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Sample records for acetylcholine transporter detected

  1. Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action.

    Ann Sluder

    Full Text Available The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.

  2. High-resolution mass spectrometry for detecting Acetylcholine in Arabidopsis

    Murata, Jun; Watanabe, Takehiro; Sugahara, Kohtaro; Yamagaki, Tohru; Takahashi, Toshio

    2015-01-01

    Acetylcholine (ACh) was first identified a century ago, and has long been known as a neurotransmitter in animals. However, it has been shown recently that the occurrence of ACh is widespread among various non-animal species including higher plants. Although previous reports suggest that various plant species are capable of responding to exogenously applied ACh, the molecular basis for ACh biosynthesis and regulatory mechanisms mediated by endogenous ACh are largely unclear. This is partly bec...

  3. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system.

    Schäfer, M K; Eiden, L E; Weihe, E

    1998-05-01

    The peripheral sympathetic and parasympathetic cholinergic innervation was investigated with antibodies directed against the C-terminus of the rat vesicular acetylcholine transporter. Immunohistochemistry for the vesicular acetylcholine transporter resulted in considerably more detailed visualization of cholinergic terminal fields in the peripheral nervous system than reported previously and was well suited to also identify cholinergic perikarya. Vesicular acetylcholine transporter immunoreactivity completely delineated the preganglionic sympathetic terminals in pre- and paravertebral sympathetic ganglia, and in the adrenal medulla as well as postganglionic cholinergic neurons in the paravertebral chain. Cholinergic terminals of sudomotor and vasomotor nerves of skeletal muscle were optimally visualized. Mixed peripheral ganglia, including periprostatic and uterovaginal ganglia, exhibited extensive preganglionic cholinergic innervation of both noradrenergic and cholinergic postganglionic principal neurons which were intermingled in these ganglia. Varicose vesicular acetylcholine transporter-positive fibres and terminals, representing the cranial parasympathetic innervation of the cerebral vasculature, of salivary and lacrimal glands, of the eye, of the respiratory tract and of the upper digestive tract innervated various target structures including seromucous gland epithelium and myoepithelium, respiratory epithelium, and smooth muscle of the tracheobronchial tree. The only macrovascular elements receiving vesicular acetylcholine transporter-positive innervation were the cerebral arteries. The microvasculature throughout the viscera, with the exception of lymphoid tissues, the liver and kidney, received vesicular acetylcholine transporter-positive innervation while the microvasculature of limb and trunk skeletal muscle appeared to be the only relevant somatic target of vesicular acetylcholine transporter innervation. Vesicular acetylcholine transporter

  4. Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission.

    Monica S Guzman; Xavier De Jaeger; Sanda Raulic; Souza, Ivana A; Li, Alex X.; Susanne Schmid; Menon, Ravi S.; Gainetdinov, Raul R.; Caron, Marc G.; Robert Bartha; Prado, Vania F.; Prado, Marco A. M.

    2011-01-01

    Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles. Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for striatal-dependent...

  5. Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission.

    Guzman, Monica S; De Jaeger, Xavier; Raulic, Sanda; Souza, Ivana A; Li, Alex X; Schmid, Susanne; Menon, Ravi S; Gainetdinov, Raul R; Caron, Marc G; Bartha, Robert; Prado, Vania F; Prado, Marco A M

    2011-11-01

    Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles. Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for striatal-dependent behaviour in mice by selective elimination of the vesicular acetylcholine transporter (VAChT) from striatal cholinergic neurons. Analysis of several behavioural parameters indicates that elimination of VAChT had only marginal consequences in striatum-related tasks and did not affect spontaneous locomotion, cocaine-induced hyperactivity, or its reward properties. However, dopaminergic sensitivity of medium spiny neurons (MSN) and the behavioural outputs in response to direct dopaminergic agonists were enhanced, likely due to increased expression/function of dopamine receptors in the striatum. These observations indicate that previous functions attributed to striatal cholinergic neurons in spontaneous locomotor activity and in the rewarding responses to cocaine are mediated by glutamate and not by acetylcholine release. Our experiments demonstrate how one population of neurons can use two distinct neurotransmitters to differentially regulate a given circuitry. The data also raise the possibility of using VAChT as a target to boost dopaminergic function and decrease high striatal cholinergic activity, common neurochemical alterations in individuals affected with Parkinson's disease. PMID:22087075

  6. Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission.

    Monica S Guzman

    2011-11-01

    Full Text Available Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles. Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for striatal-dependent behaviour in mice by selective elimination of the vesicular acetylcholine transporter (VAChT from striatal cholinergic neurons. Analysis of several behavioural parameters indicates that elimination of VAChT had only marginal consequences in striatum-related tasks and did not affect spontaneous locomotion, cocaine-induced hyperactivity, or its reward properties. However, dopaminergic sensitivity of medium spiny neurons (MSN and the behavioural outputs in response to direct dopaminergic agonists were enhanced, likely due to increased expression/function of dopamine receptors in the striatum. These observations indicate that previous functions attributed to striatal cholinergic neurons in spontaneous locomotor activity and in the rewarding responses to cocaine are mediated by glutamate and not by acetylcholine release. Our experiments demonstrate how one population of neurons can use two distinct neurotransmitters to differentially regulate a given circuitry. The data also raise the possibility of using VAChT as a target to boost dopaminergic function and decrease high striatal cholinergic activity, common neurochemical alterations in individuals affected with Parkinson's disease.

  7. Effects of Acetylcholine, Cytochalasin B and Amiprophos methyl on Phloem Transport in Radish (Raphanus sativas)

    Chong-Jun Yang; Zhi-Xi Zhai; Yu-Hai Guo; Peng Gao

    2007-01-01

    We investigated the role of the "sieve tube-companion cell complex" lining the tube periphery, particularly the microfilament and microtubule, in assisting the pushing of phloem sap flow. We made a simple phloem transport system with a living radish plant, in which the conducting channel was exposed for local treatment with chemicals that are effective in modulating protoplasmic movement (acetylcholine, (ACh) a neurotransmitter in animals and insects; cytochalasin B, (CB) a specific inhibitor of many cellular responses that are mediated by microfilament systems and amiprophos-methyl, (APM) a specific inhibitor of many cellular responses that are mediated by microtubule systems). Their effects on phloem transport were estimated by two experimental devices: (i) a comparison of changes in the amount of assimilates in terms of carbohydrates and 14C-labeled photosynthetic production that is left in the leaf blade of treated plants; and (ii) distribution patterns of 14C-labeled leaf assimilates in the phloem transport system. The results indicate that CB and APM markedly inhibited the transfer of photosynthetic product from leaf to root via the leaf vein, while ACh enhanced the transfer of photosynthetic product in low concentrations (5.0×10-4 mol/L) but inhibited it in higher concentrations (2.0×10-3 mol/L) from leaf to root via the leaf vein. Autoradiograph imaging clearly reveals that ACh treatment is more effective than the control, and both CB and APM treatments effectively inhibit the passage of radioactive assimilates. All of the results support the postulation that the peripheral protoplasm in the sieve tube serves not only as a passive semi-permeable membrane, but is also directly involved in phloem transport.

  8. Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric Detection

    Yvette Michotte

    2008-08-01

    Full Text Available Liquid chromatography with amperometric detection remains the most widely used method for acetylcholine quantification in microdialysis samples. Separation of acetylcholine from choline and other matrix components on a microbore chromatographic column (1 mm internal diameter, conversion of acetylcholine in an immobilized enzyme reactor and detection of the produced hydrogen peroxide on a horseradish peroxidase redox polymer coated glassy carbon electrode, achieves sufficient sensitivity for acetylcholine quantification in rat brain microdialysates. However, a thourough validation within the concentration range required for this application has not been carried out before. Furthermore, a rapid degradation of the chromatographic columns and enzyme systems have been reported. In the present study an ion-pair liquid chromatography assay with amperometric detection was validated and its long-term stability evaluated. Working at pH 6.5 dramatically increased chromatographic stability without a loss in sensitivity compared to higher pH values. The lower limit of quantification of the method was 0.3 nM. At this concentration the repeatability was 15.7%, the inter-day precision 8.7% and the accuracy 103.6%. The chromatographic column was stable over 4 months, the immobilized enzyme reactor up to 2-3 months and the enzyme coating of the amperometric detector up to 1-2 months. The concentration of acetylcholine in 30 μl microdialysates obtained under basal conditions from the hippocampus of freely moving rats was 0.40 ± 0.12 nM (mean ± SD, n = 30. The present method is therefore suitable for acetylcholine determination in rat brain microdialysates.

  9. Evaluation of radioiodinated (-)-o-iodovesamicol as a radiotracer for mapping the vesicular acetylcholine transporter

    We evaluated the potencies of radioiodinated (-)-o-iodovesamicol [(-)-oIV] as a selective vesicular acetylcholine transporter (VAChT) mapping agent. (-)-[125I]oIV exhibited significant accumulation (about 2.8% of the injected dose) in rat brain. The regional brain distribution of radioactivity was similar for both (-)-[125I]oIV and (-)-[3H]vesamicol. The accumulation of (-)-[125I]oIV in the brain was significant reduced by post-administration of unlabeled vesamicol (0.5 μmol/kg-1) and (-)-oIV (0.5 μmol/kg-1). On the other hand, the post-administration of sigma ligands hardly affected the accumulation of (-)-[125I]oIV in the brain. These studies showed that (-)-[125I]oIV, as well as [3H]vesamicol, bound to VAChT with high affinity in the rat brain. Furthermore, (-)-[125I]oIV binding in the ipsilateral cortex to the lesion was significantly reduced by 17.0%, compared with that in the contralateral cortex in a unilateral nucleus basalis magnocellularis (NBM)-lesioned rat. These results suggested that radioiodinated (-)-oIV may potentially be useful for the diagnosis of cholinergic neurodegenerative disorders. (author)

  10. (E)-[125I]-5-AOIBV: a SPECT radioligand for the vesicular acetylcholine transporter

    The premise that, over the course of Alzheimer's disease (AD), changes in the levels of the vesicular acetylcholine transporter (VAChT) occur in parallel with changes to other cholinergic marker proteins provides the basis for the applicability of benzovesamicol derivatives as radioligands for AD studies by single photon emission computed tomography or positron emission tomography. We report the synthesis of enantiopure benzovesamicol derivatives: (R,R) or (S,S)-(E)-2-hydroxy-5-(3-iodoprop-2-en-1-oxy)-3- (4-phenylpiperidino)tetralin [(R,R)-AOIBV: Kd=0.45 nM or (S,S)-5-AOIBV: Kd=4.3 nM] and their corresponding tributyltin precursors for radioiodination. (R,R or S,S)-5-AOIBV was labeled with iodine-125 from their corresponding n-tributyltin precursors. Both compounds were obtained with radiochemical and optical purity greater than 97% and in radiochemical yields ranging 34-36%. To determine if these compounds could provide an advantage when compared to [125I]-iodo benzovesamicol (IBVM), IBVM was also labeled and used as the reference compound in all ex vivo experiments. Ex vivo biodistribution experiments in rats revealed that [125I]-(R,R)-5-AOIBV displayed the most suitable pharmacological profile as the radioactivity distribution corresponded well with the known VAChT brain density. Moreover, pre-injection of vesamicol prevented the uptake of [125I]-(R,R)-5-AOIBV in striatum, cortex and hippocampus, demonstrating selectivity for the VAChT. However, even if time activity curves of [125I]-(R,R)-5-AOIBV confirmed that this compound could be used to visualize the VAChT in vivo, at each point of the kinetic study, [125I]-(R,R)-5-AOIBV showed a lower specific binding compared to [125I]-IBVM. These results made [125I]-( R,R)-5-AOIBV inferior to [125I]-IBVM for the VAChT exploration in vivo

  11. Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse

    Koepsell Hermann

    2006-04-01

    Full Text Available Abstract Background It has been proposed that serotonin (5-HT-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh released from the epithelium. We recently demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs, which are also expressed by airway epithelial cells. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs. Methods We tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry. Results Epithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 ± 10 % of the content of the epithelium-denuded trachea, n = 9 than in wild-type mice (16.8 ± 3.6 %, n = 11. In wild-type mice, 5-HT (1 μM caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 μM in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M2/M3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 μM significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle. Conclusion The doubling of airway epithelial ACh content in OCT1/2-/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1 a non

  12. Cholinergic activation of the murine trachealis muscle via non-vesicular acetylcholine release involving low-affinity choline transporters.

    Nassenstein, Christina; Wiegand, Silke; Lips, Katrin S; Li, Guanfeng; Klein, Jochen; Kummer, Wolfgang

    2015-11-01

    In addition to quantal, vesicular release of acetylcholine (ACh), there is also non-quantal release at the motor endplate which is insufficient to evoke postsynaptic responses unless acetylcholinesterase (AChE) is inhibited. We here addressed potential non-quantal release in the mouse trachea by organ bath experiments and (immuno)histochemical methods. Electrical field stimulation (EFS) of nerve terminals elicited tracheal constriction that is largely due to ACh release. Classical enzyme histochemistry demonstrated acetylcholinesterase (AChE) activity in nerve fibers in the muscle and butyrylcholinesterase (BChE) activity in the smooth muscle cells. Acute inhibition of both esterases by eserine significantly raised tracheal tone which was fully sensitive to atropine. This effect was reduced, but not abolished, in AChE, but not in BChE gene-deficient mice. The eserine-induced increase in tracheal tone was unaffected by vesamicol (10(-5)M), an inhibitor of the vesicular acetylcholine transporter, and by corticosterone (10(-4)M), an inhibitor of organic cation transporters. Hemicholinium-3, in low concentrations an inhibitor of the high-affinity choline transporter-1 (CHT1), completely abrogated the eserine effects when applied in high concentrations (10(-4)M) pointing towards an involvement of low-affinity choline transporters. To evaluate the cellular sources of non-quantal ACh release in the trachea, expression of low-affinity choline transporter-like family (CTL1-5) was evaluated by RT-PCR analysis. Even though these transporters were largely abundant in the epithelium, denudation of airway epithelial cells had no effect on eserine-induced tracheal contraction, indicating a non-quantal release of ACh from non-epithelial sources in the airways. These data provide evidence for an epithelium-independent non-vesicular, non-quantal ACh release in the mouse trachea involving low-affinity choline transporters. PMID:26278668

  13. Nicotinic acetylcholine receptor: subunit structure, functional binding sites, and ion transport properties

    The structure of the nicotinic acetylcholine receptor has been highly conserved during animal evolution, and in all the species and tissues studied so far, including mammals, it is a pseudosymmetric, pentameric complex of related subunits with very similar physical properties. All subunits of these nicotinic receptors were derived from a common ancestral gene, probably by way of gene duplications occurring very early in animal evolution. 45 refs., 8 figs., 2 tabs

  14. Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes

    1984-01-01

    Coated vesicles are present in the myoplasm of embryonic chick myotubes grown in vitro. They are most numerous beneath regions of the surface membrane that contain a high density of acetylcholine receptors (AChR). Prolonged exposure of myotubes to saline extract of chick brain increases the number of intracellular AChR and the number of coated vesicles. This suggests that coated vesicles contain AChR, and this hypothesis was tested with horseradish peroxidase-alpha-bungarotoxin (HRP-alpha BTX...

  15. Clinical significance of detection of antibodies to fetal and adult acetylcholine receptors in myasthenia gravis

    Qi-Guang Shi; Zhi-Hong Wang; Xiao-Wei Ma; Da-Qi Zhang; Chun-Sheng Yang; Fu-Dong Shi; Li Yang

    2012-01-01

    Objective To evaluate the frequency,distribution and clinical significance of the antibodies to the fetal and/or adult acetylcholine receptor (AChR) in patients with myasthenia gravis (MG).Methods AChR antibodies were detected by cell-based assay in the serum of ocular MG (OMG) (n =90) and generalized MG (GMG) patients (n =110).The fetaltype (2α∶ β∶ γ∶ δ) and adult-type (2α∶ β∶ ε∶ δ) AChR were used as antigens,and their relevance to disease presentation was assessed.Results The overall frequencies of anti-adult and anti-fetal AChR antibodies were similar in all 200 patients examined,with 14 having serum specific to the AChR-γ subunit,and 22 to the AChR-ε subunit.The overall sensitivity when using the fetal and adult AChR antibodies was higher than that when using the fetal AChR antibody only (P =0.015).Compared with OMG patients,the mean age at disease onset and the positive ratio of antibodies to both isoforms of the AChR were significantly higher in patients who subsequently progressed to GMG.Older patients and patients with both anti-fetal and anti-adult AChR antibodies had a greater risk for developing generalized disease [odds ratio (OR),1.03;95% confidence interval (CI),1.01-1.06 and OR,5.09;95% CI,2.23-11.62].Conclusion Using both fetal-and adulttype AChRs as the antigens may be more sensitive than using either subtype.Patients with serum specific to both isoforms are at a greater risk of progressing to GMG.Patients with disease onset at an advanced age appear to have a higher frequency of GMG conversion.

  16. Automated production of [¹⁸F]VAT suitable for clinical PET study of vesicular acetylcholine transporter.

    Yue, Xuyi; Bognar, Christopher; Zhang, Xiang; Gaehle, Gregory G; Moerlein, Stephen M; Perlmutter, Joel S; Tu, Zhude

    2016-01-01

    Automated production of a promising radiopharmaceutical (-)-(1-(8-(2-[(18)F]fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-piperidin-4-yl)(4-fluorophenyl)methanone ([(18)F]VAT) for the vesicular acetylcholine transporter(VAChT) was achieved using a two-step procedure in a current Good Manufacturing Practices fashion. The production of [(18)F]VAT was accomplished in approximately 140 min, with radiochemical yield of ~15.0% (decay corrected), specific activity>111 GBq/µmol, radiochemical purity>99% and mass of VAT ~3.4 μg/batch (n>10). The radiopharmaceutical product meets all quality control criteria for human use, and is suitable for clinical PET studies of VAChT. PMID:26408913

  17. Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors

    Rubio, Fernando; Kamp, Lisa; Carpino, Justin; Faltin, Erin; Loftin, Keith A.; Molgó, Jordi; Aráoz, Rómulo

    2014-01-01

    Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.

  18. Molecular imaging of acetylcholine vesicular transporter (VAChT) in demented patients with Alzheimer's disease (AD) by IBVM-SPECT

    Alzheimer's disease (A.D.) is characterized by a premature decline of cholinergic neurons. The 5-I.B.V.M. is an analogue of the Vesamicol that binds to the presynaptic vesicular acetylcholine transporter (VAChT). The exploration of this target should be useful to make an early diagnosis of A.D.. Our first aim was to propose a method of non invasive VAChT quantification according to 5-I.B.V.M. kinetic. 5-I.B.V.M. was injected to four A.D. patients (age = 77 ± 3.9 years and M.M.S.E. = 24.5 ± 1.02) were included in this methodological study. The single-photon emission computed tomography (SPECT) images were obtained at five, 20, 35 and 50 minutes, at then at three, five and 22 hours after intravenous injection of 5-I.B.V.M. (185 MBq). The time activity curves were obtained after SPECT images co registration on a MRI masque. Specific volume of interest (S.P.E. SPEcific) were manually drawn on striatum, pons, thalamus and para-hippocampic gyrus including hippocampus; reference volumes of interest (R.E.F. = REFerence) were drawn on frontal and occipital cerebral cortex. On the basis of uptake kinetic, two modeling approaches were considered: transient equilibrium model for reversible ligand (binding potential (B.P.) (S.P.E. - R.E.F.)/R.E.F.) and Patlak graphical analysis for irreversible tracers (slope given by Ki/DVref where Ki is the influx constant and DVref is the distribution volume of the reference region). We observed an inflection or a steady state of the activity curves in the different regions studied between 250 and 1400 minutes, what seems to confirm that the tracer is little reversible. B.P. values obtained at 21 hours with occipital areas as reference and Ki/DVref values were respectively 4.62 ± 0.42 and 0.07 ± 0.01. The S.P.E. classification according to B.P. and Ki/DVref values were similar to the classification according to the compartmental analysis (Kuhl 1994). The transient equilibrium model with late acquisition seems the more suitable because I

  19. Mice deficient for striatal Vesicular Acetylcholine Transporter (VAChT) display impaired short-term but normal long-term object recognition memory.

