Dolezal VladimirDepartment of Neurochemistry, Institute of Physiology, Academy of Sciences of the Czech Republic, CZ-14220 Prague Czech RepublicPhone : Fax : Send E-Mail to Dolezal Vladimir
Apolipoprotein E4 (apoE4) is the most prevalent genetic risk factor for Alzheimer's disease. We utilized apoE4-targeted replacement mice (approved by the Tel Aviv University Animal Care Committee) to investigate whether cholinergic dysfunction, which increases during aging and is a hallmark of Alzheimer's disease, is accentuated by apoE4. This revealed that levels of the pre-synaptic cholinergic marker, vesicular acetylcholine transporter in the hippocampus and the corresponding electrically evoked release of acetylcholine, are similar in 4-month-old apoE4 and apolipoprotein E3 (apoE3) mice. Both parameters decrease with age. This decrease is, however, significantly more pronounced in the apoE4 mice. The levels of cholinacetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) were similar in the hippocampus of young apoE4 and apoE3 mice and decreased during aging. For ChAT, this decrease was similar in the apoE4 and apoE3 mice, whereas it was more pronounced in the apoE4 mice, regarding their corresponding AChE and BuChE levels. The level of muscarinic receptors was higher in the apoE4 than in the apoE3 mice at 4 months and increased to similar levels with age. However, the relative representation of the M1 receptor subtype decreased during aging in apoE4 mice. These results demonstrate impairment of the evoked release of acetylcholine in hippocampus by apoE4 in 12-month-old mice but not in 4-month-old mice. The levels of ChAT and the extent of the M2 receptor-mediated autoregulation of ACh release were similar in the adult mice, suggesting that the apoE4-related inhibition of hippocampal ACh release in these mice is not driven by these parameters. Evoked ACh release from hippocampal and cortical slices is similar in 4-month-old apoE4 and apoE3 mice but is specifically and significantly reduced in hippocampus, but not cortex, of 12-month-old apoE4 mice. This effect is accompanied by decreased VAChT levels. These findings show that the hipocampal cholinergic nerve terminals are specifically affected by apoE4 and that this effect is age dependent.
Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the alpha3beta2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved beta-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at alpha4beta2 and alpha3beta2-nAChRs (IC50 ~0.17 and >3 muM, respectively) expressed in Xenopus oocytes. In contrast, at alpha7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 muM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with alpha3beta2-nAChRs, while it inhibited cell growth via alpha7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at alpha7 and alpha3beta2-nAChRs.
Transgenic APPswe/PS1dE9 mice modeling Alzheimer's disease demonstrate ongoing accumulation of beta-amyloid fragments resulting in formation of amyloid plaques that starts at the age of 4-5 months. Buildup of beta-amyloid fragments is accompanied by impairment of muscarinic transmission that becomes detectable at this age, well before the appearance of cognitive deficits that manifest around the age of 12 months. We have recently demonstrated that long-term feeding of trangenic mice with specific isocaloric fish oil-based diets improves specific behavioral parameters. Now we report on the influence of short-term feeding (3 weeks) of three isocaloric diets supplemented with Fortasyn (containing fish oil and ingredients supporting membrane renewal), the plant sterol stigmasterol together with fish oil, and stigmasterol alone on markers of cholinergic neurotransmission in the hippocampus of 5-month-old transgenic mice and their wild-type littermates. Transgenic mice fed normal diet demostrated increase in ChAT activity and attenuation of carbachol-stimulated GTP-gamma(35)S binding compared to wild-type mice. None of the tested diets compared to control diet influenced the activities of ChAT, AChE, BuChE, muscarinic receptor density or carbachol-stimulated GTP-gamma(35)S binding in wild-type mice. In contrast, all experimental diets increased the potency of carbachol in stimulating GTP-gamma(35)S binding in trangenic mice to the level found in wild-type animals. Only the Fortasyn diet increased markers of cholinergic synapses in transgenic mice. Our data demonstrate that even short-term feeding of transgenic mice with chow containing specific lipid-based dietary supplements can influence markers of cholinergic synapses and rectify impaired muscarinic signal transduction that develops in transgenic mice.
Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.
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.
Muscarinc receptor-mediated signaling takes part in many physiological functions ranging from complex higher nervous activity to vegetative responses. Specificity of action of the natural muscarinic agonist acetylcholine is effected by action on five muscarinic receptor subtypes with particular tissue and cellular localization, and coupling preference with different G-proteins and their signaling pathways. In addition to physiological roles it is also implicated in pathologic events like promotion of carcinoma cells growth, early pathogenesis of neurodegenerative diseases in the central nervous system like Alzheimer s disease and Parkinson s disease, schizophrenia, intoxications resulting in drug addiction, or overactive bladder in the periphery. All of these disturbances demonstrate involvement of specific muscarinic receptor subtypes and point to the importance to develop selective pharmacotherapeutic interventions. Because of the high homology of the orthosteric binding site of muscarinic receptor subtypes there is virtually no subtype selective agonist that binds to this site. Activation of specific receptor subtypes may be achieved by developing allosteric modulators of acetylcholine binding, since ectopic binding domains on the receptor are less conserved compared to the orthosteric site. Potentiation of the effects of acetylcholine by allosteric modulators would be beneficial in cases where acetylcholine release is reduced due to pathological conditions. When presynaptic function is severly compromised, the utilization of ectopic agonists can be a thinkable solution.
