H]NMS. Nonspecific binding was defined by 10 µM atropine. Values represent the means ± standard error of four experiments conducted in triplicate.
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Antagonism of agonist-induced stimulation of PI hydrolysis by Xanomeline pretreatment in CHO cells stably expressing human M1 muscarinic acetylcholine receptors.
2013Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Jan Jakubík, Vladimír Doležal, Esam E. El-fakahanyAbstract:Cells were pretreated with increasing concentrations of Xanomeline for (A) 1 h followed by washing and incubation in agonist-free media for an additional 23 h, or (B) 24 h followed by washing. Cells were subsequently incubated for 1 h at 37°C in the absence (asterisks) or presence of carbachol at 1 µM (open circles) or 10 mM (closed circles); oxotremorine at 0.1 µM (open triangles) or 1 mM (closed triangles); or Xanomeline at 0.03 µM (open diamonds) or 0.1 mM (closed diamonds). Maximal carbachol-induced PI response in untreated cells was (A) 17000±3700 dpm, (B) 20000±1000 dpm. Values represent the means ± standard error of two to four experiments conducted in triplicate.
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Effects of Xanomeline pretreatment, followed by washout, on binding of receptor-saturating concentrations of [3H]NMS and [3H]QNB in CHO cells stably expressing human M1 muscarinic acetylcholine receptors.
2013Co-Authors: Marianne K O Grant, Kayla C De Lorme, Meredith J Noetzel, Jan Jakubík, Vladimír Doležal, Esam E. El-fakahanyAbstract:The binding of (A) 2.9 nM [3H]NMS or (B) 1.4 nM [3H]QNB was measured in the presence of increasing concentrations of Xanomeline in naïve cells (closed squares), or after pretreating with increasing concentrations of Xanomeline for 1 h (open circles) or 24 h (open diamonds) followed by washing and immediate use in the binding assay, or after pretreating with increasing concentrations of agonist for 1 h followed by washing and incubation in agonist-free media for an additional 23 h before use in the binding assay (closed circles). Nonspecific binding was defined by 10 µM atropine. Values represent the means ± standard error of three to four experiments conducted in triplicate.