    Palmer, Daniel; Creighton, Samantha; Prado, Vania F; Prado, Marco A M; Choleris, Elena; Winters, Boyer D

    2016-09-15

    Substantial evidence implicates Acetylcholine (ACh) in the acquisition of object memories. While most research has focused on the role of the cholinergic basal forebrain and its cortical targets, there are additional cholinergic networks that may contribute to object recognition. The striatum contains an independent cholinergic network comprised of interneurons. In the current study, we investigated the role of this cholinergic signalling in object recognition using mice deficient for Vesicular Acetylcholine Transporter (VAChT) within interneurons of the striatum. We tested whether these striatal VAChT(D2-Cre-flox/flox) mice would display normal short-term (5 or 15min retention delay) and long-term (3h retention delay) object recognition memory. In a home cage object recognition task, male and female VAChT(D2-Cre-flox/flox) mice were impaired selectively with a 15min retention delay. When tested on an object location task, VAChT(D2-Cre-flox/flox) mice displayed intact spatial memory. Finally, when object recognition was tested in a Y-shaped apparatus, designed to minimize the influence of spatial and contextual cues, only females displayed impaired recognition with a 5min retention delay, but when males were challenged with a 15min retention delay, they were also impaired; neither males nor females were impaired with the 3h delay. The pattern of results suggests that striatal cholinergic transmission plays a role in the short-term memory for object features, but not spatial location. PMID:27233822

  20. Effects of proton irradiation of the lumbar intumescence on intra-axonal transport of acetylcholine and cholinergic enzymes in rat sciatic nerve

    The content and intra-axonal transport of acetylcholine (ACh) and the cholinergic enzymes cholineacetyl-transferase (CAT) and ACh-esterase (AChE) in sciatic nerve were investigated in rats following single dose proton irradiation of the lumbar intumescence of the spinal cord with 60 Gy or 200 Gy. One, 7 or 30 days after irradiation nerve-crush operations were performed 12 hours before killing and the levels of ACh and enzyme activities in nerve segments relative to the crushes were estimated by biologic (ACh) to chemical (enzyme) methods. The results indicate that alterations in intra-neuronal dynamics of ACh and related enzymes are not a major cause for the development of neurologic symptoms of the motor system after irradiation, and that descending myelinated axons are of minor importance for the regulation of cholinergic substances in rat motor nerves. (Auth.)

  1. Positron emission tomography imaging of (2R,3R)-5-[18F]fluoroethoxybenzovesamicol in rat and monkey brain: a radioligand for the vesicular acetylcholine transporter

    Introduction: The regional brain distribution of (2R,3R)-5-[18F]fluoroethoxy-benzovesamicol ((-)-[18F]FEOBV), a radioligand for the vesicular acetylcholine transporter (VAChT), was examined in vivo in mice, rats and rhesus monkeys. Methods: Regional brain distributions of (-)-[18F]FEOBV in mice were determined using ex vivo dissection. MicroPET imaging was used to determine the regional brain pharmacokinetics of the radioligand in rat and rhesus monkey brains. Results: In all three species, clear heterogeneous regional brain distributions were obtained, with the rank order of brain tissues (striatum>thalamus>cortex>cerebellum) consistent with the distribution of cholinergic nerve terminals containing the VAChT. Conclusions: (-)-[18F]FEOBV remains a viable candidate for further development as an in vivo imaging agent for positron emission tomography (PET) studies of the VAChT in the human brain.

  2. Ion Transport in Human Pancreatic Duct Epithelium, Capan-1 Cells, Is Regulated by Secretin, VIP, Acetylcholine, and Purinergic Receptors

    Wang, Jing; Novak, Ivana

    2013-01-01

    , purinergic receptors, and determine their effects on ion transport. METHODS: Human adenocarcinoma cell line Capan-1 cells were grown on permeable supports and set in Ussing chambers for electrophysiological recordings. Transepithelial voltage (Vte), resistance, and short-circuit currents (Isc) were measured...

  3. Review of Transportation Mode Detection techniques

    Jacopo Biancat

    2014-10-01

    Full Text Available This paper reviews the works found in the literature in the field of Transportation Mode Detection (TMD which is a subfield of Activity Recognition aiming at indentifying (i.e. classifying the mean of transportation a person is using. The solutions found in literature have different characteristics according to the device for which the solution was tailored (smartphones or other systems such as, e.g., GPS loggers and to the algorithm used for the classification task. This may vary a lot according to the number and type of input used (e.g. accelerations, GPS, maps information or GIS - Geographical Information System information and to the identified classes of transportation mode. These two aspects are the most relevant to consider when evaluating and comparing the accuracies claimed by each work. A comparison of the works is proposed taking into account the characteristics discussed above. In general the accelerometer is the most widely used sensor for TMD applications, as it limits battery consumption and captures relevant features for detecting motion. Indeed a key challenge in TMD is to detect different motorized classes such as bus, car, train and metro because they share common characteristics (such as e.g. the average speed and accelerations which make hard identifying suitable features for the classification algorithm. Identifying the “walk” and “stationary” transportation modes is a simpler task because they are characterized by distinct features.

  4. Towards Indoor Transportation Mode Detection using Mobile Sensing

    Prentow, Thor Siiger; Blunck, Henrik; Kjærgaard, Mikkel Baun; Stisen, Allan

    2015-01-01

    Transportation mode detection is a growing field of research, in which a varietyof methods have been developed for detecting transportation modes foremost foroutdoor travels. It has been employed in application areas such as publictransportation, environmental footprint profiling, and context-aware mobileassistants. For indoor travels the problem of transportation mode detection has receivedcomparatively little attention, even though diverse transportation modes, suchas biking, electric vehic...

  5. Combined α7 nicotinic acetylcholine receptor agonism and partial serotonin transporter inhibition produce antidepressant-like effects in the mouse forced swim and tail suspension tests

    Andreasen, Jesper T; Redrobe, John P; Nielsen, Elsebet Ø

    2012-01-01

    Emerging evidence points to an involvement of nicotinic acetylcholine receptors (nAChRs) in major depression. Nicotine improves symptoms of depression in humans and shows antidepressant-like effects in rodents. Monoamine release is facilitated by nAChR stimulation, and nicotine-evoked serotonin (...

  6. Morphogenetic roles of acetylcholine.

    Lauder, J. M.; Schambra, U B

    1999-01-01

    In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. In developing tissues and primitive organisms, neurotransmitters subserve growth regulatory and morphogenetic functions. Accumulated evidence suggests that acetylcholine, (ACh), released from growing axons, regulates growth, differentiation, and plasticity of developing central nervous system neurons. In addition to intrinsic cholinergic neurons, the cerebral cortex and hippocampus receive ...

  7. Metabolism of acetylcholine in human erythrocytes

    In order to examine the possible role of erythrocyte acetylcholinesterase in the maintenance of membrane phospholipid content and membrane fluidity, experiments were performed to monitor the activity of the enzyme and follow the fate of one of its hydrolytic products, choline. Intact human erythrocytes were incubated with acetylcholine (choline methyl-14C). The incubation resulted in the hydrolysis of acetylcholine to acetate and choline; the reaction was catalyzed by membrane acetylcholinesterase. The studies demonstrate the further metabolism of choline. Experiments were carried out to determine rate of hydrolysis of acetylcholine, uptake of choline, identification of intracellular metabolites of choline, and identification of radiolabeled membrane components. Erythrocytes at a 25% hematocrit were incubated in an isoosmotic bicarbonate buffer pH 7.4, containing glucose, adenosine, streptomycin and penicillin with 0.3 μCi of acetylcholine (choline methyl-14C), for 24 hours. Aliquots of the erythrocyte suspension were taken throughout for analysis. Erythrocytes were washed free of excess substrate, lysed, and the hemolysate was extracted for choline and its metabolites. Blank samples containing incubation buffer and radiolabeled acetylcholine only, and erythrocyte hemolysate extracts were analyzed for choline content, the difference between blank samples and hemolysate extracts was the amount of choline originating from acetylcholine and attributable to acetylcholinesterase activity. The conversion of choline to 14C-betaine is noted after several minutes of incubation; at 30 minutes, more than 80% of 14C-choline is taken up and after several hours, detectable levels of radiolabeled S-adenosylmethionine were present in the hemolysate extract

  8. New Insights on Plant Cell Elongation: A Role for Acetylcholine

    Gian-Pietro Di Sansebastiano

    2014-03-01

    Full Text Available We investigated the effect of auxin and acetylcholine on the expression of the tomato expansin gene LeEXPA2, a specific expansin gene expressed in elongating tomato hypocotyl segments. Since auxin interferes with clathrin-mediated endocytosis, in order to regulate cellular and developmental responses we produced protoplasts from tomato elongating hypocotyls and followed the endocytotic marker, FM4-64, internalization in response to treatments. Tomato protoplasts were observed during auxin and acetylcholine treatments after transient expression of chimerical markers of volume-control related compartments such as vacuoles. Here we describe the contribution of auxin and acetylcholine to LeEXPA2 expression regulation and we support the hypothesis that a possible subcellular target of acetylcholine signal is the vesicular transport, shedding some light on the characterization of this small molecule as local mediator in the plant physiological response.

  9. New insights on plant cell elongation: a role for acetylcholine.

    Di Sansebastiano, Gian-Pietro; Fornaciari, Silvia; Barozzi, Fabrizio; Piro, Gabriella; Arru, Laura

    2014-01-01

    We investigated the effect of auxin and acetylcholine on the expression of the tomato expansin gene LeEXPA2, a specific expansin gene expressed in elongating tomato hypocotyl segments. Since auxin interferes with clathrin-mediated endocytosis, in order to regulate cellular and developmental responses we produced protoplasts from tomato elongating hypocotyls and followed the endocytotic marker, FM4-64, internalization in response to treatments. Tomato protoplasts were observed during auxin and acetylcholine treatments after transient expression of chimerical markers of volume-control related compartments such as vacuoles. Here we describe the contribution of auxin and acetylcholine to LeEXPA2 expression regulation and we support the hypothesis that a possible subcellular target of acetylcholine signal is the vesicular transport, shedding some light on the characterization of this small molecule as local mediator in the plant physiological response. PMID:24642879

  10. Novel acetylcholine and carbamoylcholine analogues

    Hansen, Camilla Petrycer; Jensen, Anders Asbjørn; Christensen, Jeppe K.;

    2008-01-01

    A series of carbamoylcholine and acetylcholine analogues were synthesized and characterized pharmacologically at neuronal nicotinic acetylcholine receptors (nAChRs). Several of the compounds displayed low nanomolar binding affinities to the alpha 4beta 2 nAChR and pronounced selectivity for this ...

  11. Contamination detection device for radioactive material transporting device

    The present invention provides a device for detecting leakage of radioactive materials and clogging of a vial in a radioactive material transporting device. Namely, a transporting tube pneumatically transports the vial containing a sample of radioactive materials. A fine flow tube is laid in adjacent with and along the transporting tube. A pump flows a detection liquid prepared by a preparation device to the fine flow tube. A flow-cell type spectrophotometer is disposed to the fine flow tube. The detection liquid is preferably containing elements having a valency changeable by undergoing radiation irradiation, which is a type used, for example, for a Fricke dosimeter. With such a constitution, the position of the radioactive contamination of the inside of the pneumatic tube and clogging of a vial can be detected only by flowing radiation sensitive detection liquid to the fine flow tube. In addition, the detection liquid is not activated only changing the valency of the contained elements. (N.H.)

  12. Community core detection in transportation networks

    De Leo, Vincenzo; Cerina, Federica; Mureddu, Mario; Secchi, Luca; Chessa, Alessandro

    2013-01-01

    This work analyzes methods for the identification and the stability under perturbation of a territorial community structure with specific reference to transportation networks. We considered networks of commuters for a city and an insular region. In both cases, we have studied the distribution of commuters' trips (i.e., home-to-work trips and viceversa). The identification and stability of the communities' cores are linked to the land-use distribution within the zone system, and therefore their proper definition may be useful to transport planners.

  13. Community core detection in transportation networks.

    De Leo, Vincenzo; Santoboni, Giovanni; Cerina, Federica; Mureddu, Mario; Secchi, Luca; Chessa, Alessandro

    2013-10-01

    This work analyzes methods for the identification and the stability under perturbation of a territorial community structure with specific reference to transportation networks. We considered networks of commuters for a city and an insular region. In both cases, we have studied the distribution of commuters' trips (i.e., home-to-work trips and vice versa). The identification and stability of the communities' cores are linked to the land-use distribution within the zone system, and therefore their proper definition may be useful to transport planners. PMID:24229228

  14. Community core detection in transportation networks

    De Leo, Vincenzo; Santoboni, Giovanni; Cerina, Federica; Mureddu, Mario; Secchi, Luca; Chessa, Alessandro

    2013-10-01

    This work analyzes methods for the identification and the stability under perturbation of a territorial community structure with specific reference to transportation networks. We considered networks of commuters for a city and an insular region. In both cases, we have studied the distribution of commuters’ trips (i.e., home-to-work trips and vice versa). The identification and stability of the communities’ cores are linked to the land-use distribution within the zone system, and therefore their proper definition may be useful to transport planners.

  15. Community core detection in transportation networks

    Leo, Vincenzo; Santoboni, Giovanni; Cerina, Federica; Mureddu, Mario; Secchi, Luca; Chessa, Alessandro

    2013-01-01

    This work analyses methods for the identification and the stability under perturbation of a territorial community structure with specific reference to transportation networks. We considered networks of commuters for a city and an insular region. In both cases, we have studied the distribution of commuters' trips (i.e., home-to-work trips and viceversa). The identification and stability of the communities' cores are linked to the land-use distribution within the zone system, and therefore thei...

  16. Cholinergic neurotransmission in human corpus cavernosum. II. Acetylcholine synthesis

    Physiological and histochemical evidence indicates that cholinergic nerves may participate in mediating penile erection. Acetylcholine synthesis and release was studied in isolated human corporal tissue. Human corpus cavernosum incubated with [3H]choline accumulated [3H]choline and synthesized [3H]acethylcholine in an concentration-dependent manner. [3H]Acetylcholine accumulation by the tissue was inhibited by hemicholinium-3, a specific antagonist of the high-affinity choline transport in cholinergic nerves. Transmural electrical field stimulation caused release of [3H]acetylcholine which was significantly diminished by inhibiting neurotransmission with calcium-free physiological salt solution or tetrodotoxin. These observations provide biochemical and physiological evidence for the existence of cholinergic innervation in human corpus cavernosum

  17. Immunisation with Torpedo acetylcholine receptor.

    Elfman, L

    1984-01-01

    Acetylcholine mediates the transfer of information between neurons in the electric organ of, for example, Torpedo as well as in vertebrate skeletal muscle. The nicotinic acetylcholine receptor complex translates the binding of acetylcholine into ion permeability changes. This leads to an action potential in the muscle fibre. The nicotinic acetylcholine receptor protein has been purified from Torpedo by use of affinity chromatography. The receptor is an intrinsic membrane glycoprotein composed of five polypeptide chains. When various animals are immunised with the receptor they demonstrate clinical signs of severe muscle weakness coincident with high antibody titres in their sera. The symptoms resemble those found in the autoimmune neuromuscular disease myasthenia gravis in humans. This animal model has constituted a unique model for studying autoimmune diseases. This paper reviews some of the work using Torpedo acetylcholine receptor in order to increase the understanding of the motor nervous system function and myasthenia gravis. It is now known that the nicotinic acetylcholine receptor protein is the antigen involved in myasthenia gravis. The mechanism of immune damage involves a direct block of the receptor function. This depends on the presence of antibodies which crosslink the postsynaptic receptors leading to their degradation. The questions to be answered in the future are; (a) what initiates or triggers the autoimmune response, (b) how do the antibodies cause the symptoms--is there a steric hindrance of the interaction of acetylcholine and the receptor, (c) why is there not a strict relationship between antibody titre and severity of symptoms, and (d) why are some muscles affected and other spared? With help of the experimental model, answers to these questions may result in improved strategies for the treatment of the autoimmune disease myasthenia gravis. PMID:6097937

  18. PCR detection of groundwater bacteria associated with colloidal transport

    Colloidal transport may increase the amount of contaminant material than that which could be transported by water flow alone. The role of colloids in groundwater contaminant transport is complicated and may involve many different processes, including sorption of elements onto colloidal particles, coagulation/dissolution, adsorption onto solid surfaces, filtration, and migration. Bacteria are known to concentrate minerals and influence the transport of compounds in aqueous environments and may also serve as organic colloids, thereby influencing subsurface transport of radionuclides and other contaminants. The initial phase of the project consisted of assembling a list of bacteria capable of sequestering or facilitating mineral transport. The development and optimization of the PCR amplification assay for the detection of the organisms of interest, and the examination of regional groundwaters for those organisms, are presented for subsequent research

  19. PCR detection of groundwater bacteria associated with colloidal transport

    Cruz-Perez, P.; Stetzenbach, L.D.; Alvarez, A.J.

    1996-02-29

    Colloidal transport may increase the amount of contaminant material than that which could be transported by water flow alone. The role of colloids in groundwater contaminant transport is complicated and may involve many different processes, including sorption of elements onto colloidal particles, coagulation/dissolution, adsorption onto solid surfaces, filtration, and migration. Bacteria are known to concentrate minerals and influence the transport of compounds in aqueous environments and may also serve as organic colloids, thereby influencing subsurface transport of radionuclides and other contaminants. The initial phase of the project consisted of assembling a list of bacteria capable of sequestering or facilitating mineral transport. The development and optimization of the PCR amplification assay for the detection of the organisms of interest, and the examination of regional groundwaters for those organisms, are presented for subsequent research.

  20. Transports Regulators of Networks with Junctions Detected by Durations Functions

    Aubin, Jean-Pierre

    2013-01-01

    This study advocates a mathematical framework of ''transport relations'' on a network. They single out a subset of ''traffic states'' described by time, duration, position and other traffic attributes (called ''monads'' for short). Duration evolutions are non-negative, decreasing toward zero for incoming durations, increasing from zero for outgoing durations, allowing the detection of ''junction states'' defined as traffic states with ''zero duration''. A ''junction relation'' (crossroads, sy...

  1. Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release

    Alexandra eAcevedo-Rodriguez; Lifen eZhang; Fuwen eZhou; Suzhen eGong; Howard eGu; Mariella eDe Biasi; Fu-Ming eZhou; Dani, John A.

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) potently regulate dopamine (DA) release in the striatum and alter cocaine’s ability to reinforce behaviors. Since cocaine is a weak nAChR inhibitor, we hypothesized that cocaine may alter DA release by inhibiting the nAChRs in DA terminals in the striatum and thus contribute to cocaine's reinforcing properties primarily associated with the inhibition of DA transporters. We found that biologically relevant concentrations of cocaine can mildly inhibit...

  2. Tubular crystals of acetylcholine receptor

    1984-01-01

    Well-ordered tubular crystals of acetylcholine receptor were obtained from suspensions of Torpedo marmorata receptor-rich vesicles. They are composed of pairs of oppositely oriented molecules arranged on the surface lattice with the symmetry of the plane group p2 (average unit cell dimensions: a = 90 A, b = 162 A, gamma = 117 degrees). The receptor in this lattice has an asymmetric distribution of mass around its perimeter, yet a regular pentagonal shape; thus its five transmembrane subunits ...

  3. Acetylcholine functionally reorganizes neocortical microcircuits

    Runfeldt, Melissa J.; Sadovsky, Alexander J.; MacLean, Jason N.

    2014-01-01

    Sensory information is processed and transmitted through the synaptic structure of local cortical circuits, but it is unclear how modulation of this architecture influences the cortical representation of sensory stimuli. Acetylcholine (ACh) promotes attention and arousal and is thought to increase the signal-to-noise ratio of sensory input in primary sensory cortices. Using high-speed two-photon calcium imaging in a thalamocortical somatosensory slice preparation, we recorded action potential...

  4. Enzyme-linked DNA dendrimer nanosensors for acetylcholine

    Walsh, Ryan; Morales, Jennifer M.; Skipwith, Christopher G.; Ruckh, Timothy T.; Clark, Heather A.

    2015-10-01

    It is currently difficult to measure small dynamics of molecules in the brain with high spatial and temporal resolution while connecting them to the bigger picture of brain function. A step towards understanding the underlying neural networks of the brain is the ability to sense discrete changes of acetylcholine within a synapse. Here we show an efficient method for generating acetylcholine-detecting nanosensors based on DNA dendrimer scaffolds that incorporate butyrylcholinesterase and fluorescein in a nanoscale arrangement. These nanosensors are selective for acetylcholine and reversibly respond to levels of acetylcholine in the neurophysiological range. This DNA dendrimer architecture has the potential to overcome current obstacles to sensing in the synaptic environment, including the nanoscale size constraints of the synapse and the ability to quantify the spatio-temporal fluctuations of neurotransmitter release. By combining the control of nanosensor architecture with the strategic placement of fluorescent reporters and enzymes, this novel nanosensor platform can facilitate the development of new selective imaging tools for neuroscience.

  5. Dose protocols of acetylcholine test in Chinese

    向定成; 龚志华; 何建新; 洪长江; 邱建; 马骏

    2004-01-01

    @@ Acetylcholine test has been widely used clinically in several countries as a practical test provoking coronary artery spasm.1-3 Although it has also been launched recently in a few hospitals in China, the dose protocol for acetylcholine test used in these hospitals were from abroad.4,5 This study was aimed at developing a dose protocol for acetylcholine test suitable for Chinese people.