Title: On homology modeling of the M2 muscarinic acetylcholine receptor subtype Jakubik J, Randakova A, Dolezal V Ref: J Comput Aided Mol Des, 27:525, 2013 : PubMed
Twelve homology models of the human M2 muscarinic receptor using different sets of templates have been designed using the Prime program or the modeller program and compared to crystallographic structure (PDB:3UON). The best models were obtained using single template of the closest published structure, the M3 muscarinic receptor (PDB:4DAJ). Adding more (structurally distant) templates led to worse models. Data document a key role of the template in homology modeling. The models differ substantially. The quality checks built into the programs do not correlate with the RMSDs to the crystallographic structure and cannot be used to select the best model. Re-docking of the antagonists present in crystallographic structure and relative binding energy estimation by calculating MM/GBSA in Prime and the binding energy function in YASARA suggested it could be possible to evaluate the quality of the orthosteric binding site based on the prediction of relative binding energies. Although estimation of relative binding energies distinguishes between relatively good and bad models it does not indicate the best one. On the other hand, visual inspection of the models for known features and knowledge-based analysis of the intramolecular interactions allows an experimenter to select overall best models manually.
The overproduction of beta-amyloid (Abeta) fragments in transgenic APPswe/PS1dE9 mice results in formation of amyloid deposits in the cerebral cortex and hippocampus starting around four months of age and leading to cognitive impairment much later. We have previously found an age and transgene-dependent weakening of muscarinic receptor-mediated transmission that was not present in young (6-10-week-old) animals but preceded both amyloid deposits and cognitive deficits. Now we investigated immediate and prolonged in vitro effects of non-aggregated Abeta(1-42) on coupling of individual muscarinic receptor subtypes expressed in CHO (Chinese hamster ovary) cells and their underlying mechanisms. Immediate application of 1 muM Abeta(1-42) had no effect on the binding of the muscarinic antagonist N-methylscopolamine or the agonist carbachol. In contrast, 4-day treatment of CHO cells expressing the M1 muscarinic receptor with 100 nM Abeta(1-42) significantly changed the binding characteristics of the muscarinic agonist carbachol and reduced the extent of the M1 receptor-stimulated breakdown of phosphatidylinositol while it did not demonstrate overt toxic effects. The treatment had no influence on the expression of either G-proteins or muscarinic receptors. In concert, we found no change in the gene expression of muscarinic receptor subtypes and gene or protein expression of the G(s), G(q/11), and G(i/o) G-proteins in the cerebral cortex of young adult APPswe/PS1dE9 mice that demonstrate high concentrations of soluble Abeta(1-42) and impaired muscarinic receptor-mediated G-protein activation. Our results provide strong evidence that the initial injurious effects of Abeta(1-42) on M1 muscarinic receptor-mediated transmissionis is due to compromised coupling of the receptor with G(q/11) G-protein.
Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex "collides" with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G(q/11), while even-numbered receptors prefer coupling to G(i/o). We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the alpha-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [(3)H]-N-methylscopolamine. Antibodies directed against the C-terminus of the alpha-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M(1) and M(3) receptors also pre-couple with non-preferential G(i/o) G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.
Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake alpha-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 muM, ws-LYNX1 competed with (125)I-alpha-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing alpha7 nAChRs to 1 muM ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on alpha4beta2 and alpha3beta2 nAChRs. Increasing ws-LYNX1 concentration to 10 muM caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.
Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M(1) and M(4) 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 M(1) muscarinic receptor. Pretreatment of CHO cells that stably express the M(1) 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 [(3)H]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, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization.
We investigated the functional characteristics of pre- and postsynaptic cholinergic transmission in APPswe/PS1dE9 double transgenic mice at a young age (7-10 weeks) before the onset of amyloid plaque formation and at adult age (5-6 months) at its onset. We compared brain slices from cerebral cortex and hippocampus with amyloid deposits to slices from striatum with no amyloid plaques by 6 months of age. In young transgenic mice we found no impairments of preformed and newly synthesized [(3)H]-ACh release, indicating intact releasing machinery and release turnover, respectively. Adult transgenic mice displayed a significant increase in preformed [(3)H]-ACh release in cortex but a decrease in hippocampus and striatum. The extent of presynaptic muscarinic autoregulation was unchanged. Evoked release of newly synthesized [(3)H]-ACh was significantly reduced in the cortex and hippocampus but unchanged in the striatum. Carbachol-induced G-protein activation in cortical membranes displayed decreased potency but normal efficacy in adult animals and no changes in young animals. These results indicate that functional pre- and postsynaptic cholinergic deficits are not present in APPswe/PS1dE9 transgenic mice before 10 weeks of age, but develop along with beta-amyloid accumulation in the brain.
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 microM) 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]GTPgammaS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [35S]GTPgammaS binding at M2 and M4 receptors. In contrast, 0.1 microM 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]GTPgammaS 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 microM) but not at high concentrations (10 microM). 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.
Choline is an essential nutrient necessary for synthesis of membrane phospholipids, cell signalling molecules and acetylcholine. The aim of this study was to detect and characterize the choline transporter-like 1 (CTL1/SLC44A1) protein in CNS tissues and the hybrid neuroblastoma x glioma cell line NG108-15, which synthesizes acetylcholine and has high affinity choline transport but does not express the cholinergic high affinity choline transporter 1. The presence of CTL1 protein in NG108-15 cells was confirmed using our antibody G103 which recognizes the C-terminal domain of human CTL1. Three different cognate small interfering RNAs were used to decrease CTL1 mRNA in NG108-15 cells, causing lowered CTL1 protein expression, choline uptake and cell growth. None of the small interfering RNAs influenced carnitine transport, demonstrating the absence of major non-specific effects. In parental C6 cells knockdown of CTL1 also reduced high affinity choline transport. Our results support the concept that CTL1 protein is necessary for the high affinity choline transport which supplies choline for cell growth. The presence of CTL1 protein in rat and human CNS regions, where it is found in neuronal, glial and endothelial cells, suggests that malfunction of this transporter could have important implications in nervous system development and repair following injury, and in neurodegenerative diseases.
Iodinated [125I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ-specific binding was monitored. Relatively high levels of [125I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [125I]WTX-binding sites. Specific [125I]WTX binding was partially inhibited by both alpha-cobratoxin, a blocker of the alpha7 and muscle-type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a Kd of 2.0+/-0.8 microM and was inhibited by alpha-cobratoxin but not by a short-chain alpha-neurotoxin antagonist of the muscle-type nAChR, suggesting a specific interaction with the alpha7-type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 microM did not inhibit binding of [3H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [3H]N-methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G-protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G-proteins with the M1, M4, and M5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs.
Title: Importance and prospects for design of selective muscarinic agonists Jakubik J, Michal P, Machova E, Dolezal V Ref: Physiol Res, 57 Suppl 3:S39, 2008 : PubMed
There are five subtypes of muscarinic receptors that serve various important physiological functions in the central nervous system and the periphery. Mental functions like attention, learning, and memory are attributed to the muscarinic M1 subtype. These functions decline during natural aging and an early deficit is typical for Alzheimer s disease. In addition, stimulation of the M1 receptor increases non-amyloidogenic processing of the amyloid precursor protein and thus prevents accumulation of noxious beta-amyloid fragments. The selectivity of classical muscarinic agonists among receptor subtypes is very low due to the highly conserved nature of the orthosteric binding site among receptor subtypes. Herein we summarize some recent studies with the functionally-selective M1 agonist xanomeline that indicate complex pharmacological profile of this drug that includes interactions with and activation of receptor from both orthosteric and ectopic binding sites, and the time-dependent changes of ligand binding and receptor activation. These findings point to potential profitability of exploitation of ectopic ligands in the search for truly selective muscarinic receptor agonists.
We assessed the integrity of cholinergic neurotransmission in parietal cortex of young adult (7 months) and aged (17 months) transgenic APPswe/PS1dE9 female mice compared to littermate controls. Choline acetyltransferase and acetylcholinesterase activity declined age-dependently in both genotypes, whereas both age- and genotype-dependent decline was found in butyrylcholinesterase activity, vesicular acetylcholine transporter density, muscarinic receptors and carbachol stimulated binding of GTP gamma S in membranes as a functional indicator of muscarinic receptor coupling to G-proteins. Notably, vesicular acetylcholine transporter levels and muscarinic receptor-G-protein coupling were impaired in transgenic mice already at the age of 7 months compared to wild type littermates. Thus, brain amyloid accumulation in this mouse model is accompanied by a serious deterioration of muscarinic transmission already before the mice manifest significant cognitive deficits.
Title: Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of presynaptic M(2) and M(4) muscarinic receptors in rat brain Machova E, Jakubik J, El-Fakahany EE, Dolezal V Ref: Journal of Pharmacology & Experimental Therapeutics, 322:316, 2007 : PubMed
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 microM xanomeline had no effect on evoked [(3)H]ACh release or its inhibition by 10 microM carbachol. In contrast, preincubation with 1, 10, or 100 microM 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 microM xanomeline) or partially (at 100 microM xanomeline) blocked by 1 microM N-methylscopolamine. Neither presence of N-methylscopolamine during 100 microM 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 alpha(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.
Title: Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M(1) and M(2) muscarinic acetylcholine receptors Jakubik J, El-Fakahany EE, Dolezal V Ref: Molecular Pharmacology, 70:656, 2006 : PubMed
Xanomeline is a functionally selective M(1)/M(4) muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M(1) receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M(1) and M(2) receptors expressed in Chinese hamster ovary cells and [(3)H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M(2) receptor with similar affinity as at the M(1) receptor (K(I) = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M(2) receptors was markedly slower than to M(1) receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5'-O-(3-[(35)S]thio)triphosphate binding at M(1) receptors, whereas at M(2) receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation for 1 h. Development of xanomeline agonistic effects at the M(2) receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M(1) than at M(2) receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time- and concentration-dependent and vary between the M(1) and the M(2) receptor.
Title: Determinants of positive cooperativity between strychnine-like allosteric modulators and N-methylscopolamine at muscarinic receptors Jakubik J, Dolezal V Ref: Journal of Molecular Neuroscience, 30:111, 2006 : PubMed
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.