  6. Acetylcholine in plants: presence, metabolism and mechanism of action

    Tretyn, Andrzej; Kendrick, Richard E.

    1991-01-01

    Acetylcholine (ACh) has been detected in representatives of many taxonomic groups throughout the plant kingdom. The site of its synthesis in plants is probably young leaves. In some plant species choline acetyltransferase (CHAT) activity has been found. This enzyme showing properties similar to animal CHAT, probably participates in ACh synthesis from its precursors, choline and acetyl-Coenzyme A. Acetylcholinesterase (ACHE) activity has also been found in many plant tissues. Th...

  7. Nicotinic Acetylcholine Receptor (nAChR Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine.

    Zuo Jun Ren

    Full Text Available Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR, inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.

  8. Effect of organophosphorus insecticides on phosphorylation of the M2 muscarinic acetylcholine receptor

    Shuyin Li; Liming Zou; Carry Pope

    2008-01-01

    BACKGROUND: Organophosphorus insecticides may promote the accumulation of acetylcholine at synapses and the neuromuscular junction by inhibiting acetylcholinesterase activity to cause disturbance of neural signal conduction and induce a toxic reaction. Organophosphorus insecticides may act on M2 muscarinic acetylcholine receptors, whose combination with G proteins is regulated by phosphorylation of G protein-coupled receptor kinase 2.OBJECTIVE: To investigate the effects of organophosphorus insecticides on the phosphorylation of G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptors and to reveal other possible actions of organophosphorus insecticides.DESIGN, TIME AND SETTING: An observational study, which was performed in the Central Laboratory of Shenyang Medical College, and Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University from June 2002 to December 2004.METHODS: The M2 muscarinic acetylcholine receptor was extracted and purified from pig brain using affinity chromatography. Subsequently, the purified M2 muscarinic acetylcholine receptor, G protein-coupled receptor kinase 2, and [OP32] ATP were incubated with different concentrations of paraoxon and chlorpyrifos oxon together. The mixture then underwent polyacrylamide gel electrophoresis, and the gel film was dried and radioactively autographed to detect phosphorylation of the M2 muscarinic acetylcholine receptor. Finally, the radio-labeled phosphorylated M2 receptor protein band was excised for counting with an isotope liquid scintillation counter.MAIN OUTCOME MEASURES: Effects of chlorpyrifos oxon, paraoxon, chlorpyrifos, and parathion in different concentrations on the phosphorylation of the M2 muscarinic acetylcholine receptor; effects of chlorpyrifos oxon on the phosphorylation of the adrenergic receptor.CONCLUSION: Different kinds of organophosphorus insecticides have different effects on the phosphorylation of the G protein

  9. Acoustic leak-detection system for railroad transportation security

    Womble, P. C.; Spadaro, J.; Harrison, M. A.; Barzilov, A.; Harper, D.; Hopper, L.; Houchins, E.; Lemoff, B.; Martin, R.; McGrath, C.; Moore, R.; Novikov, I.; Paschal, J.; Rogers, S.

    2007-04-01

    Pressurized rail tank cars transport large volumes of volatile liquids and gases throughout the country, much of which is hazardous and/or flammable. These gases, once released in the atmosphere, can wreak havoc with the environment and local populations. We developed a system which can non-intrusively and non-invasively detect and locate pinhole-sized leaks in pressurized rail tank cars using acoustic sensors. The sound waves from a leak are produced by turbulence from the gas leaking to the atmosphere. For example, a 500 μm hole in an air tank pressurized to 689 kPa produces a broad audio frequency spectrum with a peak near 40 kHz. This signal is detectable at 10 meters with a sound pressure level of 25 dB. We are able to locate a leak source using triangulation techniques. The prototype of the system consists of a network of acoustic sensors and is located approximately 10 meters from the center of the rail-line. The prototype has two types of acoustic sensors, each with different narrow frequency response band: 40 kHz and 80 kHz. The prototype is connected to the Internet using WiFi (802.11g) transceiver and can be remotely operated from anywhere in the world. The paper discusses the construction, operation and performance of the system.

  10. neu-VISION: an explosives detection system for transportation security

    Warman, Kieffer; Penn, David

    2008-04-01

    Terrorists were targeting commercial airliners long before the 9/11 attacks on the World Trade Center and the Pentagon. Despite heightened security measures, commercial airliners remain an attractive target for terrorists, as evidenced by the August 2006 terrorist plot to destroy as many as ten aircraft in mid-flight from the United Kingdom to the United States. As a response to the security threat air carriers are now required to screen 100-percent of all checked baggage for explosives. The scale of this task is enormous and the Transportation Security Administration has deployed thousands of detection systems. Although this has resulted in improved security, the performance of the installed systems is not ideal. Further improvements are needed and can only be made with new technologies that ensure a flexible Concept of Operations and provide superior detection along with low false alarm rates and excellent dependability. To address security needs Applied Signal Technology, Inc. is developing an innovative and practical solution to meet the performance demands of aviation security. The neu-VISION TM system is expected to provide explosives detection performance for checked baggage that both complements and surpasses currently deployed performance. The neu-VISION TM system leverages a 5 year R&D program developing the Associated Particle Imaging (API) technique; a neutron based non-intrusive material identification and imaging technique. The superior performance afforded by this neutron interrogation technique delivers false alarm rates much lower than deployed technologies and "sees through" dense, heavy materials. Small quantities of explosive material are identified even in the cluttered environments.

  11. Nicotinic acetylcholine receptors mediate lung cancer growth

    PaulDGardner

    2013-09-01

    Full Text Available Ion channels modulate ion flux across cell membranes, activate signal transduction pathways, and influence cellular transport – vital biological functions that are inexorably linked to cellular processes that go awry during carcinogenesis. Indeed, deregulation of ion channel function has been implicated in cancer-related phenomena such as unrestrained cell proliferation and apoptotic evasion. As the prototype for ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs have been extensively studied in the context of neuronal cells but accumulating evidence also indicate a role for nAChRs in carcinogenesis. Recently, variants in the nAChR genes CHRNA3, CHRNA5, and CHRNB4 have been implicated in nicotine dependence and lung cancer susceptibility. Here, we silenced the expression of these three genes to investigate their function in lung cancer. We show that these genes are necessary for the viability of small cell lung carcinomas (SCLC, the most aggressive type of lung cancer. Furthermore, we show that nicotine promotes SCLC cell viability whereas an α3β4-selective antagonist, α-conotoxin AuIB, inhibits it. Our findings posit a mechanism whereby signaling via α3/α5/β4-containing nAChRs promotes lung carcinogenesis.

  12. Optical detection of spin transport in non-magnetic metals

    Fohr, Frederik; Kaltenborn, Steffen; Hamrle, Jaroslav; Schultheiss, Helmut; Serga, Alexander A.; Schneider, Hans Christian; Hillebrands, Burkard [Fachbereich Physik, Landesforschungszentrum OPTIMAS, TU Kaiserslautern, 67663 Kaiserslautern (Germany); Fukuma, Yasuhiro; Wang, Le; Otani, YoshiChika [ASI RIKEN, ISSP, University of Tokyo (Japan)

    2011-07-01

    We detect the dynamic magnetization in non-magnetic metal wedges, composed of silver, copper and platinum and grown on top of a Ni{sub 80}Fe{sub 20} layer. The Ni{sub 80}Fe{sub 20} layer is excited externally by the RF field of a coplanar waveguide (CPW), and generates the dynamic magnetization in the wedge layer via the spin pumping effect. The inelastically scattered light is collected as a function of the local wedge thickness and analysed by Brillouin light scattering (BLS) microscopy. The BLS signal originates from the metal wedges due to inelastic scattering from the spin polarization as well as from the magnetic layer below the wedge. To separate both contributions of the signal experimentally, reference samples are prepared with an interlayer between Ni{sub 81}Fe{sub 19} and the respective metal wedge to block the spin pumping. By comparing the experimental results to a macroscopic spin-transport model we determine the transverse relaxation time of the pumped spin current which is much smaller than the longitudinal relaxation time.

  13. Vadose Zone Sampling Methods for Detection of Preferential Pesticides Transport

    Peranginangin, N.; Richards, B. K.; Steenhuis, T. S.

    2003-12-01

    Leaching of agricultural applied chemicals through the vadose zone is a major cause for the occurrence of agrichemicals in groundwater. Accurate soil water sampling methods are needed to ensure meaningful monitoring results, especially for soils that have significant preferential flow paths. The purpose of this study was to assess the capability and the effectiveness of various soil water sampling methods in detecting preferential transport of pesticides in a strongly-structured silty clay loam (Hudson series) soil. Soil water sampling devices tested were wick pan and gravity pan lysimeters, tile lines, porous ceramic cups, and pipe lysimeters; all installed at 45 to105 cm depth below the ground surface. A reasonable worse-case scenario was tested by applying a simulated rain storm soon after pesticides were sprayed at agronomic rates. Herbicides atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) and 2,4-D (2,4-dichloro-phenoxyacetic acid) were chosen as model compounds. Chloride (KCl) tracer was used to determine spatial and temporal distribution of non-reactive solute and water as well as a basis for determining the retardation in pesticides movement. Results show that observed pesticide mobility was much greater than would be predicted by uniform flow. Under relatively high soil moisture conditions, gravity and wick pan lysimeters had comparably good collection efficiencies, whereas the wick samplers had an advantage over gravity driven sampler when the soil moisture content was below field capacity. Pipe lysimeters had breakthrough patterns that were similar to pan samplers. At small plot scale, tile line samplers tended to underestimate solute concentration because of water dilution around the samplers. The use of porous cup samplers performed poorly because of their sensitivity to local profile characteristics: only by chance can they intercept and sample the preferential flow paths that are critical to transport. Wick sampler had the least

  14. Early fluorescence signals detect transitions at mammalian serotonin transporters.

    Li, Ming; Lester, Henry A.

    2002-01-01

    The mammalian serotonin transporters rSERT or hSERT were expressed in oocytes and labeled with sulforhodamine-MTS. The endogenous Cys-109 residue contributes most of the signal, and the labeled transporter shows normal function. The SERT fluorescence decreases in the presence of 5-HT and also depends on the inorganic substrates of SERT. The fluorescence also increases with membrane depolarization. During voltage-jump experiments, fluorescence relaxations show little inactivation or history de...

  15. Using Smart Phone Sensors to Detect Transportation Modes

    Hao Xia; Yanyou Qiao; Jun Jian; Yuanfei Chang

    2014-01-01

    The proliferation of mobile smart devices has led to a rapid increase of location-based services, many of which are amassing large datasets of user trajectory information. Unfortunately, current trajectory information is not yet sufficiently rich to support classification of user transportation modes. In this paper, we propose a method that employs both the Global Positioning System and accelerometer data from smart devices to classify user outdoor transportation modes. The classified modes i...

  16. Uptake of 3H-choline and synthesis of 3H-acetylcholine by human penile corpus cavernosum

    The neuroeffectors which relax penile smooth muscle and lead to erection are unknown; physiological studies of human corpus cavernosum, in vitro, have suggested a significant role of cholinergic neurotransmission. To further characterize the importance of cholinergic nerves, biopsies of human corpus cavernosum were obtained at the time of penile prosthesis implantation. Tissues were incubated in 3H-choline (10-5M, 80 Ci/mmol) in oxygenated physiological salt solution at 370C, pH 7.4 for 1 hour. Radiolabelled compounds were extracted with perchloric acid (0.4 M) and acetylcholine and choline were separated by HPLC; 14C-acetylcholine was used as internal standard. 3H-choline was accumulated by the tissues (20 +/- 1.9 fmol/mg), and 3H-acetylcholine was synthesized (4.0 +/- 1.1 fmol/mg). In control experiments, heating of the tissue blocked synthesis of 3H-acetylcholine. Inhibition of high affinity choline transport by hemicholinium-3 (10-5M) diminished tissue accumulation of 3H-choline and significantly reduced the synthesis of 3H-acetylcholine (0.5 +/ 0.2 fmol/mg, p < 0.05). These results provide direct evidence of neuronal accumulation of choline and enzymatic conversion to acetylcholine in human corpus cavernosum. Taken together with the physiological studies, it can be concluded that cholinergic neurotransmission in human corpus cavernosum plays a role in penile erection

  17. Acetylcholine synthesis and possible functions during sea urchin development

    C Angelini

    2009-06-01

    Full Text Available Cholinergic neurotransmitter system molecules were found to play a role during fertilisation and early cell cycles of a large number of invertebrate and vertebrate organisms. In this study, we investigated the presence and possible function of choline acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholine in gametes of the sea urchin, Paracentrotus lividus, through localisation and functional studies. ChAT-like molecules were detected in oocytes, mature eggs and zygotes with indirect immunofluorescence methods. Positive immunoreactivity was found in the ovarian egg cytoplasm and surface as well as at the zygote surface. This suggests the eggs' capacity to autonomously synthesise acetylcholine (ACh, the signal molecule of the cholinergic system. Acetylcholinesterase (AChE, the lytic enzyme of acetylcholine was also found in ovarian eggs, with a similar distribution; however, it disappeared after fertilisation. Ultrastructural ChAT localisation in sperms, which was carried out with the immuno-gold method, showed immunoreactivity in the acrosome of unreacted sperms and at the head surface of reacted sperms. In order to verify a functional role of ACh during fertilization and sea urchin development, in vivo experiments were performed. Exposure of the eggs before fertilisation to 1 mM ACh + 1 ?M eserine caused an incomplete membrane depolarisation and consequently enhanced polyspermy, while lower concentrations of ACh caused developmental anomalies. The exposure of zygotes to 0,045 AChE Units/mL of sea water caused developmental anomalies as well, in 50% of the embryos. Altogether, these findings and other previously obtained results, suggest that the cholinergic system may subserve two different tasks during development, according to which particular type of ACh receptor is active during each temporal window. The first function, taking place in the course of fertilisation is a result of autonomously synthesised ACh in sperms, while the

  18. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Jakubík, Jan; El-Fakahany, E. E.

    2010-01-01

    Roč. 3, č. 9 (2010), s. 2838-2860. ISSN 1424-8247 R&D Projects: GA ČR GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic acetylcholine receptors * allosteric modulation * Alzheimer ´s disease Subject RIV: CE - Biochemistry

  19. Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors.

    Rozman, Klara Bulc; Araoz, Romulo; Sepcić, Kristina; Molgo, Jordi; Suput, Dusan

    2010-09-01

    Nicotinic acetylcholine receptors are implicated in different nervous system-related disorders, and their modulation could improve existing therapy of these diseases. Parazoanthoxanthin A (ParaA) is a fluorescent pigment of the group of zoanthoxanthins. Since it is a potent acetylcholinesterase inhibitor, it may also bind to nicotinic acetylcholine receptors (nAChRs). For this reason its effect on Torpedo nAChR (alpha1(2)betagammadelta) transplanted to Xenopus laevis oocytes was evaluated, using the voltage-clamp technique. ParaA dose-dependently reduced the acetylcholine-induced currents. This effect was fully reversible only at lower concentrations. ParaA also reduced the Hill coefficient and the time to peak current, indicating a channel blocking mode of action. On the other hand, the combined effect of ParaA and d-tubocurarine (d-TC) on acetylcholine-induced currents exhibited only partial additivity, assuming a competitive mode of action of ParaA on nAChR. These results indicate a dual mode of action of ParaA on the Torpedo AChR. PMID:20230806

  20. Towards Indoor Transportation Mode Detection using Mobile Sensing

    Prentow, Thor Siiger; Blunck, Henrik; Kjærgaard, Mikkel Baun;

    2015-01-01

    ) such methods at a large hospital complex. The evaluation presented here utilizes Wi-Fi and accelerometer data collected through smartphones carried by several hospital workers throughout four days of work routines. The results show that the methods can distinguish between six common modes of transportation...

  1. Acetylcholine affects osteocytic MLO-Y4 cells via acetylcholine receptors.

    Ma, Yuanyuan; Li, Xianxian; Fu, Jing; Li, Yue; Gao, Li; Yang, Ling; Zhang, Ping; Shen, Jiefei; Wang, Hang

    2014-03-25

    The identification of the neuronal control of bone remodeling has become one of the many significant recent advances in bone biology. Cholinergic activity has recently been shown to favor bone mass accrual by complex cellular regulatory networks. Here, we identified the gene expression of the muscarinic and nicotinic acetylcholine receptors (m- and nAChRs) in mice tibia tissue and in osteocytic MLO-Y4 cells. Acetylcholine, which is a classical neurotransmitter and an osteo-neuromediator, not only influences the mRNA expression of the AChR subunits but also significantly induces the proliferation and viability of osteocytes. Moreover, acetylcholine treatment caused the reciprocal regulation of RANKL and OPG mRNA expression, which resulted in a significant increase in the mRNA ratio of RANKL:OPG in osteocytes via acetylcholine receptors. The expression of neuropeptide Y and reelin, which are two neurogenic markers, was also modulated by acetylcholine via m- and nAChRs in MLO-Y4 cells. These results indicated that osteocytic acetylcholine receptors might be a new valuable mediator for cell functions and even for bone remodeling. PMID:24508663

  2. The Diversity and Scale Matter: Ubiquitous Transportation Mode Detection using Single Cell Tower Information

    AbdelAziz, Ali Mohamed; Youssef, Moustafa

    2015-01-01

    Detecting the transportation mode of a user is important for a wide range of applications. While a number of recent systems addressed the transportation mode detection problem using the ubiquitous mobile phones, these studies either leverage GPS, the inertial sensors, and/or multiple cell towers information. However, these different phone sensors have high energy consumption, limited to a small subset of phones (e.g. high-end phones or phones that support neighbouring cell tower information),...

  3. Using smart phone sensors to detect transportation modes.

    Xia, Hao; Qiao, Yanyou; Jian, Jun; Chang, Yuanfei

    2014-01-01

    The proliferation of mobile smart devices has led to a rapid increase of location-based services, many of which are amassing large datasets of user trajectory information. Unfortunately, current trajectory information is not yet sufficiently rich to support classification of user transportation modes. In this paper, we propose a method that employs both the Global Positioning System and accelerometer data from smart devices to classify user outdoor transportation modes. The classified modes include walking, bicycling, and motorized transport, in addition to the motionless (stationary) state, for which we provide new depth analysis. In our classification, stationary mode has two sub-modes: stay (remaining in the same place for a prolonged time period; e.g., in a parked vehicle) and wait (remaining at a location for a short period; e.g., waiting at a red traffic light). These two sub-modes present different semantics for data mining applications. We use support vector machines with parameters that are optimized for pattern recognition. In addition, we employ ant colony optimization to reduce the dimension of features and analyze their relative importance. The resulting classification system achieves an accuracy rate of 96.31% when applied to a dataset obtained from 18 mobile users. PMID:25375756

  4. Using Smart Phone Sensors to Detect Transportation Modes

    Hao Xia

    2014-11-01

    Full Text Available The proliferation of mobile smart devices has led to a rapid increase of location-based services, many of which are amassing large datasets of user trajectory information. Unfortunately, current trajectory information is not yet sufficiently rich to support classification of user transportation modes. In this paper, we propose a method that employs both the Global Positioning System and accelerometer data from smart devices to classify user outdoor transportation modes. The classified modes include walking, bicycling, and motorized transport, in addition to the motionless (stationary state, for which we provide new depth analysis. In our classification, stationary mode has two sub-modes: stay (remaining in the same place for a prolonged time period; e.g., in a parked vehicle and wait (remaining at a location for a short period; e.g., waiting at a red traffic light. These two sub-modes present different semantics for data mining applications. We use support vector machines with parameters that are optimized for pattern recognition. In addition, we employ ant colony optimization to reduce the dimension of features and analyze their relative importance. The resulting classification system achieves an accuracy rate of 96.31% when applied to a dataset obtained from 18 mobile users.

  5. Application of neutron/gamma transport codes for the design of explosive detection systems

    Applications of neutron and gamma transport codes to the design of nuclear techniques for detecting concealed explosives material are discussed. The methodology of integrating radiation transport computations in the development, optimization and analysis phases of these new technologies is discussed. Transport and Monte Carlo codes are used for proof of concepts, guide the system integration, reduce the extend of experimental program and provide insight into the physical problem involved. The paper concentrates on detection techniques based on thermal and fast neutron interactions in the interrogated object. (authors). 6 refs., 1 tab., 5 figs

  6. Conflict Detection and Resolution for Future Air Transportation Management

    Krozel, Jimmy; Peters, Mark E.; Hunter, George

    1997-01-01

    With a Free Flight policy, the emphasis for air traffic control is shifting from active control to passive air traffic management with a policy of intervention by exception. Aircraft will be allowed to fly user preferred routes, as long as safety Alert Zones are not violated. If there is a potential conflict, two (or more) aircraft must be able to arrive at a solution for conflict resolution without controller intervention. Thus, decision aid tools are needed in Free Flight to detect and resolve conflicts, and several problems must be solved to develop such tools. In this report, we analyze and solve problems of proximity management, conflict detection, and conflict resolution under a Free Flight policy. For proximity management, we establish a system based on Delaunay Triangulations of aircraft at constant flight levels. Such a system provides a means for analyzing the neighbor relationships between aircraft and the nearby free space around air traffic which can be utilized later in conflict resolution. For conflict detection, we perform both 2-dimensional and 3-dimensional analyses based on the penetration of the Protected Airspace Zone. Both deterministic and non-deterministic analyses are performed. We investigate several types of conflict warnings including tactical warnings prior to penetrating the Protected Airspace Zone, methods based on the reachability overlap of both aircraft, and conflict probability maps to establish strategic Alert Zones around aircraft.