Title: The increase of choline acetyltransferase activity by docosahexaenoic acid in NG108-15 cells grown in serum-free medium is independent of its effect on cell growth Machova E, Malkova B, Lisa V, Novakova J, Dolezal V Ref: Neurochem Res, 31:1239, 2006 : PubMed
We investigated the influence of the polyunsaturated docosahexaenoic acid (22:6n-3; DHA) on the constitutive expression of choline acetyltransferase (ChAT) in native and induced expression in differentiated cholinergic cells NG108-15 grown in serum-free medium. Elimination of serum-derived trophic support resulted in growth arrest and a strong decrease of ChAT activity. In either conditions, DHA largely rescued general indicators of cell growth and function, and partially prevented the decrease of ChAT activity. However, the maximal effect on general cell state in native and differentiated cells, and ChAT activity in native cells, was reached at or below 10 mumol/l of DHA. In contrast, maximal induction of ChAT activity in differentiated cells required about six times higher concentrations of DHA. These data thus demonstrate stimulatory effect of DHA on ChAT activity that is independent of its general cell protective properties.
Title: Docosahexaenoic acid supports cell growth and expression of choline acetyltransferase and muscarinic receptors in NG108-15 cell line Machova E, Novakova J, Lisa V, Dolezal V Ref: Journal of Molecular Neuroscience, 30:25, 2006 : PubMed
A large body of evidence indicates that adequate intake of polyunsaturated fatty acids is essential for brain development in early ontogenesis and positively impacts various pathological states connected with aging, as well as other neurodegenerative diseases (Jump, 2002; Bazan, 2003; Ruxton et al., 2004). In the present experiments, we investigated the possible effects of polyunsaturated docosahexanoic acid (DHA [22:6, n = 3]) on the expression of cholinergic phenotype-represented by choline acetyltransferase (ChAT) activity and a number of surface muscarinic receptors-as well as on cell growth in the cholinergic cell line NG108-15(Hamprecht, 1977; Hamprecht et al., 1985). However, chemical composition of different batches of sera is neither stable nor defined, and this fact complicates investigations on in vitro effects of substances that are natural constituents of serum. To avoid this restraint we employed defined medium in which fatty acid-free bovine albumin as a carrier of DHA replaced serum. Growth of most cell lines, as well as cells in primary cultures, depends strictly on the presence of serum in growth medium. As expected, withdrawal of serum resulted in growth arrest exemplified by a decrease in protein content compared with control cells grown in the presence of serum and also caused a decrease in ChAT activity (Fig. 1, lower left). DHA, at a concentration of 10 mumol/L, largely prevented both growth arrest in defined medium with fatty acid-free bovine albumin as a carrier of DHA and the attenuation of ChAT activity. DHA at concentrations 10 times higher had no further effect. At a concentration of 100 mumol/L, DHA also significantly increased the number of surface muscarinic receptors compared with cells grown in serum-containing as well as serum-free medium (Fig. 1, upper right). These data demonstrate the ability of DHA at low micromolar concentrations to support cell growth and expression of ChAT activity. Although it is not possible to stipulate a mechanism of action on the expression of ChAT and muscarinic receptors, a plausible explanation could be prevention of apoptosis, evidenced by a sharp decrease in executive caspase-3 activity (Fig. 1, lower right). Apoptosis is a process with a high requirement for energy. An improved metabolic state of cells consequent to suppression of apoptosis might thus better fulfill requirements for protein synthesis and targeting.
Title: Asparagine, valine, and threonine in the third extracellular loop of muscarinic receptor have essential roles in the positive cooperativity of strychnine-like allosteric modulators Jakubik J, Krejci A, Dolezal V Ref: Journal of Pharmacology & Experimental Therapeutics, 313:688, 2005 : PubMed
We have investigated allosteric interactions of four closely related strychnine-like substances: Wieland-Gumlich aldehyde (WGA), propargyl Wieland-Gumlich aldehyde, strychnine, and brucine with N-methylscopolamine (NMS) on M(3) subtype of muscarinic receptor genetically modified in the second or the third extracellular loop to corresponding loops of M(2) subtype (M(3)o2 and M(3)o3 chimera). The M(3)o2 chimeric receptor The exhibited no change in either affinity of strychnine, brucine, and WGA or in cooperativity of brucine or WGA, whereas both parameters for propargyl-WGA changed. In contrast, there was a change in affinity of all tested modulators (except for brucine) and in their cooperativity in the M(3)o3 chimera. Directions of affinity changes in both chimeras were always toward values of the donor M(2) subtype, but changes in cooperativity were variable. Compared with the native M(3) receptor, strychnine displayed a slight increase in positive cooperativity and propargyl-WGA a robust decrease in negative cooperativity at M(3)o2 chimera. Similar changes were found in the M(3)o3 chimera. Interestingly, cooperativity of brucine and WGA at the M(3)o3 chimera changed from negative to positive. This is the first evidence of constitution of positive cooperativity of WGA by switching sequences of two parental receptors, both exhibiting negative cooperativity. Gradual replacement of individual amino acids revealed that only three residues (NVT of the o3 loop of the M(2) receptor) are involved in this effect. Data suggest that these amino acids are essential for propagation of a conformation change resulting in positive cooperativity induced by these modulators.
Title: Chronic treatment with amyloid beta(1-42) inhibits non-cholinergic high-affinity choline transport in NG108-15 cells through protein kinase C signaling Novakova J, Mikasova L, Machova E, Lisa V, Dolezal V Ref: Brain Research, 1062:101, 2005 : PubMed
We investigated the influence of the amyloid-beta-peptide(1-42) on hemicholinum-3-sensitive high-affinity choline uptake in NG108-15 cells. RT-PCR analysis revealed the presence of mRNA for a choline transporter-like protein but not for cholinergic high-affinity choline transporter. Differentiation of cells increased both hemicholinum-3-sensitive choline uptake and high-affinity hemicholinium-3 binding. This transport was not influenced by tenfold excess of carnitine. Continuous presence of submicromolar concentrations of amyloid-beta-peptide(1-42) during differentiation resulted in a decrease of both choline uptake and hemicholinium-3 binding. These effects were not present when amyloid-beta-peptide(1-42) was added 5 min prior to measurements. Neither differentiation nor amyloid-beta-peptide(1-42) treatment changed levels of choline transporter-like protein mRNA. Protein kinase C inhibition by staurosporine or its inactivation by continuous presence of tetradecanoyl phorbol acetate prevented the inhibitory effect of amyloid-beta-peptide(1-42) treatment on choline uptake. Activation of protein kinase C by tetradecanoyl phorbol acetate during measurement had inhibitory effect on choline uptake in control but not amyloid-beta-peptide(1-42)-treated cells. The concentration of amyloid-beta-peptide(1-42) maximally effective on hemicholinium-3-sensitive choline uptake had no effect on cell growth, oxidative activity, membrane integrity, number of surface muscarinic receptors, caspase-3 and -8 activities, or uptake of deoxyglucose. Results demonstrate that long-term treatment with non-toxic concentrations of amyloid-beta-peptide(1-42) downregulates choline uptake presumably mediated by a choline transporter-like protein through activation of protein kinase C signaling. The decrease of choline uptake may have relevance to the pathogenesis of Alzheimer's disease.
Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.
Title: Multiple promoters drive tissue-specific expression of the human M muscarinic acetylcholine receptor gene Krejci A, Bruce AW, Dolezal V, Tucek S, Buckley NJ Ref: Journal of Neurochemistry, 91:88, 2004 : PubMed
Despite the wealth of information on the functional and pharmacological properties of the M2 muscarinic receptor, we know relatively little of structure and regulation of the M2 receptor gene. Here, we describe the organisation of the human M2 gene and its promoters. Four exons are present in the 5' untranslated region of the human M2 mRNA distributed over 146 kb on chromosome 7 which produce eight different splice variants in the IMR-32 neuroblastoma cell line. The unexpectedly large size of this gene indicates that transcription initiates much further upstream of the coding region than earlier studies had indicated. We present evidence that there are three distinct human M2 promoters. Analysis of endogenous transcripts revealed that promoter 2 is preferentially used in neuroblastoma cells, whereas promoter 1 in cardiac cells. All promoters are highly conserved across human, mouse, rat and pig. They contain multiple start sites and none possess a TATA-box. In addition, we describe another M2 promoter that is specific for rat. We show that GATA-4 transcription factor binds to two sites within the regulatory regions of the M2 gene using reporter gene assays, electromobility shift assays and mutational analysis.
Title: Roles of external loops of muscarinic receptors in interactions between N-methylscopolamine and allosteric modulators. Tucek S, Krejci A, Lysikova M, Jakubik J, Dolezal V Ref: Cholinergic Mechanisms, CRC Press, :71, 2004 : PubMed
Expression of choline acetyltransferase (ChAT) and of the vesicular acetylcholine transporter (VAChT) is required for the acquisition and the maintenance of the cholinergic phenotype. The ChAT and VAChT genes have been demonstrated to share a common gene locus and this suggests a coordinate regulation of their expression. In the present work, we examined the effects of several differentiating treatments on the modulation of ChAT and VAChT expression at the mRNA and protein levels in growing and differentiating NG108-15 cells. In cells grown in the presence of serum, all the agents tested-retinoic acid, dexamethasone and dibutyrylcyclicAMP (dbcAMP)-induced an increase of ChAT and VAChT mRNA levels but with different efficacy. Treatment with dbcAMP plus dexamethasone resulted in the largest increase of VAChT mRNA amount while retinoic acid mostly enhanced ChAT mRNA level. However, while ChAT activity was increased by all agents, no change in the VAChT protein level was detected. In cells differentiated by serum deprivation, only retinoic acid was effective in inducing an increase of VAChT and ChAT mRNA and ChAT activity, while we observed a downregulation by dbcAMP and dexamethasone. Treatment with the antimitotic agent cytosine arabinoside led to an increase of ChAT activity which was further largely enhanced by the addition of dbcAMP plus dexamethasone, but to only a slight change in VAChT activity. We further showed that complex glycosylation processes which might play a role in targeting and/or stability of the membrane protein VAChT are deficient in these cells. Indeed, in transient transfection assays with the reporter soluble enzyme luciferase placed under regulatory and promoter regions of the VAChT gene, we observed a modulation of luciferase expression by differentiating agents. These data illustrate the complexity of the processes which participate to the expression of the ChAT and VAChT genes, both at the transcriptional and posttranslational levels.