  7. Detecting conformational change by current transport in Bacteriorhodopsin

    Alfinito, Eleonora

    2008-01-01

    Charge transport modification exhibiting an increase of conductance activated by an external green light in Bacteriorhodopsin is correlated to its conformational change. A theoretical model based on a map of the protein structure into a resistor network is implemented to account for a sequential tunneling mechanism of charge transfer through neighbouring amino-acids. The model is validated by comparison with current-voltage experiments and provides for the potential barriers involved in the charge transfer an average height of 69 meV over an interacting radius of 6 \\aa. The predictability of the model is also tested on bovine rhodopsin, the prototype of the G protein coupled receptor (GPCR) family also sensitive to the light, with results exhibiting the opposite behaviour of a decrease of conductance in the presence of light.

  8. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Esam E. El-Fakahany

    2010-08-01

    Full Text Available An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity or decrease (negative cooperativity in the binding or action of an orthosteric agonist (e.g., acetylcholine. Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer’s disease and other disorders involving impaired cognitive function.

  9. The Role of Acetylcholine in Cocaine Addiction

    Williams, Mark J.; Adinoff, Bryon

    2007-01-01

    Central nervous system cholinergic neurons arise from several discrete sources, project to multiple brain regions, and exert specific effects on reward, learning, and memory. These processes are critical for the development and persistence of addictive disorders. Although other neurotransmitters, including dopamine, glutamate, and serotonin, have been the primary focus of drug research to date, a growing preclinical literature reveals a critical role of acetylcholine (ACh) in the experience a...

  10. Transportation Mode Detection Based on Permutation Entropy and Extreme Learning Machine

    Lei Zhang

    2015-01-01

    Full Text Available With the increasing prevalence of GPS devices and mobile phones, transportation mode detection based on GPS data has been a hot topic in GPS trajectory data analysis. Transportation modes such as walking, driving, bus, and taxi denote an important characteristic of the mobile user. Longitude, latitude, speed, acceleration, and direction are usually used as features in transportation mode detection. In this paper, first, we explore the possibility of using Permutation Entropy (PE of speed, a measure of complexity and uncertainty of GPS trajectory segment, as a feature for transportation mode detection. Second, we employ Extreme Learning Machine (ELM to distinguish GPS trajectory segments of different transportation. Finally, to evaluate the performance of the proposed method, we make experiments on GeoLife dataset. Experiments results show that we can get more than 50% accuracy when only using PE as a feature to characterize trajectory sequence. PE can indeed be effectively used to detect transportation mode from GPS trajectory. The proposed method has much better accuracy and faster running time than the methods based on the other features and SVM classifier.

  11. Performance Analysis of Transport Layer Basedhybrid Covert Channel Detection Engine

    Anjan K

    2013-11-01

    Full Text Available Computer network is unpredictable due to information warfareand is prone to various attacks. Such attackson network compromiseson the most important attribute, the privacy. Most of such attacksare devised usingspecial communication channel called Covert Channel".The word Covert" stands for hidden or non-transparent.Network Covert Channel is concealed communication paths within legitimatenetworkcommunication that clearly violates security policies laiddown. Non-transparency in covert channel isalsoreferred to as trapdoor.A trapdoor is unintended design within legitimate communication whosemotto isleak information. Subliminal channel, a variant ofcovert channelworks similarly as network covert channelexcept that trapdoor is setin cryptographic algorithm. A composition of covert channel withsubliminalchannel is the Hybrid Covert Channel". Hybrid covert channelis the homogeneous orheterogeneous mixture of two or more variantsof covert channel either active at same instance or atdifferent instanceof time. Detecting such maliciouschannel activity plays a vital role inremoving threat tolegitimate network.In this paper, we introduce newdetection engine for hybrid covert channelin transportlayer visualized in TCP and SSL. A setup made onexperimental test bed (DE-HCC9 in RD Lab of ourdepartment. Thepurpose of this study is to introduce few performance metrics to evaluatedetection engineand also to understand the multi-trapdoor natureofcovert channel.

  12. A Real-Time Pothole Detection Approach for Intelligent Transportation System

    Hsiu-Wen Wang

    2015-01-01

    Full Text Available In recent years, fast economic growth and rapid technology advance have led to significant impact on the quality of traditional transport system. Intelligent transportation system (ITS, which aims to improve the transport system, has become more and more popular. Furthermore, improving the safety of traffic is an important issue of ITS, and the pothole on the road causes serious harm to drivers’ safety. Therefore, drivers’ safety may be improved with the establishment of real-time pothole detection system for sharing the pothole information. Moreover, using the mobile device to detect potholes has been more popular in recent years. This approach can detect potholes with lower cost in a comprehensive environment. This study proposes a pothole detection method based on the mobile sensing. The accelerometer data is normalized by Euler angle computation and is adopted in the pothole detection algorithm to obtain the pothole information. Moreover, the spatial interpolation method is used to reduce the location errors from global positioning system (GPS data. In experiments, the results show that the proposed approach can precisely detect potholes without false-positives, and the higher accuracy is performed by the proposed approach. Therefore, the proposed real-time pothole detection approach can be used to improve the safety of traffic for ITS.

  13. A controlled field pilot for testing near surface CO2 detection techniques and transport models

    Spangler, L.H.; Dobeck, L.M.; Repasky, K.; Nehrir, A.; Humphries, S.; Keith, C.; Shaw, J.; Rouse, J.; Cunningham, A.; Benson, S.; Oldenburg, C.M.; Lewicki, J.L.; Wells, A.; Diehl, R.; Strazisar, B.; Fessenden, J.; Rahn, Thomas; Amonette, J.; Barr, J.; Pickles, W.; Jacobson, J.; Silver, E.; Male, E.; Rauch, H.; Gullickson, K.; Trautz, R.; Kharaka, Y.; Birkholzer, J.; Wielopolski, L.

    2009-01-01

    A field facility has been developed to allow controlled studies of near surface CO2 transport and detection technologies. The key component of the facility is a shallow, slotted horizontal well divided into six zones. The scale and fluxes were designed to address large scale CO2 storage projects and desired retention rates for those projects. A wide variety of detection techniques were deployed by collaborators from 6 national labs, 2 universities, EPRI, and the USGS. Additionally, modeling of CO2 transport and concentrations in the saturated soil and in the vadose zone was conducted. An overview of these results will be presented. ?? 2009 Elsevier Ltd. All rights reserved.

  14. A photonic crystal based sensing scheme for acetylcholine and acetylcholinesterase inhibitors

    Fenzl, Christoph; Genslein, Christa; Zöpfl, Alexander; Baeumner, Antje; Hirsch, Thomas

    2015-01-01

    We present a new scheme for sensing biomolecules by combining an enzyme hydrogel with a photonic crystal hydrogel layer that responds to ionic strength and pH changes. We demonstrate this unique combination by successfully detecting acetylcholine (ACh) and acetylcholinesterase (AChE) inhibitors. Specifically, the sandwich assembly is composed of layers of photonic crystals and a polyacrylamide hydrogel functionalized with AChE. The photonic crystal film has a red color and turns dark purple w...

  15. Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes

    Deckmann, Klaus; Filipski, Katharina; Krasteva-Christ, Gabriela; Fronius, Martin; Althaus, Mike; Rafiq, Amir; Papadakis, Tamara; Renno, Liane; Jurastow, Innokentij; Wessels, Lars; Wolff, Miriam; Schütz, Burkhard; Weihe, Eberhard; Chubanov, Vladimir; Gudermann, Thomas

    2014-01-01

    We report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system. These cells exhibit structural markers of respiratory chemosensory cells (“brush cells”). They use the classical taste transduction cascade to detect potential hazardous compounds (bitter, umami, uropathogenic bacteria) and release acetylcholine in response. They lie next to sensory ...

  16. Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine Binding Protein as a Structural Surrogate

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette; Balle, Thomas

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for development of drugs a...

  17. Facilitation of acetylcholine signaling by the dithiocarbamate fungicide propineb.

    Marinovich, Marina; Viviani, Barbara; Capra, Valerie; Corsini, Emanuela; Anselmi, Laura; D'Agostino, Gianluigi; Di Nucci, Amalia; Binaglia, Marco; Tonini, Marcello; Galli, Corrado L

    2002-01-01

    Dithiocarbamates (DTCs) are used mainly in agriculture as pesticides and as alcohol deterrent drugs. Neurological complications as well as movement disorders characterized by plastic rigidity, muscle twitch and paralysis are the prevailing symptoms in chronically exposed animals and humans. We investigated whether propineb interfered with peripheral cholinergic transmission in various isolated model systems. In electrically stimulated longitudinal muscle-myenteric plexus preparations (LMMPs), propineb (0.01-1000 nM) concentration-dependently enhanced the amplitude of both neurogenic twitch contractions and tritiated acetylcholine ([3H]ACh) release. The maximum percent increase was achieved by 10 nM propineb and was 19% and 14%, respectively. The effect on twitch contractions was partially antagonized by hexamethonium, a ganglionic nicotinic receptor blocker. In unstimulated LMMPs, propineb (10 pM, 10 nM, 10 microM) did not affect contractions to applied acetylcholine (ACh; 1 nM-10 microM), a finding indicating that propineb has no anticholinesterase activity. In human neuroblastoma cells (SH-SY5Y), propineb facilitated ACh release evoked by KCl depolarization. The increase in ACh release was not associated with detectable alterations of intracellular Ca2+([Ca2+]i) homeostasis. Binding studies carried out with alpha-bungarotoxin in striated muscle cells (L6) failed to demonstrate any influence of propineb on both affinity and capacity of skeletal muscle nicotinic receptors. In conclusion, propineb was found to interfere with cholinergic transmission in LMMPs and SH-SY5Y cells. In LMMPs, the potentiation of cholinergic transmission is partly dependent on the activation of ganglionic nicotinic receptors. Other targets relevant to cholinergic transmission seem not to be affected by propineb. PMID:11800594

  18. The effect of ketamine on intraspinal acetylcholine release

    Abelson, Klas S P; Goldkuhl, Renée Röstlinger; Nylund, Anders;

    2006-01-01

    The general anaesthetic ketamine affects the central cholinergic system in several manners, but its effect on spinal acetylcholine release, which may be an important transmitter in spinal antinociception, is unknown. This study aimed to investigate the effect of ketamine on spinal acetylcholine...... release. Microdialysis probes were placed intraspinally in male rats, and acetylcholine was quantified with HPLC. Anaesthesia was switched from isoflurane (1.3%) to ketamine (150 mg/kg h), which resulted in a 500% increased acetylcholine release. The increase was attenuated during nicotinic receptor...... blockade (50 microM mecamylamine). The nicotinic receptor agonist epibatidine (175 microM) produced a ten-fold higher relative increase of acetylcholine release during isoflurane anaesthesia compared to ketamine anaesthesia (270% to 27%). Intraspinal administration of ketamine and norketamine both...

  19. Expression and function of nicotinic acetylcholine receptors in stem cells

    Carlos M. Carballosa

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  20. Acetylcholine determination of microdialysates of fetal neocortex grafts that induce recovery of learning.

    Miranda, M I; Bermúdez-Rattoni, F

    1998-03-01

    The microdialysis technique for acetylcholine (ACh) first became possible when sensitive and specific assays for ACh (pmol/sample range) were developed [G. Damsma, B.H.C. Westerink, P. de Boer, J.B. de Vries, A.S. Horn, Determination of basal acetylcholine release in freely moving rats by transstriatal dialysis coupled to on-line HPLC analysis: pharmacological aspects, Life Sci. 43 (1988) 1161-1168; G. Damsma, B.H.C. Westerink, A. Imperato, H. Rollema, J.B. de Vries, A. S. Horn, Automated brain dialysis of acetylcholine in freely moving rats: detection of basal acetylcholine, Life Sci. 41 (1987) 873-876; P.E. Potter, J.L. Meek, N.H. Neff, Acetylcholine and choline in neural tissue measured by HPLC with electrochemical detection, J. Neurochem. 41 (1983) 188-194; B.H.C. Westerink, G. Damsma, Determination of acetylcholine in microdialysates by HPLC and electrochemical detection, Neurosci. Protocols 20 (1993) 1-9.]. In the present protocol, the microdialysis technique was used to correlate ACh release with the recovery of the ability to acquire a conditioning taste aversion (CTA), by fetal brain grafts in insular cortex (IC) lesioned rats [M.I. Miranda, A.M. Lopez-Colome, F. Bermúdez Rattoni, Recovery of conditional taste aversion induced by fetal neocortex grafts. In vivo correlation of acetylcholine levels, Brain Res. 759 (1997) 141-148]. Three groups of IC lesioned rats showing disrupted CTA received cell suspension grafts of fetal tissue dissected from either the IC or occipital cortex (OC) of 16-day-old rat fetuses. One of the groups of IC-grafted animals was tested after 15 days post-graft; the other groups, IC- and OC-grafted animals, were tested after a recovery time of 45 days, as well as the groups of lesioned and unoperated animals used as control. After the CTA test, guide cannulas were stereotaxically implanted into the IC of all groups. Two days later, microdialysis was performed to determine the extracellular levels of ACh inside the graft. The

  1. Performance of Four Transport and Storage Systems for Molecular Detection of Multidrug-Resistant Tuberculosis

    Rabodoarivelo, Marie Sylvianne; Imperiale, Bélen; andrianiavomikotroka, Rina; Brandao, Angela; Kumar, Parveen; Singh, Sarman; Ferrazoli, Lucilaine; Morcillo, Nora; Rasolofo, Voahangy; Palomino, Juan Carlos; Vandamme, Peter; Martin, Anandi

    2015-01-01

    Background Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus. Methods Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing. Results For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%. Conclusion The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings. PMID:26431352

  2. The Detection of Transport Land-Use Data Using Crowdsourcing Taxi Trajectory

    Ai, T.; Yang, W.

    2016-06-01

    This study tries to explore the question of transport land-use change detection by large volume of vehicle trajectory data, presenting a method based on Deluanay triangulation. The whole method includes three steps. The first one is to pre-process the vehicle trajectory data including the point anomaly removing and the conversion of trajectory point to track line. Secondly, construct Deluanay triangulation within the vehicle trajectory line to detect neighborhood relation. Considering the case that some of the trajectory segments are too long, we use a interpolation measure to add more points for the improved triangulation. Thirdly, extract the transport road by cutting short triangle edge and organizing the polygon topology. We have conducted the experiment of transport land-use change discovery using the data of taxi track in Beijing City. We extract not only the transport land-use area but also the semantic information such as the transformation speed, the traffic jam distribution, the main vehicle movement direction and others. Compared with the existed transport network data, such as OpenStreet Map, our method is proved to be quick and accurate.

  3. Chemical stimulation of adherent cells by localized application of acetylcholine from a microfluidic system.

    Zibek, Susanne; Hagmeyer, Britta; Stett, Alfred; Stelzle, Martin

    2010-01-01

    Chemical stimulation of cells is inherently cell type selective in contrast to electro-stimulation. The availability of a system for localized application of minute amounts of chemical stimulants could be useful for dose related response studies to test new compounds. It could also bring forward the development of a novel type of neuroprostheses. In an experimental setup microdroplets of an acetylcholine solution were ejected from a fluidic microsystem and applied to the bottom of a nanoporous membrane. The solution traveled through the pores to the top of the membrane on which TE671 cells were cultivated. Calcium imaging was used to visualize cellular response with temporal and spatial resolution. Experimental demonstration of chemical stimulation for both threshold gated stimulation as well as accumulated dose-response was achieved by either employing acetylcholine as chemical stimulant or applying calcein uptake, respectively. Numerical modeling and simulation of transport mechanisms involved were employed to gain a theoretical understanding of the influence of pore size, concentration of stimulant and droplet volume on the spatial-temporal distribution of stimulant and on the cellular response. Diffusion, pressure driven flow and evaporation effects were taken into account. Fast stimulation kinetic is achieved with pores of 0.82 μm diameter, whereas sustained substance delivery is obtained with nanoporous membranes. In all cases threshold concentrations ranging from 0.01 to 0.015 μM acetylcholine independent of pore size were determined. PMID:21151808

  4. Chemical Stimulation of Adherent Cells by Localized Application of Acetylcholine from a Microfluidic System

    Zibek, Susanne; Hagmeyer, Britta; Stett, Alfred; Stelzle, Martin

    2010-01-01

    Chemical stimulation of cells is inherently cell type selective in contrast to electro-stimulation. The availability of a system for localized application of minute amounts of chemical stimulants could be useful for dose related response studies to test new compounds. It could also bring forward the development of a novel type of neuroprostheses. In an experimental setup microdroplets of an acetylcholine solution were ejected from a fluidic microsystem and applied to the bottom of a nanoporous membrane. The solution traveled through the pores to the top of the membrane on which TE671 cells were cultivated. Calcium imaging was used to visualize cellular response with temporal and spatial resolution. Experimental demonstration of chemical stimulation for both threshold gated stimulation as well as accumulated dose–response was achieved by either employing acetylcholine as chemical stimulant or applying calcein uptake, respectively. Numerical modeling and simulation of transport mechanisms involved were employed to gain a theoretical understanding of the influence of pore size, concentration of stimulant and droplet volume on the spatial-temporal distribution of stimulant and on the cellular response. Diffusion, pressure driven flow and evaporation effects were taken into account. Fast stimulation kinetic is achieved with pores of 0.82 μm diameter, whereas sustained substance delivery is obtained with nanoporous membranes. In all cases threshold concentrations ranging from 0.01 to 0.015 μM acetylcholine independent of pore size were determined. PMID:21151808

  5. Structural model of nicotinic acetylcholine receptor isotypes bound to acetylcholine and nicotine

    Abagyan Ruben

    2002-01-01

    Full Text Available Abstract Background Nicotine is a psychoactive drug presenting a diverse array of biological activities, some positive, such as enhancement of cognitive performances, others negative, such as addiction liability. Ligands that discriminate between the different isotypes of nicotinic acetylcholine receptors (nAChRs could present improved pharmacology and toxicity profile. Results Based on the recent crystal structure of a soluble acetylcholine binding protein from snails, we have built atomic models of acetylcholine and nicotine bound to the pocket of four different human nAChR subtypes. The structures of the docked ligands correlate with available biochemical data, and reveal that the determinants for isotype selectivity are relying essentially on four residues, providing diversity of the ligand binding pocket both in terms of Van der Waals boundary, and electrostatic potential. We used our models to screen in silico a large compound database and identify a new ligand candidate that could display subtype selectivity. Conclusion The nAChR-agonist models should be useful for the design of nAChR agonists with diverse specificity profiles.

  6. Spin transport, magnetoresistance, and electrically detected magnetic resonance in amorphous hydrogenated silicon nitride

    Mutch, Michael J.; Lenahan, Patrick M.; King, Sean W.

    2016-08-01

    We report on a study of spin transport via electrically detected magnetic resonance (EDMR) and near-zero field magnetoresistance (MR) in silicon nitride films. Silicon nitrides have long been important materials in solid state electronics. Although electronic transport in these materials is not well understood, electron paramagnetic resonance studies have identified a single dominating paramagnetic defect and have also provided physical and chemical descriptions of the defects, called K centers. Our EDMR and MR measurements clearly link the near-zero field MR response to the K centers and also indicate that K center energy levels are approximately 3.1 eV above the a-SiN:H valence band edge. In addition, our results suggest an approach for the study of defect mediated spin-transport in inorganic amorphous insulators via variable electric field and variable frequency EDMR and MR which may be widely applicable.

  7. Equipment of high sensitivity to detect smuggled radioactive materials transported across the ''east-west'' border

    An equipment specially developed for the customs radiation control is described. Its sensitivity is higher than requirements of western countries. The equipment ensures an alarm when a radioactive source (both shielded or not) is found in the controlled area, localizes and identifies the source detected, and provides the radiation protection of customs personnel. Most of devices have a non-volatile memory where the radiation situation history is stored and then transferred to PC. The equipment may be used by personnel of special services for secret detection of radioactive materials. Some Belarussian and Russian documents specifying measures to prevent an unauthorized transportation of radioactive materials are discussed. (author)

  8. The α7 nicotinic acetylcholine receptor complex

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of diseases ranging from schizophrenia and Alzheimer's disease to chronic pain and inflammatory diseases. Focusing on the central nervous system, we describe how endogenous and experimental compounds and...... compounds in vivo is highly dependent on α7 nAChR-interacting proteins, such as RIC-3 and lynx1, which modulate expression and function of the receptor. These regulatory proteins are often not expressed in in vitro models used to study α7 nAChR function, and it is not known to what extent they are involved...... in diseases such as schizophrenia and Alzheimer's disease. Furthermore, α7 nAChR agonists and allosteric modulators differentially alter expression and functionality of the α7 nAChR with repeated administration, which suggests that there may be fundamentally different outcomes of long...

  9. Inhibition of acetylcholine synthesis in vitro

    In order to better understand diseases that stem from deficiencies in cholinergic activity, reproducible in vitro and in vivo models displaying cholinergic hypofunction are desirable. This necessitates the availability of specific inhibitors. This paper examines the design, synthesis and evaluation of quinuclidinyl compounds with structural features previously reported, but with certain key differences. Structure activity studies with in vitro assay systems are presented. In a few studies, choline was held constant and acetyl-CoA concentration was varied, but with a constant amount of (14C) - acetyl CoA. Acetylcholine synthesis and CO2 production from labelled glucose were measured in cerebral cortex slices from male rats after decapitation. The nanomoles of ACh and CO2 produced from (14C) -glucose were calculated from glucose specific activity. Results are presented

  10. Cocaine Inhibition of Nicotinic Acetylcholine ReceptorsInfluences Dopamine Release

    Alexandra eAcevedo-Rodriguez

    2014-09-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs potently regulate dopamine (DA release in the striatum and alter cocaine’s ability to reinforce behaviors. Since cocaine is a weak nAChR inhibitor, we hypothesized that cocaine may alter DA release by inhibiting the nAChRs in DA terminals in the striatum and thus contribute to cocaine's reinforcing properties primarily associated with the inhibition of DA transporters. We found that biologically relevant concentrations of cocaine can mildly inhibit nAChR-mediated currents in midbrain DA neurons and consequently alter DA release in the dorsal and ventral striatum. At very high concentrations, cocaine also inhibits voltage-gated Na channels in DA neurons. Furthermore, our results show that partial inhibition of nAChRs by cocaine reduces evoked DA release. This diminution of DA release via nAChR inhibition more strongly influences release evoked at low or tonic stimulation frequencies than at higher (phasic stimulation frequencies, particularly in the dorsolateral striatum. This cocaine-induced shift favoring phasic DA release may contribute to the enhanced saliency and motivational value of cocaine-associated memories and behaviors.

  11. Role of acetylcholine on plant root-shoot signal transduction

    2003-01-01

    The role of acetylcholine (ACh) on plant root- shoot communication was investigated using the root-split system of Vicia faba L. In the experiments, slight osmotic stress caused the decrease of ACh content in root tips and the xylem sap transported up per time unit from root tip to the shoot when the water potential of the shoot was kept unchanged. It also caused the decrease of ACh content in the abaxial epidermis. The decrease was highly correlative to the changes of transpiration rate, suggesting that the decrease of ACh content probably functions as a signal to regulate stomatal behavior. The effect of osmotic stress might be mainly through the inhibition of the ACh synthesis in root tip; thus further influences the ACh content in root tip, xylem sap and abaxial epidermis and resulting in the changes of stomatal behavior. These results provide new evidence that plants transduce positive and negative signals among roots and shoots to coordinate stomatal behavior and adapt to variable environments.

  12. Acoustic wave detection of chemical species electrokinetically transported within a capillary tube.

    Li, Paul C H; Prasad, Ronald

    2003-06-01

    For the first time, we report the acoustic wave detection of chemical species being transported in a capillary tube to a region where acoustic coupling occurs. The measured parameter was a change in phase, which was originally only attributed to a change in solution density as the analyte passed by the detection region. Accordingly, we report the detection of change in phase as various chemical species (e.g. Cy5 dye, Cy5-derivatized glycine and underivatized glycine) were introduced into and migrated along a capillary tube through electrokinetic processes. To improve detection sensitivity, we modified various experimental parameters, such as run buffer concentration, capillary wall thickness and transducer frequency. Although acoustic wave detection was feasible, the peak width and detection limit were inadequate as compared to conventional detection methods for HPLC or CE. Nevertheless, the effects of various physical and chemical relaxation processes on acoustic wave absorption were discussed, and this has shed some light on explaining some observations, which cannot be explained by density differences alone. Accordingly, the acoustic wave method is suggested to investigate these processes, as studied in ultrasonic relaxation spectroscopy, in a flow system. PMID:12866892

  13. Detection and characterization of uranium-humic complexes during 1D transport studies

    Lesher, Emily K. [Colorado School of Mines, Golden, CO (United States). Civil and Environmental Engineering; Honeyman, Bruce D. [Colorado School of Mines, Golden, CO (United States). Civil and Environmental Engineering; Ranville, James F. [Colorado School of Mines, Golden, CO (United States). Dept. of Chemistry and Geochemistry

    2013-05-01

    The speciation and transport of uranium (VI) through porous media is highly dependent on solution conditions, the presence of complexing ligands, and the nature of the porous media. The dependency on many variables makes prediction of U transport in bench-scale experiments and in the field difficult. In particular, the identification of colloidal U phases poses a technical challenge. Transport of U in the presence and absence of natural organic matter (Suwannee River humic acid, SRHA) through silica sand and hematite coated silica sand was tested at pH 4 and 5 using static columns, where flow is controlled by gravity and residence time between advective pore volume exchanges can be strictly controlled. The column effluents were characterized by traditional techniques including ICPMS quantification of total [U] and [Fe], TOC analysis of [DOC], and pH analysis, and also by non-traditional techniques: flow field flow fractionation with online ICPMS detection (FlFFF-ICPMS) and specific UV absorbance (SUVA) characterization of effluent fractions. Key results include that the transport of U through the columns was enhanced by pre-equilibration with SRHA, and previously deposited U was remobilized by the addition of SRHA. The advanced techniques yielded important insights on the mechanisms of transport: FlFFF-ICPMS identified a U-SRHA complex as the mobile U species and directly quantified relative amounts of the complex, while specific UV absorbance (SUVA) measurements indicated a composition-based fractionation onto the porous media.

  14. Chemical stimulation of adherent cells by localized application of acetylcholine from a microfluidic system

    Susanne Zibek

    2010-11-01

    Numerical modelling and simulation of transport mechanisms involved were employed to gain a theoretical understanding of the influence of pore size, concentration of stimulant and droplet volume on the spatial-temporal distribution of stimulant and on the cellular response. Diffusion, pressure driven flow and evaporation effects were taken into account. Fast stimulation kinetic is achieved with pores of 0.82 µm diameter, whereas sustained substance delivery is obtained with nanoporous membranes. In all cases threshold concentrations ranging from 0.01 to 0.015 µM acetylcholine independent of pore size were determined.

  15. Hybrid light transport model based bioluminescence tomography reconstruction for early gastric cancer detection

    Chen, Xueli; Liang, Jimin; Hu, Hao; Qu, Xiaochao; Yang, Defu; Chen, Duofang; Zhu, Shouping; Tian, Jie

    2012-03-01

    Gastric cancer is the second cause of cancer-related death in the world, and it remains difficult to cure because it has been in late-stage once that is found. Early gastric cancer detection becomes an effective approach to decrease the gastric cancer mortality. Bioluminescence tomography (BLT) has been applied to detect early liver cancer and prostate cancer metastasis. However, the gastric cancer commonly originates from the gastric mucosa and grows outwards. The bioluminescent light will pass through a non-scattering region constructed by gastric pouch when it transports in tissues. Thus, the current BLT reconstruction algorithms based on the approximation model of radiative transfer equation are not optimal to handle this problem. To address the gastric cancer specific problem, this paper presents a novel reconstruction algorithm that uses a hybrid light transport model to describe the bioluminescent light propagation in tissues. The radiosity theory integrated with the diffusion equation to form the hybrid light transport model is utilized to describe light propagation in the non-scattering region. After the finite element discretization, the hybrid light transport model is converted into a minimization problem which fuses an l1 norm based regularization term to reveal the sparsity of bioluminescent source distribution. The performance of the reconstruction algorithm is first demonstrated with a digital mouse based simulation with the reconstruction error less than 1mm. An in situ gastric cancer-bearing nude mouse based experiment is then conducted. The primary result reveals the ability of the novel BLT reconstruction algorithm in early gastric cancer detection.

  16. Methodologic aspects of acetylcholine-evoked relaxation of rabbit aorta.

    Hansen, K; Nedergaard, O A

    1999-08-01

    The acetylcholine-evoked relaxation of rabbit isolated thoracic aorta precontracted by phenylephrine was studied. Phenylephrine caused a steady contraction that was maintained for 6 h. In the presence of calcium disodium ethylenediaminetetraacetate (EDTA) and ascorbic acid the contraction decreased with time. N(G)-Nitro-L-arginine abolished the inhibitory effect of EDTA and ascorbic acid. Acetylcholine evoked a rapid concentration-dependent relaxation that recovered spontaneously and slowly, but fully, with time. Relaxation evoked by equieffective concentrations of carbachol and acetylcholine had the same time course. Cumulative addition of acetylcholine (10(-7)-3 x 10(-5) M) caused a marked relaxation that was reverted slightly at high concentrations. The relaxation was the same with rings derived from the upper, middle, and lower part of the thoracic aorta. Two consecutive concentration-response curves for acetylcholine obtained at a 2-h interval demonstrated a slight development of tachyphylaxis. The relaxation was inversely related to precontractile tension evoked by phenylephrine when expressed as a percentage, but independent when expressed as g tension. Storage of aorta in cold salt solution for 24 h did not alter the relaxation. EDTA and ascorbic acid did not alter the relaxation. It is concluded that (1) EDTA and ascorbic acid can not be used with impunity to stabilize catecholamines used as preconstriction agents; (2) the reversal of the acetylcholine-evoked relaxation is not due to hydrolysis of acetylcholine; (3) the relaxation is uniform in all segments of thoracic aorta; (4) cold storage of aorta does not alter the relaxation; and (5) acetylcholine releases the same amount of relaxing factor, irrespective of the precontractile tension. PMID:10691020

  17. Passive mass transport for direct and quantitative SERS detection using purified silica encapsulated metal nanoparticles

    Shrestha, Binaya Kumar

    This thesis focuses on understanding implications of nanomaterial quality control and mass transport through internally etched silica coated nanoparticles for direct and quantitative molecular detection using surface enhanced Raman scattering (SERS). Prior to use, bare nanoparticles (partially or uncoated with silica) are removal using column chromatography to improve the quality of these nanomaterials and their SERS reproducibility. Separation of silica coated nanoparticles with two different diameters is achieved using Surfactant-free size exclusion chromatography with modest fractionation. Next, selective molecular transport is modeled and monitored using SERS and evaluated as a function of solution ionic strength, pH, and polarity. Molecular detection is achieved when the analytes first partition through the silica membrane then interact with the metal surface at short distances (i.e., less than 2 nm). The SERS intensities of unique molecular vibrational modes for a given molecule increases as the number of molecules that bind to the metal surface increases and are enhanced via both chemical and electromagnetic enhancement mechanisms as long as the vibrational mode has a component of polarizability tensor along the surface normal. SERS signals increase linearly with molecular concentration until the three-dimensional SERS-active volume is saturated with molecules. Implications of molecular orientation as well as surface selection rules on SERS intensities of molecular vibrational modes are studied to improve quantitative and reproducible SERS detection using internally etched Ag Au SiO2 nanoparticles. Using the unique vibrational modes, SERS intensities for p-aminothiophenol as a function of metal core compositions and plasmonics are studied. By understanding molecular transport mechanisms through internally etched silica matrices coated on metal nanoparticles, important experimental and materials design parameters are learned, which can be subsequently applied

  18. Crucial technologies of oil-transporting pipe leak detection and location based on wavelet and chaos

    Zhang, S. Q.; Jin, S. J.; Yang, F. L.; Wang, X. Q.; Bai, Q. Y.

    2006-03-01

    By detecting pressure and flux simultaneously, the leak of an oil-transporting pipe can be found and diagnosed synthetically. The flux of pipes is measured by an ultrasonic flow meter; considering the precision of the flux measured, a method based on the character of intermittent chaos of the Duffing system to detect weak signals under strong noise is introduced. The ultrasonic signals with a certain frequency could be extracted accurately from the complicated strong noise. So the flux of pipes can be computed precisely by an accurate ultrasonic signal. The location of a leak position is mainly determined by the time difference between the negative pressure waves measured by the pressure sensors located at both ends of the oil-transporting pipe. A singular point of a negative pressure wave can be judged accurately by a coefficient feature of the local extreme values of the wavelet transform. So, the precise location of the leak position of the oil-transporting pipe can be found. It can be shown by results of experiments that the precision of the leak location has been improved effectively, which can be about 1%.

  19. Identification of petrogenic produced water components as acetylcholine esterase inhibitors.

    Froment, Jean; Langford, Katherine; Tollefsen, Knut Erik; Bråte, Inger Lise N; Brooks, Steven J; Thomas, Kevin V

    2016-08-01

    Effect-directed analysis (EDA) was applied to identify acetylcholine esterase (AChE) inhibitors in produced water. Common produced water components from oil production activities, such as polycyclic aromatic hydrocarbons (PAHs), alkylphenols, and naphthenic acids were tested for AChE inhibition using a simple mixture of PAHs and naphthenic acids. Produced water samples collected from two offshore platforms in the Norwegian sector of the North Sea were extracted by solid phase extraction and fractionated by open-column liquid solid chromatography and high-performance liquid chromatography (HPLC) before being tested using a high-throughput and automated AChE assay. The HPLC fractions causing the strongest AChE inhibition were analysed by gas chromatography coupled to a high-resolution time-of-flight mass spectrometry (GC-HR-ToF-MS). Butylated hydroxytoluene and 4-phenyl-1,2-dihydronaphthalene were identified as two produced water components capable of inhibiting AChE at low concentrations. In order to assess the potential presence of such compounds discharged into aquatic ecosystems, AChE activity in fish tissues was measured. Saithe (Pollachius virens) caught near two offshore platforms showed lower enzymatic activity than those collected from a reference location. Target analysis of saithe did not detected the presence of these two putative AChE inhibitors and suggest that additional compounds such as PAHs, naphthenic acids and yet un-identified compounds may also contribute to the purported AChE inhibition observed in saithe. PMID:27176761

  20. Influence of atmospheric transport patterns on xenon detections at the CTBTO radionuclide network

    Krysta, Monika; Kusmierczyk-Michulec, Jolanta

    2016-04-01

    In order to fulfil its task of monitoring for signals emanating from nuclear explosions, Comprehensive Nuclear-Test-Ban Treaty Organization (CTBTO) operates global International Monitoring System (IMS) comprising seismic, infrasound, hydroacoustic and radionuclide measurement networks. At present, 24 among 80 radionuclide stations foreseen by the Comprehensive Nuclear-Test-Ban Treaty (CTBT) are equipped with certified noble gas measurement systems. Over a past couple of years these systems collected a rich set of measurements of radioactive isotopes of xenon. Atmospheric transport modelling simulations are crucial to an assessment of the origin of xenon detected at the IMS stations. Numerous studies undertaken in the past enabled linking these detections to non Treaty-relevant activities and identifying main contributors. Presence and quantity of xenon isotopes at the stations is hence a result of an interplay of emission patterns and atmospheric circulation. In this presentation we analyse the presence or absence of radioactive xenon at selected stations from an angle of such an interplay. We attempt to classify the stations according to similarity of detection patterns, examine seasonality in those patterns and link them to large scale or local meteorological phenomena. The studies are undertaken using crude hypotheses on emission patterns from known sources and atmospheric transport modelling simulations prepared with the FLEXPART model.

  1. A transportable fast neutron and dual gamma-ray system for the detection of illicit materials

    A transportable FNGR radiography system has been simulated using the MCNPX Monte Carlo code. The system is envisaged to be applied to the material characterisation of a suspicious bulky object, in view of identifying illegal materials. The system combines a neutron and two gamma-ray sources achieving characterisation of the material of the object through two ratios, namely 137Cs/DD and 60Co/DD. Hence, the system discriminates materials of similar or even the same of either of the two ratios. The proposed unit complies with radiation protection requirements achieving a safe occupational environment. - Highlights: → Transportable radiography system. → Neutron- and dual energy photon-beams available. → Discrimination of materials. → Detection of illicit materials.

  2. A transportable fast neutron and dual gamma-ray system for the detection of illicit materials

    Fantidis, J.G. [Laboratory of Nuclear Technology, School of Engineering, ' Democritus' University of Thrace, Vas. Sofias 12, Xanthi 67100 (Greece); Nicolaou, G.E., E-mail: nicolaou@ee.duth.gr [Laboratory of Nuclear Technology, School of Engineering, ' Democritus' University of Thrace, Vas. Sofias 12, Xanthi 67100 (Greece)

    2011-08-21

    A transportable FNGR radiography system has been simulated using the MCNPX Monte Carlo code. The system is envisaged to be applied to the material characterisation of a suspicious bulky object, in view of identifying illegal materials. The system combines a neutron and two gamma-ray sources achieving characterisation of the material of the object through two ratios, namely {sup 137}Cs/DD and {sup 60}Co/DD. Hence, the system discriminates materials of similar or even the same of either of the two ratios. The proposed unit complies with radiation protection requirements achieving a safe occupational environment. - Highlights: > Transportable radiography system. > Neutron- and dual energy photon-beams available. > Discrimination of materials. > Detection of illicit materials.

  3. The ATR noninvasive detection of transported medicinal ions and the performance of newly designed iontophoresis instruments

    Ueda, Toyotoshi; Watanabe, Yukio; Suzuki, Harue

    2005-02-01

    The attenuated total reflection and near-infrared diffusive-reflection methods are proposed as safe and powerful ways to detect and measure the quantity of medication transported by iontophoresis. Especially, the former method can evaluate the quantity of such negative ions as L-ascorbyl-2-phosphate in the top (horny) layer of epidermis (about 1 μm under the skin surface) using, respectively, characteristic ion's bands. Factors making iontophoresis more effective are discussed from the points of electric currents, duty ratio, frequency of superposing intermittent current, simultaneous supersonic perforation, etc. The use of intermittent direct current superposed by 40 kHz pulsed current and pulse irradiation of supersonic waves accelerated drastically the disappearing rate of transported ions from the horny layer with a life of 10 h to 1 min. This technique may be applied to a new and powerful drug delivery system into topical deep tissues.

  4. Supporting data for 'Vortex detection and quantum transport in mesoscopic graphene Josephson-junction arrays'

    Richardson, C. L.; Edkins, S. D.; Berdiyorov, G. R.; Chua, C. J.; Griffiths, J. P.; Jones, G. A. C.; Buitelaar, M.R.; Narayan, V; Sfigakis, F.; Smith, C. G.; Covaci, L.; Connolly, M. R.

    2015-01-01

    The files listed below are raw experimental data from 'Vortex detection and quantum transport in mesoscopic graphene Josephson-junction arrays' by C. L. Richardson et al., to appear in Phys. Rev. B. The three-part files (filename, filenameHdr and filenameSD) are in tab-separated .txt format, produced by CryoMeas (written by C. J. B. Ford). Only a subset of the Fig. 6 dataset is included. 20130514-040 (device A, Fig. 2(a) and 3) 20130515-041 (device A, Fig. 2(a) and 3) 20130809-066 (...

  5. New Sensor Cable for the Detection and Location of Leaks in Pipelines for Transportation of Hydrocarbons

    E.Orduña-Reyes

    2012-08-01

    Full Text Available At present, hydrocarbon leaks, generated mainly by corrosion of pipelines, cause large economic losses for Mexico.These leaks constitute a problem of serious consequences in Mexico and in other countries in the world. This workdescribes the results of the tests conducted on a new sensor cable for the detection and location of leaks in pipelinesfor transportation of hydrocarbons. When a liquid or gas enters in contact with the wall of the sensor cable, it causes ashort circuit in the wires; changing the measurement of the resistance may detect and locate the leak. The new sensorcable that is presented in this article has advantages over cables with similar characteristic made in other countries.The use of this sensor cable in pipelines of PEMEX will avoid economic losses, environmental damage and risks ofpossible explosions to the population. The experimental results demonstrate these advantages.

  6. Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine-Binding Protein as a Structural Surrogate.