Title: Differentiation of NG108-15 cells induced by the combined presence of dbcAMP and dexamethasone brings about the expression of N and P/Q types of calcium channels and the inhibitory influence of muscarinic receptors on calcium influx Dolezal V, Lisa V, Diebler MF, Kasparova J, Tucek S Ref: Brain Research, 910:134, 2001 : PubMed
Differentiation of cholinergic cell line NG108-15 induced by a combination of dibutyryl cyclic AMP (dbcAMP) and dexamethasone enhances the cholinergic phenotype of the cells more than that induced by either agent alone. We investigated the effect of treatment with dbcAMP and dexamethasone on potassium depolarization-evoked influx of calcium and its regulation by the muscarinic agonist carbachol. Depolarization of control cells and of cells differentiated in the presence of dbcAMP or dexamethasone alone, or in the combined presence of dbcAMP and dexamethasone induced, respectively, 2.2-, 4.3-, 2.7- and 10.7-fold increases of the resting [Ca(2+)](i). Dexamethasone alone and the combination of dbcAMP and dexamethasone augmented the number of muscarinic receptors by 25 and 40%, respectively. Inhibitors of N (omega-conotoxin GVIA) or P/Q (omega-agatoxin TK) calcium channels had no effect on Ca(2+) influx in control cells, whereas in cells differentiated in the combined presence of dbcAMP and dexamethasone they significantly diminished the influx of Ca(2+) by 20 and 5%, respectively. Carbachol attenuated calcium influx in differentiated cells in an atropine-insensitive manner if it was present during stimulation. This effect of carbachol was probably due to an open-channel block of L type channels. In the presence of nifedipine, carbachol attenuated the influx of Ca(2+) into cells differentiated with dbcAMP and dexamethasone by 20% in an atropine-sensitive way. Data show that differentiation of NG108-15 cells by dbcAMP and dexamethasone promotes the expression of functional nifedipine-insensitive N and P/Q types of Ca(2+) channels and that the nifedipine-insensitive calcium influx becomes subject to inhibitory regulation by muscarinic receptors.
Title: Chronic exposure of NG108-15 cells to amyloid beta peptide (A beta(1-42)) abolishes calcium influx via N-type calcium channels Kasparova J, Lisa V, Tucek S, Dolezal V Ref: Neurochem Res, 26:1079, 2001 : PubMed
We investigated whether amyloid-beta-peptide (A beta(1-42)) has an effect on the elevations of the intracellular concentration of Ca2+ ions ([Ca2+]i) induced by depolarizations of NG108-15 cells and on related Ca2+ channels. A beta(1-42) (10-1000 nM) had no immediate effect on depolarization-induced [Ca2+]i elevations. [Ca2+]i increases were slightly diminished in cells grown in the presence of 100 or 1000 nM A beta(1-42). Nifedipine (1 microM) reduced these elevations equally in cells grown in the absence or presence of A beta(1-42). In contrast, the ability of omega-conotoxin GVIA to diminish the depolarization-induced [Ca2+]i responses became lost in cells grown in the presence of 100 nM A beta(1-42). This indicates that the influx of calcium through the N-type Ca2+ channels was compromised by the chronic exposure of cells to a submicromolar concentration of A beta(1-42), presumably because of impairement of their function or diminished expression. This may be important in the pathogeny of Alzheimer's dementia in view of the pivotal role of N-type Ca2+ channels in neurotransmitter release.
Title: Calcium channels involved in the inhibition of acetylcholine release by presynaptic muscarinic receptors in rat striatum Dolezal V, Tucek S Ref: British Journal of Pharmacology, 127:1627, 1999 : PubMed
1. The mechanism of the inhibitory action of presynaptic muscarinic receptors on the release of acetylcholine from striatal cholinergic neurons is not known. We investigated how the electrically stimulated release of [3H]-acetylcholine from superfused rat striatal slices and its inhibition by carbachol are affected by specific inhibitors of voltage-operated calcium channels of the L-type (nifedipine), N-type (omega-conotoxin GVIA) and P/Q-type (omega-agatoxin IVA). 2. The evoked release of [3H]-acetylcholine was not diminished by nifedipine but was lowered by omega-conotoxin GVIA and by omega-agatoxin IVA, indicating that both the N- and the P/Q-type (but not the L-type) channels are involved in the release. The N-type channels were responsible for approximately two thirds of the release. The release was >97% blocked when both omega-toxins acted together. 3. The inhibition of [3H]-acetylcholine release by carbachol was not substantially affected by the blockade of the L- or P/Q-type channels. It was diminished but not eliminated by the blockade of the N-type channels. 4. In experiments on slices in which cholinesterases had been inhibited by paraoxon, inhibition of [3H]-acetylcholine release by endogenous acetylcholine accumulating in the tissue could be demonstrated by the enhancement of the release after the addition of atropine. The inhibition was higher in slices with functional N-type than with functional P/Q-type channels. 5. We conclude that both the N- and the P/Q-type calcium channels contribute to the stimulation-evoked release of acetylcholine in rat striatum, that the quantitative contribution of the N-type channels is higher, and that the inhibitory muscarinic receptors are more closely coupled with the N-type than with the P/Q-type calcium channels.