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette S; Balle, Thomas

    2016-06-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in the control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for the development of drugs against a number of mental health disorders and for marketed smoking cessation aids. Unfortunately, drug discovery has been hampered by difficulties in obtaining sufficiently selective compounds. Together with functional complexity of the receptors, this has made it difficult to obtain drugs with sufficiently high-target to off-target affinity ratios. The recent and ongoing progress in structural studies holds promise to help understand structure-function relationships of nAChR drugs at the atomic level. This will undoubtedly lead to the design of more efficient drugs with fewer side effects. As a high-resolution structure of a nAChR is yet to be determined, structural studies are to a large extent based on acetylcholine-binding proteins (AChBPs) that despite low overall sequence identity display a high degree of conservation of overall structure and amino acids at the ligand-binding site. Further, AChBPs reproduce relative binding affinities of ligands at nAChRs. Over the past decade, AChBPs have been used extensively as models for nAChRs and have aided the understanding of drug receptor interactions at nAChRs significantly. PMID:26572235

  7. m1 Acetylcholine Receptor Expression is Decreased in Hippocampal CA1 region of Aged Epileptic Animals

    Cavarsan, Clarissa Fantin; Avanzi, Renata Della Torre; Queiroz, Claudio Marcos; Xavier, Gilberto Fernando; Mello, Luiz Eugênio; Covolan, Luciene

    2011-01-01

    In the present study, we investigated the possible additive effects of epilepsy and aging on the expression of m1 muscarinic acetylcholine receptors (AChR) in the rat hippocampus. Young (3 months) and Aged (20 months) male, Wistar rats were treated with pilocarpine to induce status epilepticus (SE). Immunohistochemical procedure for m1 AChR detection was performed 2 months after pilocarpine-induced SE. In the CA1 pyramidal region m1 AChR staining was significantly decreased in aged epileptic ...

  8. Interaction of ibogaine with human α3β4-nicotinic acetylcholine receptors in different conformational states

    Arias, Hugo R.; Rosenberg, Avraham; Targowska-Duda, Katarzyna M.; Feuerbach, Dominik; Yuan, Xiao Juan; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W.

    2010-01-01

    The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca2+ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (±)-epibatidine-induced Ca2+ influx in hα3β4 AChRs with ~9-fold higher potency than that for PCP, (b) [3H]ibogaine binds to a single s...

  9. Simulation of atmospheric krypton-85 transport to assess the detectability of clandestine nuclear reprocessing

    Ross, Jens Ole

    2010-02-02

    The radioactive noble gas krypton-85 is released into the atmosphere during reprocessing of spent nuclear fuel or irradiated breeding targets. This is a necessary step for plutonium separation. Therefore the {sup 85}Kr signature of reprocessing could possibly be used for the detection of undeclared nuclear facilities producing nuclear weaponusable material. The {sup 85}Kr content of the atmosphere has grown over the last decades as the emissions from military and civilian nuclear industry could not be compensated by the decay with a half-life of 10.76 years. In this study, the global {sup 85}Kr background distribution due to emissions of known reprocessing facilities for the period from 1971 until 2006 was simulated using the atmospheric general circulation model ECHAM5 applying the newest available annual emission data. The convective tracer transport scheme and the operator splitting for the physical calculations in the model were modified in order to guarantee physically correct results for tracer point sources, in particular non negative concentrations. An on-line routine controlling the {sup 85}Kr -budget in the model enforced exact mass conservation. The results of the simulation were evaluated by extensive comparison with measurements performed by the German Federal Office for Radiation Protection with very good agreement at most observation sites except those in the direct vicinity of {sup 85}Kr sources. Of particular interest for the {sup 85}Kr detection potential was the variability of {sup 85}Kr background concentrations which was evaluated for the first time in a global model. In addition, the interhemispheric transport as simulated by ECHAM5 was analyzed using a two-box model providing a mean exchange time of τ {sub ex} = 10.5 months. The analysis of τ{sub ex} over simulated 35 years indicates that in years with strong South Asian or African Monsoon the interhemispheric transport is faster during the monsoon season. A correlation analysis of

  10. Defect chemistry and electronic transport in low-κ dielectrics studied with electrically detected magnetic resonance

    Mutch, Michael J.; Lenahan, Patrick M.; King, Sean W.

    2016-03-01

    Defect mediated electronic transport phenomena in low-κ dielectric films are of great technological interest for state-of-the-art and next generation microprocessors. At the present time, the leading low-κ interlayer dielectrics and etch-stop layers are based upon a-SiOC:H and a-SiCN:H, respectively. In this study, we utilize electrically detected magnetic resonance (EDMR), a derivative of electron paramagnetic resonance, to provide physical insight into electronic transport, as well as the nature and origin of defects in dense and porous a-SiOC:H and dense a-SiCN:H films. Resonance measurements are performed before and after the removal of sacrificial porogens via UV treatments to understand the role of specific defect centers in electronic transport in a-SiOC:H systems, and the nature of defects created by UV treatments. Unfortunately, a-SiOC:H and a-SiCN:H EDMR spectra are relatively broad and featureless. These featureless spectra are consistent with fairly complex a-SiOC:H and a-SiCN:H systems. We argue that physical insight may be gleaned from featureless spectra via multiple frequency EDMR. Baseline multiple frequency EDMR measurements are performed in a-Si:H and a-C:H to illustrate the nature of line broadening mechanisms of silicon and carbon related defects.

  11. α4 nicotinic acetylcholine receptor modulated by galantamine on nigrostriatal terminals regulates dopamine receptor-mediated rotational behavior.

    Inden, Masatoshi; Takata, Kazuyuki; Yanagisawa, Daijiro; Ashihara, Eishi; Tooyama, Ikuo; Shimohama, Shun; Kitamura, Yoshihisa

    2016-03-01

    Galantamine, an acetylcholine esterase (AChE) inhibitor used to treat dementia symptoms, also acts as an allosteric potentiating ligand (APL) at nicotinic acetylcholine receptors (nAChRs). This study was designed to evaluate the allosteric effect of galantamine on nAChR regulation of nigrostrial dopaminergic neuronal function in the hemiparkinsonian rat model established by unilateral nigral 6-hydroxydopamine (6-OHDA) injection. Methamphetamine, a dopamine releaser, induced ipsilateral rotation, whereas dopamine agonists apomorphine (a non-selective dopamine receptor agonist), SKF38393 (a selective dopamine D1 receptor agonist), and quinpirole (a selective dopamine D2 receptor agonist) induced contralateral rotation. When 6-OHDA-injected rats were co-treated with nomifensine, a dopamine transporter inhibitor, a more pronounced and a remarkable effect of nicotine and galantamine was observed. Under these conditions, the combination of nomifensine with nicotine or galantamine induced the ipsilateral rotation similar to the methamphetamine-induced rotational behavior, indicating that nicotine and galantamine also induce dopamine release from striatal terminals. Both nicotine- and galantamine-induced rotations were significantly blocked by flupenthixol (an antagonist of both D1 and D2 dopamine receptors) and mecamylamine (an antagonist of nAChRs), suggesting that galantamine modulation of nAChRs on striatal dopaminergic terminals regulates dopamine receptor-mediated movement. Immunohistochemical staining showed that α4 nAChRs were highly expressed on striatal dopaminergic terminals, while no α7 nAChRs were detected. Pretreatment with the α4 nAChR antagonist dihydroxy-β-erythroidine significantly inhibited nicotine- and galantamine-induced rotational behaviors, whereas pretreatment with the α7 nAChR antagonist methyllycaconitine was ineffective. Moreover, the α4 nAChR agonist ABT-418 induced ipsilateral rotation, while the α7 nAChR agonist PNU282987 had no

  12. Electrical detection of spin transport in Si two-dimensional electron gas systems

    Chang, Li-Te; Fischer, Inga Anita; Tang, Jianshi; Wang, Chiu-Yen; Yu, Guoqiang; Fan, Yabin; Murata, Koichi; Nie, Tianxiao; Oehme, Michael; Schulze, Jörg; Wang, Kang L.

    2016-09-01

    Spin transport in a semiconductor-based two-dimensional electron gas (2DEG) system has been attractive in spintronics for more than ten years. The inherent advantages of high-mobility channel and enhanced spin–orbital interaction promise a long spin diffusion length and efficient spin manipulation, which are essential for the application of spintronics devices. However, the difficulty of making high-quality ferromagnetic (FM) contacts to the buried 2DEG channel in the heterostructure systems limits the potential developments in functional devices. In this paper, we experimentally demonstrate electrical detection of spin transport in a high-mobility 2DEG system using FM Mn-germanosilicide (Mn(Si0.7Ge0.3)x) end contacts, which is the first report of spin injection and detection in a 2DEG confined in a Si/SiGe modulation doped quantum well structure (MODQW). The extracted spin diffusion length and lifetime are l sf = 4.5 μm and {τ }{{s}}=16 {{ns}} at 1.9 K respectively. Our results provide a promising approach for spin injection into 2DEG system in the Si-based MODQW, which may lead to innovative spintronic applications such as spin-based transistor, logic, and memory devices.

  13. Transport

    Transport is one of the major causes of environmental damage in Austria. Energy consumption, pollutants emissions, noise emissions, use of surfaces, sealing of surfaces, dissection of ecosystems and impact on landscape are the most significant environmental impacts caused by it. An overview of the transport development of passengers and freight in Austria is presented. Especially the energy consumption growth, carbon dioxide and nitrogen oxide emissions by type of transport, and the emissions development (HC, particle and carbon monoxide) of goods and passengers transport are analyzed covering the years 1980 - 1999. The health cost resulting from transport-related air pollution in Austria is given and measures to be taken for an effective control of the transport sector are mentioned. Figs. 8, Table 1. (nevyjel)

  14. Transport simulation and image reconstruction for fast-neutron detection of explosives and narcotics

    Micklich, B.J.; Fink, C.L.; Sagalovsky, L.

    1995-07-01

    Fast-neutron inspection techniques show considerable promise for explosive and narcotics detection. A key advantage of using fast neutrons is their sensitivity to low-Z elements (carbon, nitrogen, and oxygen), which are the primary constituents of these materials. We are currently investigating two interrogation methods in detail: Fast-Neutron Transmission Spectroscopy (FNTS) and Pulsed Fast-Neutron Analysis (PFNA). FNTS is being studied for explosives and narcotics detection in luggage and small containers for which the transmission ratio is greater than about 0.01. The Monte-Carlo radiation transport code MCNP is being used to simulate neutron transmission through a series of phantoms for a few (3-5) projection angles and modest (2 cm) resolution. Areal densities along projection rays are unfolded from the transmission data. Elemental abundances are obtained for individual voxels by tomographic reconstruction, and these reconstructed elemental images are combined to provide indications of the presence or absence of explosives or narcotics. PFNA techniques are being investigated for detection of narcotics in cargo containers because of the good penetration of the fast neutrons and the low attenuation of the resulting high-energy gamma-ray signatures. Analytic models and Monte-Carlo simulations are being used to explore the range of capabilities of PFNA techniques and to provide insight into systems engineering issues. Results of studies from both FNTS and PFNA techniques are presented.

  15. Organophosphate acetylcholine esterase inhibitor poisoning from a home-made shampoo

    Sadaka, Yair; Broides, Arnon; Tzion, Raffi Lev; Lifshitz, Matitiahu

    2011-01-01

    Organophosphate acetylcholine esterase inhibitor poisoning is a major health problem in children. We report an unusual cause of organophosphate acetylcholine esterase inhibitor poisoning. Two children were admitted to the pediatric intensive care unit due to organophosphate acetylcholine esterase inhibitor poisoning after exposure from a home-made shampoo that was used for the treatment of head lice. Owing to no obvious source of poisoning, the diagnosis of organophosphate acetylcholine ester...

  16. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Meyyammai Swaminathan

    2014-06-01

    Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

  17. Expression of the α7 nicotinic acetylcholine receptor in human lung cells

    Schuller Hildegard M

    2005-04-01

    Full Text Available Abstract Background We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the α7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK, is a high affinity agonist for the α7 nicotinic acetylcholine receptor. In the present study, our goal was to determine the extent of α7 mRNA and protein expression in the human lung. Methods Experiments were done using reverse transcription polymerase chain reaction (RT-PCR, a nuclease protection assay and western blotting using membrane proteins. Results We detected mRNA for the neuronal nicotinic acetylcholine receptor α7 receptor in seven small cell lung cancer (SCLC cell lines, in two pulmonary adenocarcinoma cell lines, in cultured normal human small airway epithelial cells (SAEC, one carcinoid cell line, three squamous cell lines and tissue samples from nine patients with various types of lung cancer. A nuclease protection assay showed prominent levels of α7 in the NCI-H82 SCLC cell line while α7 was not detected in SAEC, suggesting that α7 mRNA levels may be higher in SCLC compared to normal cells. Using a specific antibody to the α7 nicotinic receptor, protein expression of α7 was determined. All SCLC cell lines except NCI-H187 expressed protein for the α7 receptor. In the non-SCLC cells and normal cells that express the α7 nAChR mRNA, only in SAEC, A549 and NCI-H226 was expression of the α7 nicotinic receptor protein shown. When NCI-H69 SCLC cell line was exposed to 100 pm NNK, protein expression of the α7 receptor was increased at 60 and 150 min. Conclusion Expression of mRNA for the neuronal nicotinic acetylcholine receptor α7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441 as well as normal

  18. Sensitive and transportable gadolinium-core plastic scintillator sphere for neutron detection and counting

    Dumazert, Jonathan; Coulon, Romain; Carrel, Frédérick; Corre, Gwenolé; Normand, Stéphane; Méchin, Laurence; Hamel, Matthieu

    2016-08-01

    Neutron detection forms a critical branch of nuclear-related issues, currently driven by the search for competitive alternative technologies to neutron counters based on the helium-3 isotope. The deployment of plastic scintillators shows a high potential for efficient detectors, safer and more reliable than liquids, more easily scalable and cost-effective than inorganic. In the meantime, natural gadolinium, through its 155 and mostly 157 isotopes, presents an exceptionally high interaction probability with thermal neutrons. This paper introduces a dual system including a metal gadolinium core inserted at the center of a high-scale plastic scintillator sphere. Incident fast neutrons are thermalized by the scintillator shell and then may be captured with a significant probability by gadolinium 155 and 157 nuclei in the core. The deposition of a sufficient fraction of the capture high-energy prompt gamma signature inside the scintillator shell will then allow discrimination from background radiations by energy threshold, and therefore neutron detection. The scaling of the system with the Monte Carlo MCNPX2.7 code was carried out according to a tradeoff between the moderation of incident fast neutrons and the probability of slow neutron capture by a moderate-cost metal gadolinium core. Based on the parameters extracted from simulation, a first laboratory prototype for the assessment of the detection method principle has been synthetized. The robustness and sensitivity of the neutron detection principle are then assessed by counting measurement experiments. Experimental results confirm the potential for a stable, highly sensitive, transportable and cost-efficient neutron detector and orientate future investigation toward promising axes.

  19. Piezo-microfluidic transport system for multi-targets biochip detections

    Li, Chia-Chin; Wang, Pei-Wen; Lee, Chih-Kung

    2016-03-01

    Detecting minute trace of interferon-gamma and various bio-markers by using a single biochip was adopted as a platform to examine the technology advancements presented. As bio-detection faces the restriction that only very small quantity of specimen is available, ways to make the best use of the sample available are a must. Since samples concentration will affect the binding rate of an immunoassay, the testing order will become an influencing factor if multiple biomarkers testing situation are needed by using only a single trace of sample. More specifically, if we test disease A first and then detect disease B using the sample just been measured by testing disease A, we most likely will get different results if we reverse the testing order. With an attempt to examine and maybe resolve the issues mentioned above, a micro-fluid control system was developed. The design requirements not only ask for microfluidic control but also demand the system developed has the potential to be integrated within the biochip once its performance is verified. A piezo-vibrating system that can generate traveling waves for microfluidic control was chosen due to its versatility and large force to volume ratio. A simulation software COMSOL was adopted first to predict the microfluidic behavior of the two-mode excited piezo-microfluidic transport system. Secondly, fluorescent particles was used to analyze the microfluidic behavior of system fabricated based on the simulation. Finally, Electrochemistry Impedance Spectroscopy (EIS) was implemented to verify the performance and extendibility of this newly developed system for multi-target detections.

  20. Generation, transport and detection of valley-polarized electrons in diamond.

    Isberg, Jan; Gabrysch, Markus; Hammersberg, Johan; Majdi, Saman; Kovi, Kiran Kumar; Twitchen, Daniel J

    2013-08-01

    Standard electronic devices encode bits of information by controlling the amount of electric charge in the circuits. Alternatively, it is possible to make devices that rely on other properties of electrons than their charge. For example, spintronic devices make use of the electron spin angular momentum as a carrier of information. A new concept is valleytronics in which information is encoded by the valley quantum number of the electron. The analogy between the valley and spin degrees of freedom also implies the possibility of valley-based quantum computing. In this Article, we demonstrate for the first time generation, transport (across macroscopic distances) and detection of valley-polarized electrons in bulk diamond with a relaxation time of 300 ns at 77 K. We anticipate that these results will form the basis for the development of integrated valleytronic devices. PMID:23852401

  1. Functional partial agonism at cloned human muscarinic acetylcholine receptors

    Bräuner-Osborne, Hans; Ebert, B; Brann, M R;

    1996-01-01

    of maximal response, depending on the molar ratio of agonist and antagonist used. Using recombinant human muscarinic acetylcholine receptors (m1 and m5) and the functional assay, receptor selection and amplification technology (R-SAT), we have now shown that co-administration of the full agonist...

  2. Tissue-specific effects of acetylcholine in the canine heart

    Callø, Kirstine; Goodrow, Robert; Olesen, Søren-Peter;

    2013-01-01

    INTRODUCTION: Acetylcholine (ACh) release from the vagus nerve slows heart rate and atrioventricular conduction. ACh stimulates a variety of receptors and channels, including an inward rectifying current (IK,ACh). The effect of ACh in ventricle is still debated. We compare the effect of ACh on...

  3. Detecting the local transport properties and the dimensionality of transport of epitaxial graphene by a multi-point probe approach

    Barreto, Lucas; Perkins, Edward; Johannsen, Jens;

    2013-01-01

    The electronic transport properties of epitaxial monolayer graphene (MLG) and hydrogen-intercalated quasi free-standing bilayer graphene (QFBLG) on SiC(0001) are investigated by micro multi-point probes. Using a probe with 12 contacts, we perform four-point probe measurements with the possibility...... to effectively vary the contact spacing over more than one order of magnitude, allowing us to establish that the transport is purely two-dimensional. Combined with the carrier density obtained by angle-resolved photoemission spectroscopy, we find the room temperature mobility of MLG to be (870...

  4. Detection of smuggling of nuclear material covered by a legal transport of radioactive material

    Full text: One of the worst scenarios for detection of illicit trafficking of nuclear material is when a legal transport of radioactive material is used to cover the radiation of the smuggled uranium. Feasibility study was performed in the Institute of Isotopes and Surface Chemistry of the Chemical Research Centre of the Hungarian Academy of Sciences (hereinafter: Institute) in order to study the possible on site measurement techniques and approaches applicable in such cases. As the type A and type B packages always incorporate a feature such as a seal, in a realistic scenario the confiscated nuclear material is expected to be placed outside the package. The passive neutron emission of the uranium is negligible for a reasonable isotopic abundance therefore the feasibility study was concentrating on non-destructive, passive gamma- spectrometric methods. Possible application of Nal (diameter 40x40 mm3, large planar (15x15x3 mm3) and a hemispheric CdZnTe (500 mm3, and high purity Germanium detectors was investigated. During the on site measurements portable electronics, mini multichannel analyzer, palmtop and/or notebook computer were used. The shielding material of the packages was lead or depleted uranium. The smuggled material was simulated by a package of reactor fuel pellets containing low enriched or natural uranium (materials confiscated in earlier cases) and standards containing low enriched uranium. During the supposed scenario the portal monitor provides an indication of an elevated level of the environmental radioactivity. Then the responsible (e.g. customs) officer investigate the vehicle by a hand-held survey meter in order to search for peaks in dose rates. If a peak was localized, which is different from the position of the legally transported package(s) the officer requests for the expertise of the designated institutes. The following model cases provided the basic conclusion: 1. The legal transport of the radioactive material was simulated by a 4

  5. Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

    Hoffmann, Carsten; Nuber, Susanne; Zabel, Ulrike; Ziegler, Nicole; Winkler, Christiane; Hein, Peter; Berlot, Catherine H; Bünemann, Moritz; Lohse, Martin J

    2012-08-01

    Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3

  6. Polyester with Pendent Acetylcholine-Mimicking Functionalities Promotes Neurite Growth.

    Wang, Shaofei; Jeffries, Eric; Gao, Jin; Sun, Lijie; You, Zhengwei; Wang, Yadong

    2016-04-20

    Successful regeneration of nerves can benefit from biomaterials that provide a supportive biochemical and mechanical environment while also degrading with controlled inflammation and minimal scar formation. Herein, we report a neuroactive polymer functionalized by covalent attachment of the neurotransmitter acetylcholine (Ach). The polymer was readily synthesized in two steps from poly(sebacoyl diglyceride) (PSeD), which previously demonstrated biocompatibility and biodegradation in vivo. Distinct from prior acetylcholine-biomimetic polymers, PSeD-Ach contains both quaternary ammonium and free acetyl moieties, closely resembling native acetylcholine structure. The polymer structure was confirmed via (1)H nuclear magnetic resonance and Fourier-transform infrared spectroscopy. Hydrophilicity, charge, and thermal properties of PSeD-Ach were determined by tensiometer, zetasizer, differential scanning calorimetry, and thermal gravimetric analysis, respectively. PC12 cells exhibited the greatest proliferation and neurite outgrowth on PSeD-Ach and laminin substrates, with no significant difference between these groups. PSeD-Ach yielded much longer neurite outgrowth than the control polymer containing ammonium but no the acetyl group, confirming the importance of the entire acetylcholine-like moiety. Furthermore, PSeD-Ach supports adhesion of primary rat dorsal root ganglions and subsequent neurite sprouting and extension. The sprouting rate is comparable to the best conditions from previous report. Our findings are significant in that they were obtained with acetylcholine-like functionalities in 100% repeating units, a condition shown to yield significant toxicity in prior publications. Moreover, PSeD-Ach exhibited favorable mechanical and degradation properties for nerve tissue engineering application. Humidified PSeD-Ach had an elastic modulus of 76.9 kPa, close to native neural tissue, and could well recover from cyclic dynamic compression. PSeD-Ach showed a gradual in

  7. Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors.

    Jakubík, J; Bacáková, L; El-Fakahany, E E; Tucek, S

    1997-07-01

    It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in

  8. Micro and nanoscale electrochemical systems for reagent generation, coupled electrokinetic transport and enhanced detection

    Contento, Nicholas M.

    Chemical analysis is being performed in devices operated at ever decreasing length scales in order to harness the fundamental benefits of micro and nanoscale phenomena while minimizing operating footprint and sample size. The advantages of moving traditional sample or chemical processing steps (e.g. separation, detection, and reaction) into micro- and nanofluidic devices have been demonstrated, and they arise from the relatively rapid rates of heat and mass transport at small length scales. The use of electrochemical methods in micro/nanoscale systems to control and improve these processes holds great promise. Unfortunately, much is still not understood about the coupling of multiple electrode driven processes in a confined environment nor about the fundamental changes in device performance that occur as geometries approach the nanoscale regime. At the nanoscale a significant fraction of the sample volume is in close contact with the device surface, i.e. most of the sample is contained within electronic or diffusion layers associated with surface charge or surface reactions, respectively. The work presented in this thesis aims to understand some fundamental different behaviors observed in micro/nanofluidic structures, particularly those containing one or more embedded, metallic electrode structures. First, a quantitative method is devised to describe the impact of electric fields on electrochemistry in multi-electrode micro/nanofluidic systems. Next the chemical manipulation of small volumes (≤ 10-13 L) in micro/nanofluidic structures is explored by creating regions of high pH and high dissolved gas (H 2) concentration through the electrolysis of H2O. Massively parallel arrays of nanochannel electrodes, or embedded annular nanoband electrodes (EANEs), are then studied with a focus on achieving enhanced signals due to coupled electrokinetic and electrochemical effects. In EANE devices, electroosmotic flow results from the electric field generated between the

  9. Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

    Rajendran, R.; Borghi, E.; M. Falleni; F Perdoni; Tosi, D.; D.F. Lappin; L. O'Donnell; Greetham, D.; G. Ramage; C. Nile

    2015-01-01

    Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathoge...

  10. Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

    Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M.; Zakarian, Armen; Molgó, Jordi

    2012-01-01

    Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine rec...

  11. Detection of follicular transport of lidocaine and metabolism in adipose tissue in pig ear skin by DESI mass spectrometry imaging

    D'Alvise, Janina; Mortensen, Rasmus; Hansen, Steen H;

    2014-01-01

    Desorption electrospray ionization (DESI) mass spectrometry imaging is demonstrated as a detection technique for penetration experiments of drugs in skin. Lidocaine ointment was used as the model compound in ex vivo experiments with whole pig ears as the skin model. Follicular transport of...

  12. The use of neutron generators for the detection of illicit materials in the sea transportation system

    In today's society acts of terrorism must involve in some stages the illicit trafficking either of explosives, chemical agents and/or nuclear materials. Therefore society must rely on an anti-trafficking infrastructure which encompasses responsible authorities, field personnel and adequate instrumental networks. Modern inspection systems for personnel, parcel, vehicle and cargo, as noninvasive imaging techniques, are based on the use of nuclear analytical methods. The inspection systems make use of penetrating radiation (neutrons, gamma and x-rays) in a scanning geometry, with the detection of radiation either transmitted or produced in the interrogated object. Explosives and chemical agent detection systems are based on the fact that the problem of identification can be reduced to the measurement of elemental concentrations. Different nuclear analytical techniques could be used for this purpose; however the use of neutrons has some specific advantages due to the high penetrability in large payloads. Of special interest is the design and use of a transportable neutron system coupled to a gamma-ray radiographic device for inspecting large containers searching for contraband, explosives, weapons etc. The use of neutron induced reactions for non-destructive bulk elemental analysis is well documented. All neutrons, in particular fast neutrons, are well suited to explore large volume samples because of their high penetration in bulk material. Fast neutrons can be produced efficiently and economically by natural radioactive sources, small accelerators or compact electronic neutron generators, making possible the use of neutron based techniques in field applications. Gamma-rays produced by irradiating the sample with neutrons gives the elemental composition of the material, moreover, knowing the nuclear cross-sections and estimating the absorption factors in the different materials, it is possible to perform a quantitative analysis of elements in the sample even in depth

  13. Localization of muscarinic acetylcholine receptor in plant guard cells

    2001-01-01

    Acetylcholine (ACh), as an important neurotransmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. In Vicia faba L. and Pisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane of Vicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.

  14. Structures of acetylcholine picrate and methoxycarbonylcholine picrate hemihydrate

    Frydenvang, Karla Andrea; Grønborg, L; Jensen, B

    Acetylcholine picrate, C7H16NO2+.C6H2N3-O7-, Mr = 374.3, orthorhombic, Pbca, at 105 K: a = 18.799 (4), b = 7.726 (2), c = 22.878 (4) A, V = 3323 (2) A3, Z = 8, Dm(295 K, flotation) = 1.44, D chi(105 K) = 1.496 Mg m-3, mu(Mo K alpha) = 0.120 mm-1, F(000) = 1568, m.p. (hot-stage microscope) 381-382 K......(295 K, flotation) = 1.49, D chi(105 K) = 1.539 Mg m-3, mu(Mo K alpha) = 0.126 mm-1, F(000) = 836, m.p. (hot-stage microscope) 391-391.5 K, R = 0.033 for 6359 observed [I greater than or equal to 3.0 sigma(I)] reflections. The acetylcholine ion as well as the methoxycarbonylcholine ion have as first...

  15. Long release latencies are increased by acetylcholine at frog endplate

    Samigullin, D.; Bukharaeva, E. A.; Nikolsky, E.; Adámek, S.; Vyskočil, František

    2003-01-01

    Roč. 52, č. 4 (2003), s. 475-480. ISSN 0862-8408 R&D Projects: GA ČR GA305/02/1333; GA ČR GA202/02/1213 Grant ostatní: RFBR(RU) 02/04/48901 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100003 Keywords : quantal release * acetylcholine * synaptic latency Subject RIV: ED - Physiology Impact factor: 0.939, year: 2003

  16. Threonine in the selectivity filter of the acetylcholine receptor channel.

    Villarroel, A.; Sakmann, B

    1992-01-01

    The acetylcholine receptor (AChR) is a cation selective channel whose biophysical properties as well as its molecular composition are fairly well characterized. Previous studies on the rat muscle alpha-subunit indicate that a threonine residue located near the cytoplasmic side of the M2 segment is a determinant of ion flow. We have studied the role of this threonine in ionic selectivity by measuring conductance sequences for monovalent alkali cations and bionic reversal potentials of the wild...

  17. Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors

    Jakubík, Jan; Janíčková, Helena; Randáková, Alena; El-Fakahany, E. E.; Doležal, Vladimír

    2011-01-01

    Roč. 6, č. 11 (2011), e27732. E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GA305/09/0681; GA AV ČR(CZ) IAA500110703; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : acetylcholine muscarinic receptors * G proteins * subtype differences Subject RIV: ED - Physiology Impact factor: 4.092, year: 2011

  18. Alteration in cellular acetylcholine influences dauer formation in Caenorhabditis elegans

    Lee, Jeeyong; Kim, Kwang-Youl; Paik, Young-Ki

    2014-01-01

    Altered acetylcholine (Ach) homeostasis is associated with loss of viability in flies, developmental defects in mice, and cognitive deficits in human. Here, we assessed the importance of Ach in Caenorhabditis elegans development, focusing on the role of Ach during dauer formation. We found that dauer formation was disturbed in choline acetyltransferase (cha-1) and acetylcholinesterase (ace) mutants defective in Ach biosynthesis and degradation, respectively. When examined the potential role o...

  19. Leukocytic acetylcholine in chronic rejection of renal allografts

    Wilczynska, Joanna

    2011-01-01

    Leukocytes, which accumulate in graft blood vessels during fatal acute rejection of experimental renal allografts, synthesise and release acetylcholine (ACh). In this study, I tested the hypothesis that ACh produced by leukocytes accumulating in graft blood vessels contributes to the pathogenesis of chronic renal allograft vasculopathy (CAV). Kidneys were transplanted in the allogeneic Fischer 344 to Lewis rat strain combination. Isogeneic transplantations were performed in Lew...

  20. New Insights on Plant Cell Elongation: A Role for Acetylcholine

    Gian-Pietro Di Sansebastiano; Silvia Fornaciari; Fabrizio Barozzi; Gabriella Piro; Laura Arru

    2014-01-01

    We investigated the effect of auxin and acetylcholine on the expression of the tomato expansin gene LeEXPA2, a specific expansin gene expressed in elongating tomato hypocotyl segments. Since auxin interferes with clathrin-mediated endocytosis, in order to regulate cellular and developmental responses we produced protoplasts from tomato elongating hypocotyls and followed the endocytotic marker, FM4-64, internalization in response to treatments. Tomato protoplasts were observed during auxin and...

  1. Travel path and transport mode identification method using ''less-frequently-detected'' position data

    This study aims to seek method on travel path and transport mode identification in case positions of travellers are detected in low frequency. The survey in which ten test travellers with GPS logger move around Tokyo city centre was conducted. Travel path datasets of each traveller in which position data are selected every five minutes are processed from our survey data. Coverage index analysis based on the buffer analysis using GIS software is conducted. The condition and possibility to identify a path and a transport mode used are discussed

  2. Mutants of the Base Excision Repair Glycosylase, Endonuclease III: DNA Charge Transport as a First Step in Lesion Detection

    Romano, Christine A.; Sontz, Pamela A.; Barton, Jacqueline K.

    2011-01-01

    Endonuclease III (EndoIII) is a base excision repair glycosylase that targets damaged pyrimidines and contains a [4Fe-4S] cluster. We have proposed a model where BER proteins that contain redox-active [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in the detection of DNA lesions. Here, several mutants of EndoIII were prepared to probe their efficiency of DNA/protein charge transport. Cyclic voltammetry experiments on DNA-modified electrodes show that aromatic residues F30...

  3. A new family of insect muscarinic acetylcholine receptors.

    Xia, R-Y; Li, M-Q; Wu, Y-S; Qi, Y-X; Ye, G-Y; Huang, J

    2016-08-01

    Most currently used insecticides are neurotoxic chemicals that target a limited number of sites and insect cholinergic neurotransmission is the major target. A potential target for insecticide development is the muscarinic acetylcholine receptor (mAChR), which is a metabotropic G-protein-coupled receptor. Insects have A- and B-type mAChRs and the five mammalian mAChRs are close to the A-type. We isolated a cDNA (CG12796) from the fruit fly, Drosophila melanogaster. After heterologous expression in Chinese hamster ovary K1 cells, CG12796 could be activated by acetylcholine [EC50 (half maximal effective concentration), 73 nM] and the mAChR agonist oxotremorine M (EC50 , 48.2 nM) to increase intracellular Ca(2+) levels. Thus, the new mAChR is coupled to Gq/11 but not Gs and Gi/o . The classical mAChR antagonists atropine and scopolamine N-butylbromide at 100 μM completely blocked the acetylcholine-induced responses. The orthologues of CG12796 can also be found in the genomes of other insects, but not in the genomes of the honeybee or parasitoid wasps. Knockdown of CG12796 in the central nervous system had no effect on male courtship behaviours. We suggest that CG12796 represents the first recognized member of a novel mAChR class. PMID:27003873

  4. Gold nanoparticle–choline complexes can block nicotinic acetylcholine receptors

    Chur Chin

    2010-04-01

    Full Text Available Chur Chin1, In Kyeom Kim2, Dong Yoon Lim3, Ki Suk Kim4, Hyang Ae Lee4, Eun Joo Kim41Department of Pediatrics, Fatima Hospital, Daegu, Korea; 2Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Korea; 3Department of Pharmacology, School of Medicine, Chosun University, Gwangju, Korea; 4Korea Institute of Toxicology, Daejeon, KoreaAbstract: We identified a novel class of direct ion-channel blockers of ligand-gated ion channels called the gold nanoparticle–choline complex. Negatively charged gold nanoparticles (1.4 nm block ion pores by binding to the sulfur group of the cysteine loop of nicotinic acetylcholine receptors (nAChRs, and currents evoked by acetylcholine (Ach can break these bonds. The current evoked by ACh in nAChRs was blocked directly in ion pores by the gold nanoparticle–choline complex. In adrenal-gland perfusion studies, the complex also blocked nAChRs by diminishing catecholamine release by about 75%. An in vivo study showed muscle relaxation in rats after injection of the complex. These results will foster the application of gold nanoparticles as a direct ion-channel blocker. Keywords: negatively charged gold nanoparticle, choline, gold–sulfur bond, nicotinic acetylcholine receptor, direct ion-channel blocker

  5. Comparison of [3H]nicotine and [3H]acetylcholine binding in mouse brain: regional distribution

    In a continuing study of nicotine binding sites, the authors determined the relative amount of nicotine binding and acetylcholine binding in various brain regions of C57/BL and of DBA mice. Although midbrain showed the highest and cerebellum the lowest binding for both [3H]nicotine and [3H]acetylcholine, the ratio of nicotine to acetylcholine binding showed a three-fold regional variation. Acetylcholine inhibition of [3H]nicotine binding indicated that a portion of nicotine binding was not inhibited by acetylcholine. These results indicate important differences between the binding of (+/-)-[3H]nicotine and that of [3H]acetylcholine

  6. Effects of cooling on the response of the snail bursting neuron to acetylcholine.

    Nedeljković, Miodrag; Kartelija, Gordana; Radenović, Lidija

    2005-06-01

    The Br-type neuron of the snail Helix pomatia, involved in neuronal regulation of various homeostatic and adaptive mechanisms, represents an interesting model for studying effects of temperature change on neuronal activity of poikilotherms. Acetylcholine induces a transient, inward dose-dependent current in the identified Br neuron. In the work presented, we analyzed the effects of cooling on the acetylcholine-induced inward current. The amplitude of acetylcholine-induced inward current was markedly decreased after cooling, and the speed of the decay of acetylcholine response was decreased. PMID:16154950

  7. Study of acetylcholine and barium receptors in the rat duodeno-jejunum by means of labelled molecules

    The purpose of this work is the determination of the number and the localization of Acetylcholine and Barium receptors in the rat intestine. We used 'radioactive labelled' drugs to reach a high sensitiveness of detection. So we were able to point out the number of 'effective' molecules of drugs, that is to say the only ones combining with receptors. With the aid of some assumptions, we determine on the one hand the receptors localization by an assessment of the drug penetration depth before reaching their level and on the other hand the number of these receptors. (author)

  8. Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport*

    Johnson, Danielle E.; Ai, Hui-wang; Wong, Peter; Young, James D.; Campbell, Robert E.; Casey, Joseph R

    2009-01-01

    Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside and H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluor...

  9. Habituation-Like Decrease of Acetylcholine-Induced Inward Current in Helix Command Neurons: Role of Microtubule Motor Proteins.

    Vasil'yeva, Natal'ya A; Murzina, Galina B; Pivovarov, Arkady S

    2015-07-01

    The role of kinesin and dynein microtubule-associated molecular motors in the cellular mechanism of depression of acetylcholine-induced inward chloride current (ACh-current) was examined in command neurons of land snails (Helix lucorum) in response to repeated applications of ACh to neuronal soma. This pharmacological stimulation imitated the protocol of tactile stimulation evoking behavioural habituation of the defensive reaction. In this system, a dynein inhibitor (erythro-9-(2-hydroxy-3-nonyl)adenine, 50 µM) decreased the ACh-current depression rate. Kinesin Eg5 inhibitors (Eg5 inhibitor III, 10 µM and Eg5 inhibitor V, trans-24, 15 µM) reduced the degree of current depression, and Eg5 inhibitor V also reduced the initial rate of depression. The results of electrophysiological experiments in combination with mathematical modelling provided evidence of the participation of dyneins and kinesin Eg5 proteins in the radial transport of acetylcholine receptors in command neurons of H. lucorum in the cellular analogue of habituation. Furthermore, these results suggest that the reciprocal interaction between dynein and kinesin proteins located on the same vesicle can lead to reverse their usual direction of transport (dyneins-in exocytosis and kinesin Eg5-in endocytosis). PMID:25687906

  10. Synthesis of poly(ester-carbonate) with a pendant acetylcholine analog for promoting neurite growth.

    Xing, Dongming; Ma, Lie; Gao, Changyou

    2014-10-01

    The modification of biodegradable polyesters with bioactive molecules has become an important strategy for controlling neuron adhesion and neurite outgrowth in nerve regeneration. In this study we report a biodegradable poly(ester-carbonate) with a pendant acetylcholine analog, which a neurotransmitter for the enhancement of neuron adhesion and outgrowth. The acetylcholine-functionalized poly(ester-carbonate) (Ach-P(LA-ClTMC)) was prepared by copolymerizing l-lactide (LA) and 5-methyl-5-chloroethoxycarbonyl trimethylene carbonate (ClTMC), followed by quaternization with trimethylamine. The acetylcholine analog content could be modulated by changing the molar feeding fraction of ClTMC. The incorporation of the acetylcholine analog improved the hydrophilicity of the films, but the acetylcholine analog content did not significantly influence the surface morphology of the acetylcholine-functionalized films. The results of PC12 cell culture showed that the acetylcholine analog promoted cell viability and neurite outgrowth in a concentration-dependent manner. The longest length of neurite and the percentage of cells bearing neurites were obtained on the Ach-P(LA-ClTMC)-10 film. All the results indicate that the integration of the acetylcholine analog at an appropriate fraction could be an effective strategy for optimizing the existing biodegradable polyesters for nerve regeneration applications. PMID:24998182

  11. Conformationally restrained carbamoylcholine homologues. Synthesis, pharmacology at neuronal nicotinic acetylcholine receptors and biostructural considerations

    de la Fuente Revenga, M; Balle, Thomas; Jensen, Anders A.; Frølund, Bente

    2015-01-01

    Exploration of small selective ligands for the nicotinic acetylcholine receptors (nAChRs) based on acetylcholine (ACh) has led to the development of potent agonists with clear preference for the α4β2 nAChR, the most prevalent nAChR subtype in the central nervous system. In this work we present the...

  12. Transport and Non-Invasive Position Detection of Electron Beams from Laser-Plasma Accelerators

    Osterhoff, Jens; Sokollik, Thomas; Nakamura, Kei; Bakeman, Michael; Weingartner, R; Gonsalves, Anthony; Shiraishi, Satomi; Lin, Chen; vanTilborg, Jeroen; Geddes, Cameron; Schroeder, Carl; Esarey, Eric; Toth, Csaba; DeSantis, Stefano; Byrd, John; Gruner, F; Leemans, Wim

    2011-07-20

    The controlled imaging and transport of ultra-relativistic electrons from laser-plasma accelerators is of crucial importance to further use of these beams, e.g. in high peak-brightness light sources. We present our plans to realize beam transport with miniature permanent quadrupole magnets from the electron source through our THUNDER undulator. Simulation results demonstrate the importance of beam imaging by investigating the generated XUV-photon flux. In addition, first experimental findings of utilizing cavity-based monitors for non-invasive beam-position measurements in a noisy electromagnetic laser-plasma environment are discussed.