Title: Poster: Allosteric potentiation of the effects of muscarinic agonists Tucek S, Dolezal V Ref: Life Sciences, 64:561, 1999 : PubMed
Title: Influence of retinoic acid and of cyclic AMP on the expression of choline acetyltransferase and of vesicular acetylcholine transporter in NG108-15 cells Diebler MF, Tomasi M, Meunier FM, Israel M, Dolezal V Ref: Journal de Physiologie (Paris), 92:379, 1998 : PubMed
Treatment of the cholinergic cell line NG108-15 with retinoic acid or cAMP results in an increase of choline acetyltransferase activity (ChAT) whereas none of these agents influences the amount of the vesicular acetylcholine transporter (VAChT) as judged from vesamicol binding and immunoblot studies. We suggest that immaturity of posttranslational events controlling the expression of VAChT protein is responsible for the apparent absence of coregulation of ChAT and VAChT protein expression.
Title: Tacrine inhibits L-type calcium channels in the cholinergic SN56 cell line Dolezal V, Lisa V, Tucek S Ref: Journal de Physiologie (Paris), 92:426, 1998 : PubMed
Title: Positive effects of allosteric modulators on the binding properties and the function of muscarinic acetylcholine receptors Tucek S, Jakubik J, Dolezal V, El-Fakahany EE Ref: Journal de Physiologie (Paris), 92:241, 1998 : PubMed
Data are reviewed indicating that allosteric modulators can enhance the affinities of muscarinic receptors for their antagonists and agonists, that the enhancement of the affinity for agonists is relevant functionally, and that the allosterically induced conformational change also affects the interaction between the receptors and the G proteins.
Title: Effect of tacrine on intracellular calcium in cholinergic SN56 neuronal cells Dolezal V, Lisa V, Tucek S Ref: Brain Research, 769:219, 1997 : PubMed
We have found earlier that the depolarization-induced release of acetylcholine from the brain could be inhibited by tacrine (tetrahydroaminoacridine) but the mechanism of this action of tacrine was not clarified (S. Tucek, V. Dolezal, J. Neurochem. 56 (1991) 1216). We have now investigated whether tacrine has an effect on the changes in the intracellular concentration of calcium ions ([Ca2+]i) induced by depolarization. Experiments were performed on the cholinergic SN56 neuronal cell line with Fura-2 fluorescence technique of calcium imaging. The depolarization by 71 mmol/l K+ evoked minimum increases of [Ca2+]i up to day 5 in culture. Then the response gradually increased and reached a plateau after 7 days in culture. A similar time course was observed for acetylcholinesterase activity. The effect of K+ ions was concentration-dependent and the concentration of 71 mmol/l K+ evoked maximum [Ca2+]i responses. The increases of [Ca2+]i did not occur in the absence of extracellular calcium. They were mediated by high voltage-activated calcium channels of the L-type and the N-type. Nifedipine (2 micromol/l; L-type calcium channel blocker) and omega-conotoxin GVIA (100 nmol/l; N-type calcium channel blocker) diminished the response to 71 mmol/l K+ by 53% and 39%, respectively, and their effects were additive (decrease to 8% of controls). Non-selective inorganic blocker of voltage-activated calcium channels LaCl3 (0.1 mmol/l) decreased the response by 83%. Tacrine attenuated the [Ca2+]i response in a concentration-dependent manner. At a concentration of 10 micromol/l it inhibited the [Ca2+]i response by 55% and its inhibitory effect was additive with that of omega-conotoxin GVIA but not with that of nifedipine. An equimolar concentration of paraoxon, an irreversible inhibitor of cholinesterases, had no influence on [Ca2+]i response. Tacrine exhibited the same inhibitory effect when paraoxon was present. In conclusion, our data indicate that high-voltage-activated calcium channels of the L-type and the N-type are both present in the SN56 cells but that they are fully expressed only after 6-7 days in culture. Tacrine attenuates the influx of calcium by inhibiting the L-type calcium channels. This inhibitory effect is not a consequence of the anticholinesterase activity of tacrine. The finding that low micromolar concentrations of tacrine may interfere with calcium-dependent events is likely to be of importance for the evaluation of the therapeutic potential of the drug.
Title: The non-quantal release of acetylcholine from motor nerve terminals: comment on its likely size Tucek S, Dolezal V Ref: Prog Brain Res, 98:209, 1993 : PubMed
Title: Investigation of the mechanism of the effect of tacrine (tetrahydroaminoacridine) on the metabolism of acetylcholine and choline in brain cortical prisms Dolezal V, Tucek S Ref: J Neural Transm Park Dis Dement Sect, 4:303, 1992 : PubMed
The mechanism by which tacrine increases the content and synthesis of acetylcholine (ACh) in cerebrocortical prisms exposed to an irreversible inhibitor of cholinesterases and incubated under resting conditions (Dolezal and Tucek, 1991) is not known. As found in the present experiments, this effect of tacrine is only apparent if its application had been preceded by a period of preincubation, but the preincubation is ineffective if it occurs in the presence of hemicholinium-3. Apparently, choline or a choline-containing compound accumulates in the slices during the preincubation and is then utilized for the enhanced synthesis of ACh in the presence of tacrine. Tacrine did not induce a decrease in the amount of radiolabel that had been incorporated from choline into acid-insoluble compounds, which suggests that the choline which is used for the synthesis of additional ACh does not originate from choline lipids. However, tacrine was found to diminish the efflux of choline from prisms which had been preincubated with an increased concentration of choline in the medium, and from prisms incubated in the presence of hemicholinium-3. It also diminished the efflux of radioactive choline that had accumulated in the prisms during preincubation with a very low concentration of tacrine, when the prisms were subsequently incubated with 4-aminopyridine. It is proposed that the potency of tacrine to increase the content and synthesis of ACh in cerebrocortical prisms whose cholinesterases had been inhibited is due to its ability to diminish the efflux of endogenous choline from the nerve terminals.
Title: Positive and negative effects of tacrine (tetrahydroaminoacridine) and methoxytacrine on the metabolism of acetylcholine in brain cortical prisms incubated under resting conditions Dolezal V, Tucek S Ref: Journal of Neurochemistry, 56:1207, 1991 : PubMed
The effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) and 7-methoxytacrine on the metabolism of acetylcholine were investigated in experiments on prisms of rat cerebral cortex incubated in vitro in low-potassium (3 mmol/L K+) media; cholinesterases were inactivated by paraoxon to avoid any action of tacrine and methoxytacrine via their inhibition. Under "resting" conditions, tacrine and methoxytacrine increased the synthesis of unlabeled acetylcholine in the prisms; at the same time, they inhibited the uptake of [14C]choline from the medium and the synthesis of [14C]acetylcholine. The concentration of free choline was not increased by tacrine or methoxytacrine in either the tissue or the medium. The contradiction between the increased synthesis of unlabeled and the diminished synthesis of labeled acetylcholine indicates that the utilization of intracellular choline (which is presumably mobilized from intracellular choline esters) for the synthesis of acetylcholine is increased by tacrine and methoxytacrine. This conclusion is supported by the observation that the inhibition of acetylcholine synthesis during incubation with hemicholinium-3 (an inhibitor of choline transport into cholinergic nerve terminals) was overcome when tacrine was present simultaneously with hemicholinium-3. When the prisms were preincubated with [14C]choline and incubated with tacrine or methoxytacrine only after this, the amount of [14C]acetylcholine recovered in the tissue plus the medium was higher at the end of incubation with tacrine or methoxytacrine than without them, again suggesting that the drugs were able to increase the utilization of intracellular [14C]choline or its esters for acetylcholine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Title: Negative effects of tacrine (tetrahydroaminoacridine) and methoxytacrine on the metabolism of acetylcholine in brain slices incubated under conditions stimulating neurotransmitter release Tucek S, Dolezal V Ref: Journal of Neurochemistry, 56:1216, 1991 : PubMed
The effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) and 7-methoxytacrine on the metabolism of brain acetylcholine were investigated in experiments in which acetylcholine turnover was stimulated by tissue depolarization or by 4-aminopyridine. Rat cerebrocortical prisms were preincubated under "resting" conditions (Krebs-Ringer buffer with 3 mmol/L K+ and with paraoxon to inhibit cholinesterases) and then incubated in the presence of tacrine or methoxytacrine and of 50 mmol/L K+. Both drugs diminished the amount of acetylcholine released by depolarization and the amount of acetylcholine synthesized during incubation; in experiments in which [14C]choline was present in the incubation medium simultaneously with tacrine or methoxytacrine, the drugs diminished the uptake of [14C]choline by the tissue and the amount of [14C]-acetylcholine synthesized and released into the medium. In these experiments, it was not possible to distinguish whether the inhibitory effects of tacrine and methoxytacrine were primarily on the process of acetylcholine synthesis (particularly on the uptake of choline), or whether the drugs also acted directly on the process of neurotransmitter release. In subsequent experiments the prisms were preincubated with [14C]choline and only then subjected to a short depolarization in the presence of hemicholinium-3 and tacrine or methoxytacrine. Both drugs severely inhibited the release of preformed [14C]acetylcholine and prevented the diminution of tissue [14C]acetylcholine stores. Methoxytacrine was also found to diminish the release of acetylcholine induced by 4-aminopyridine while increasing the content of acetylcholine in the tissue. Tacrine and methoxytacrine had no effect on the activity of choline acetyltransferase (EC 2.3.1.6).(ABSTRACT TRUNCATED AT 250 WORDS)
Title: Effect of lanthanum on the release of acetylcholine from the myenteric plexus and on its activation by ouabain and electrical stimulation Dolezal V, Somogyi GT, Bernath S, Tucek S, Vizi ES Ref: Journal of Neurochemistry, 49:503, 1987 : PubMed
The effect of lanthanum ions (La3+) on the release of acetylcholine (ACh) from longitudinal muscle strips of the guinea pig ileum with the myenteric plexus attached was investigated. After an exposure of the tissue to 2 mM LaCl3 for 18 min the rate of ACh release was increased approximately eightfold and the increased release lasted for more than 100 min. The augmented release of ACh was accompanied by enhanced synthesis. At the end of the experiments (102 min after LaCl3 had been removed), when the release of ACh was still more than six times higher than in controls, the content of ACh was the same in La3+-treated and untreated tissues. Electrical field stimulation failed to cause a further increase in the release of ACh from La3+-pretreated preparations whereas ouabain released considerable more ACh when compared to controls. It is concluded from this difference that electrical stimulation and ouabain release ACh from different pools.
Title: Origin of Acetyl Groups of Acetylcholine in the Brain and the Role of Acetylcoenzyme A in the Control of its Synthesis Tucek S, Dolezal V, Ricny J Ref: Advances in Behavioral Biology, 25:415, 1981 : PubMed