  13. Histamine H3 receptors regulate acetylcholine release from the guinea pig ileum myenteric plexus

    The effect of selective histamine H3-receptor agonists and antagonists on the acetylcholine release from peripheral nerves was evaluated in the guinea pig longitudinal muscle-myenteric plexus preparations, preloaded with (3H)choline. In the presence of H1 and H2 blockade, histamine and (R)-α-methylhistamine inhibited the electrically-evoked acetylcholine release, being (R)-α-methylhistamine more active than histamine, but behaving as a partial agonist. The effect of histamine was completely reversed by selective H3-blocking drugs, thioperamide and impromidine, while only submaximal doses of (R)-α-methylhistamine were antagonized. Furthermore, thioperamide and impromidine enhanced the electrically-evoked acetylcholine release. On the contrary, the new H3-blocker, HST-7, was found substantially ineffective, both as histamine antagonist and as acetylcholine overflow enhancer. These data suggest that histamine exerts an inhibitory control on the acetylcholine release from intestinal cholinergic nerves through the activation of H3 receptors

  14. Novel compound, organic cation transporter 3 detection agent and organic cation transporter 3 activity inhibitor, WO2015002150 A1: a patent evaluation.

    Hu, Tao; Wang, Li; Pan, Xiaolei; Qi, Hualin

    2016-08-01

    Increasing pharmacological studies have demonstrated that organic cation transporter 3 (OCT3) plays an important role in controlling the extracellular concentrations of released monoamine neurotransmitter, suggesting that OCT3 might be a promising target in the treatment of depression. As a consequence, compounds showing inhibitory effects on the function of OCT3 have the potential for depression treatment. The current patent WO2015002150 A1 described the synthesis of 59 novel guanidine derivatives. All investigated compounds exhibited significant inhibitory effects (41.9-88.2%) on human OCT3 activity at 30 µM, using human OCT3-transfected human embryonic kidney 293 cell. Concentration-response curves (IC50 values) were determined for seven compounds with higher inhibition potency from the initial screening. IC50 values ranged from 1.9 to 24 µM. In addition, the concentration of these compound in aqueous solution with artificial membranes containing human OCT3 protein was measured. The concentration of compound 6 (SR-2045) was significantly reduced in the presence of human OCT3. Therefore, these compounds have the potential to be further developed as novel antidepressant and human OCT3 detection agent. Future investigations are needed to study the pharmacokinetic and pharmacological properties of these compounds and potential interaction with other transporters. PMID:27097290

  15. Lane Detection in Video-Based Intelligent Transportation Monitoring via Fast Extracting and Clustering of Vehicle Motion Trajectories

    Jianqiang Ren

    2014-01-01

    Full Text Available Lane detection is a crucial process in video-based transportation monitoring system. This paper proposes a novel method to detect the lane center via rapid extraction and high accuracy clustering of vehicle motion trajectories. First, we use the activity map to realize automatically the extraction of road region, the calibration of dynamic camera, and the setting of three virtual detecting lines. Secondly, the three virtual detecting lines and a local background model with traffic flow feedback are used to extract and group vehicle feature points in unit of vehicle. Then, the feature point groups are described accurately by edge weighted dynamic graph and modified by a motion-similarity Kalman filter during the sparse feature point tracking. After obtaining the vehicle trajectories, a rough k-means incremental clustering with Hausdorff distance is designed to realize the rapid online extraction of lane center with high accuracy. The use of rough set reduces effectively the accuracy decrease, which results from the trajectories that run irregularly. Experimental results prove that the proposed method can detect lane center position efficiently, the affected time of subsequent tasks can be reduced obviously, and the safety of traffic surveillance systems can be enhanced significantly.

  16. Detection of pollution transport events southeast of Mexico City using ground-based visible spectroscopy measurements of nitrogen dioxide

    M. Grutter

    2009-07-01

    Full Text Available This work presents ground based differential optical absorption spectroscopy (DOAS measurements of nitrogen dioxide (NO2 during the MILAGRO field campaign in March 2006 at the Tenango del Aire research site located to the southeast of Mexico City. The DOAS NO2 column density measurements are used in conjunction with ceilometer, meteorological and surface nitric oxide (NO, nitrogen oxides (NOx and total reactive nitrogen (NOy measurements to analyze pollution transport events to the southeast of Mexico City during the MILARGO field campaign. The study divides the data set into three case study pollution transport events that occurred at the Tenango del Aire research site. The unique data set is then used to provide an in depth analysis of example days of each of the pollution transport events. An in depth analysis of 13 March 2006, a Case One day, shows the transport of several air pollution plumes during the morning through the Tenango del Aire research site when southerly winds are present and demonstrates how DOAS tropospheric NO2 vertical column densities (VCD, surface NO2 mixing ratios and ceilometer data are used to determine the vertical homogeneity of the pollution layer. The analysis of 18 March 2006, a Case Two day, shows that when northerly winds are present for the entire day, the air at the Tenango del Aire research site is relatively clean and no major pollution plumes are detected. Case 3 days are characterized by relatively clean air throughout the morning with large DOAS NO2 enhancements detected in the afternoon. The analysis of 28 March 2006 show the DOAS NO2 enhancements are likely due to lightning activity and demonstrate how suitable ground-based DOAS measruements are for monitoring anthropogenic and natural pollution sources that reside above the mixing layer.

  17. Detection and Quantification of Neurotransmitters in Dialysates

    Zapata, Agustin; Chefer, Vladimir I.; Shippenberg, Toni S.; Denoroy, Luc

    2009-01-01

    Sensitive analytical methods are needed for the separation and quantification of neurotransmitters obtained in microdialysate studies. This unit describes methods that permit quantification of nanomolar concentrations of monoamines and their metabolites (high-pressure liquid chromatography electrochemical detection), acetylcholine (HPLC-coupled to an enzyme reactor), and amino acids (HPLC-fluorescence detection; capillary electrophoresis with laser-induced fluorescence detection).

  18. Adult celiac disease with acetylcholine receptor antibody positive myasthenia gravis

    Hugh J Freeman; Helen R Gillett; Peter M Gillett; Joel Oger

    2009-01-01

    Celiac disease has been associated with some autoimmune disorders. A 40-year-old competitive strongman with celiac disease responded to a glutenfree diet, but developed profound and generalized motor weakness with acetylcholine receptor antibody positive myasthenia gravis, a disorder reported to occur in about 1 in 5000. This possible relationship between myasthenia gravis and celiac disease was further explored in serological studies. Frozen stored serum samples from 23 acetylcholine receptor antibody positive myasthenia gravis patients with no intestinal symptoms were used to screen for celiac disease. Both endomysial and tissue transglutaminase antibodies were examined. One of 23 (or, about 4.3%) was positive for both IgA-endomysial and IgA tissue transglutaminase antibodies. Endoscopic studies subsequently showed duodenal mucosal scalloping and biopsies confirmed the histopathological changes of celiac disease. Celiac disease and myasthenia gravis may occur together more often than is currently appreciated. The presence of motor weakness in celiac disease may be a clue to occult myasthenia gravis, even in the absence of intestinal symptoms.

  19. Calcium transport by ionophorous peptides in dog and human lymphocytes detected by quin-2 fluorescence.

    Deber, C M; Hsu, L C

    1986-01-29

    Synthetic peptides of structure cyclo(Glu(OBz)-Sar-Gly-(N-R)Gly)2 (I), electrogenic Ca2+-selective carriers in phospholipid vesicle membranes, are shown to mediate the uptake of Ca2+ ions into the cytoplasm of dog and human lymphocytes. Ca2+ transport by DECYL-2E (I, R = n-decyl) - monitored by measurements of the fluorescence of an intracellular dye, quin-2 - occurred at a rate comparable to that produced by electroneutral Ca2+ ionophores ionomycin and Br-A23187. Fluorescence quenching experiments using Mn2+ suggested a greater selectivity by DECYL-2E for Ca2+/Mn2+ vs. the other two ionophores. The result that Ca2+ ions can traverse biological membranes bound in a neutral cavity consisting exclusively of peptide carbonyl ligands may imply the functional significance of binding sites of similar structures in membrane transport proteins. PMID:3947349

  20. Synthesis and characterization of valyloxy methoxy luciferin for the detection of valacyclovirase and peptide transporter.

    Walls, Zachary F; Gupta, Sheeba Varghese; Amidon, Gordon L; Lee, Kyung-Dall

    2014-10-15

    An amino acid ester derivative of luciferin (valoluc) was synthesized to mimic the transport and activation of valacyclovir. This molecule was characterized in vitro for specificity and enzymatic constants, and then assayed in two different, physiologically-relevant conditions. It was demonstrated that valoluc activation is sensitive to the same cellular factors as valacyclovir and thus has the potential to elucidate the dynamics of amino acid ester prodrug therapies in a functional, high-throughput manner. PMID:25240255

  1. Synthesis and Characterization of Valyloxy Methoxy Luciferin for the Detection of Valacyclovirase and Peptide Transporter

    Amidon, Gordon L.; Lee, Kyung-Dall

    2014-01-01

    An amino acid ester derivative of luciferin (valoluc) was synthesized to mimic the transport and activation of valacyclovir. This molecule was characterized in vitro for specificity and enzymatic constants, and then assayed in two different, physiologically-relevant conditions. It was demonstrated that valoluc activation is sensitive to the same cellular factors as valacyclovir and thus has the potential to elucidate the dynamics of amino acid ester prodrug therapies in a functional, high-throughput manner. PMID:25240255

  2. Quantitative Evaluation of an Air-monitoring Network Using Atmospheric Transport Modeling and Frequency of Detection Methods.

    Rood, Arthur S; Sondrup, A Jeffrey; Ritter, Paul D

    2016-04-01

    A methodology has been developed to quantify the performance of an air-monitoring network in terms of frequency of detection. Frequency of detection is defined as the fraction of "events" that result in a detection at either a single sampler or network of samplers. An "event" is defined as a release to the atmosphere of a specified amount of activity over a finite duration that begins on a given day and hour of the year. The methodology uses an atmospheric transport model to predict air concentrations of radionuclides at the samplers for a given release time and duration. Another metric of interest determined by the methodology is called the network intensity, which is defined as the fraction of samplers in the network that have a positive detection for a given event. The frequency of detection methodology allows for evaluation of short-term releases that include effects of short-term variability in meteorological conditions. The methodology was tested using the U.S. Department of Energy Idaho National Laboratory Site ambient air-monitoring network consisting of 37 low-volume air samplers in 31 different locations covering a 17,630 km region. Releases from six major facilities distributed over an area of 1,435 km were modeled and included three stack sources and eight ground-level sources. A Lagrangian Puff air dispersion model (CALPUFF) was used to model atmospheric transport. The model was validated using historical Sb releases and measurements. Relevant 1-wk release quantities from each emission source were calculated based on a dose of 1.9×10 mSv at a public receptor (0.01 mSv assuming release persists over a year). Important radionuclides were Am, Cs, Pu, Pu, Sr, and tritium. Results show the detection frequency was over 97.5% for the entire network considering all sources and radionuclides. Network intensity results ranged from 3.75% to 62.7%. Evaluation of individual samplers indicated some samplers were poorly located and added little to the overall

  3. Ca2+ is involved in muscarine-acetylcholine-receptor-mediated acetylcholine signal transduction in guard cells of Vicia faba L.

    MENG Fanxia; MIAO Long; ZHANG Shuqiu; LOU Chenghou

    2004-01-01

    Acetylcholine (ACh) is an important neurochemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the stomatal opening. By monitoring the changes of cytosolic Ca2+ with fluorescent probe Fluo-3 AM under the confocal microscopy,we found that exogenous ACh increased cytosolic Ca2+ concentration of guard cells of Vicia faba L. Muscarine, an agonist of muscarine acetylcholine receptor (mAChR), could do so as well. In contrast, atropine, the antagonist of mAChR abolished the ability of ACh to increase Ca2+ in guard cells.This mechanism is similar to mAChR in animals. When EGTA was used to chelate Ca2+ or ruthenium red to block Ca2+ released from vacuole respectively, the results showed that the increased cytosolic Ca2+ mainly come from intracellular Ca2+ store. The evidence supports that Ca2+ is involved in guard-cell response to ACh and that Ca2+ signal is coupled to mAChRs in ACh signal transduction in guard cells.

  4. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label

    The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]Nicotine binds at equilibrium with Keq = 0.6 μM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the α- and γ-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the α-subunit was labeled via both agonist sites but the γ-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the α-subunit confirmed that Try-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193

  5. Conceptual design and development of GEM based detecting system for tomographic tungsten focused transport monitoring

    Chernyshova, M.; Czarski, T.; Malinowski, K.; Kowalska-Strzęciwilk, E.; Poźniak, K.; Kasprowicz, G.; Zabołotny, W.; Wojeński, A.; Kolasiński, P.; Mazon, D.; Malard, P.

    2015-10-01

    Implementing tungsten as a plasma facing material in ITER and future fusion reactors will require effective monitoring of not just its level in the plasma but also its distribution. That can be successfully achieved using detectors based on Gas Electron Multiplier (GEM) technology. This work presents the conceptual design of the detecting unit for poloidal tomography to be tested at the WEST project tokamak. The current stage of the development is discussed covering aspects which include detector's spatial dimensions, gas mixtures, window materials and arrangements inside and outside the tokamak ports, details of detector's structure itself and details of the detecting module electronics. It is expected that the detecting unit under development, when implemented, will add to the safe operation of tokamak bringing the creation of sustainable nuclear fusion reactors a step closer. A shorter version of this contribution is due to be published in PoS at: 1st EPS conference on Plasma Diagnostics

  6. Conceptual design and development of GEM based detecting system for tomographic tungsten focused transport monitoring

    Implementing tungsten as a plasma facing material in ITER and future fusion reactors will require effective monitoring of not just its level in the plasma but also its distribution. That can be successfully achieved using detectors based on Gas Electron Multiplier (GEM) technology. This work presents the conceptual design of the detecting unit for poloidal tomography to be tested at the WEST project tokamak. The current stage of the development is discussed covering aspects which include detector's spatial dimensions, gas mixtures, window materials and arrangements inside and outside the tokamak ports, details of detector's structure itself and details of the detecting module electronics. It is expected that the detecting unit under development, when implemented, will add to the safe operation of tokamak bringing the creation of sustainable nuclear fusion reactors a step closer. A shorter version of this contribution is due to be published in PoS at: 1st EPS conference on Plasma Diagnostics

  7. Transcriptomic effects of depleted uranium on acetylcholine and cholesterol metabolisms in Alzheimer's disease model; Effets transcriptomiques de l'uranium appauvri sur les metabolismes de l'acetylcholine et du cholesterol chez un modele de maladie d'Alzheimer

    Lestaevel, Ph.; Bensoussan, H.; Racine, R.; Airault, F.; Gourmelon, P.; Souidi, M. [Direction de la radioprotection de l' Homme, service de radiobiologie et d' epidemiologie, laboratoire de radiotoxicologie experimentale, institut de radioprotection et de surete nucleaire, BP no 17, 92262 Fontenay-aux-Roses cedex (France)

    2011-02-15

    Some heavy metals, or aluminium, could participate in the development of Alzheimer disease (AD). Depleted uranium (DU), another heavy metal, modulates the cholinergic system and the cholesterol metabolism in the brain of rats, but without neurological disorders. The aim of this study was to determine what happens in organisms exposed to DU that will/are developing the AD. This study was thus performed on a transgenic mouse model for human amyloid precursor protein (APP), the Tg2576 strain. The possible effects of DU through drinking water (20 mg/L) over an 8-month period were analyzed on acetylcholine and cholesterol metabolisms at gene level in the cerebral cortex. The mRNA levels of choline acetyl transferase (ChAT) vesicular acetylcholine transporter (VAChT) and ATP-binding cassette transporter A1 (ABC A1) decreased in control Tg2576 mice in comparison with wild-type mice (respectively -89%, -86% and -44%, p < 0.05). Chronic exposure of Tg2576 mice to DU increased mRNA levels of ChAT (+189%, p < 0.05), VAChT (+120%, p < 0.05) and ABC A1 (+52%, p < 0.05) compared to control Tg2576 mice. Overall, these modifications of acetylcholine and cholesterol metabolisms did not lead to increased disturbances that are specific of AD, suggesting that chronic DU exposure did not worsen the pathology in this experimental model. (authors)

  8. Colloidal transport of uranium in soil: Size fractionation and characterization by field-flow fractionation-multi-detection

    The aim of this study was to characterize colloids associated with uranium by using an on-line fractionation/multi-detection technique based on asymmetrical flow field-flow fractionation (As-Fl-FFF) hyphenated with UV detector, multi angle laser light scattering (MALLS) and inductively coupling plasma-mass spectrometry (ICP-MS). Moreover, thanks to the As-Fl-FFF, the different colloidal fractions were collected and characterized by a total organic carbon analyzer (TOC). Thus it is possible to determine the nature (organic or inorganic colloids), molar mass, size (gyration and hydrodynamic radii) and quantitative uranium distribution over the whole colloidal phase. In the case of the site studied, two populations are highlighted. The first population corresponds to humic-like substances with a molar mass of (1500 ± 300) g mol-1 and a hydrodynamic diameter of (2. 0 ± 0. 2) nm. The second one has been identified as a mix of carbonated nano-particles or clays with organic particles (aggregates and/or coating of the inorganic particles) with a size range hydrodynamic diameter between 30 and 450 nm. Each population is implied in the colloidal transport of uranium: maximum 1% of the uranium content in soil leachate is transported by the colloids in the site studied, according to the depth in the soil. Indeed, humic substances are the main responsible of this transport in sub-surface conditions whereas nano-particles drive the phenomenon in depth conditions. (authors)

  9. Detection and correction of blinking bias in image correlation transport measurements of quantum dot tagged macromolecules

    Durisic, Nela; Bachir, Alexia I; Kolin, David L; Hebert, Benedict; Lagerholm, B Christoffer; Grutter, Peter; Wiseman, Paul W

    2007-01-01

    model, but the transport coefficients can have significant systematic errors in the measurements due to blinking. Image correlation measurements of the diffusing QD samples measured at different laser excitation powers and analysis of computer simulated image time series verified that the effect we...... application of the image correlation methods for measurement of the diffusion coefficient of glycosyl phosphatidylinositol-anchored proteins tagged with QDs as imaged on living fibroblasts Udgivelsesdato: 2007-Aug-15......Semiconductor nanocrystals or quantum dots (QDs) are becoming widely used as fluorescent labels for biological applications. Here we demonstrate that fluorescence fluctuation analysis of their diffusional mobility using temporal image correlation spectroscopy is highly susceptible to systematic...

  10. Online Slug Detection in Multi-phase Transportation Pipelines Using Electrical Tomography

    Pedersen, Simon; Mai, Christian; Hansen, Leif;

    2015-01-01

    Slugging flow in offshore oil & gas multi-phase transportation pipelines cause big challenges as the flow regime induces flow and pressure oscillations in the multi-phase pipelines. The negative impacts of the most severe slugs are significant and thus the elimination of slugging flow in the......) has been examined as an alternative to the traditional pressure transmitters. A lab-scaled testing facility has been constructed in order to obtain test data from a ERT transmitter with 12 probes. Different flow regimes have been generated by a pump and a compressor where 2-phase flow can be tested...

  11. Detection of smuggling of nuclear material covered by a legal transport of radioactive material

    Feasibility study was performed in order to investigate the possible on site measurement techniques and approaches applicable in cases when a legal transport of radioactive material is used to cover the radiation of the smuggled uranium. The study was concentrating on non-destructive, passive gamma-spectrometric methods. Possible application of NaI, CdZnTe, and high purity Germanium planar detectors was investigated. An important conclusion of the study was that the higher resolution of the measured spectra provides significant advantages. (author)

  12. Effects of cholinoblockers on acetylcholine content in rat striatum in neuroleptic-induced parkinsonism.

    Dagaev, S G; Kosmachev, A B; Soloveva, N E; Filko, O A; Sanotskii, V I; Dolgo-Saburov, V B

    2004-02-01

    Correction of neuroleptic-induced parkinsonism in rats with two central cholinoblockers atropine and pentifine (acetylene aminoalcohol synthesized at Institute of Toxicology) were studied by measuring the content of acetylcholine in the striatum. The content of the transmitter secretion was estimated from the content of bound acetylcholine fraction in homogenates of the above-mentioned compartment of the brain. The results indicate that atropine and pentifine in doses equally effectively preventing catalepsy in rats had different effects on acetylcholine secretion in the striatum. Hence, cholinolytics with different pharmacological selective effects differently interact with central muscarine receptor subtypes. PMID:15273765

  13. A robust method for inverse transport modelling of atmospheric emissions using blind outlier detection

    M. Martinez-Camara

    2014-05-01

    Full Text Available Emissions of harmful substances into the atmosphere are a serious environmental concern. In order to understand and predict their effects, it is necessary to estimate the exact quantity and timing of the emissions, from sensor measurements taken at different locations. There exists a number of methods for solving this problem. However, these existing methods assume Gaussian additive errors, making them extremely sensitive to outlier measurements. We first show that the errors in real-world measurement datasets come from a heavy-tailed distribution, i.e., include outliers. Hence, we propose to robustify the existing inverse methods by adding a blind outlier detection algorithm. The improved performance of our method is demonstrated on a real dataset and compared to previously proposed methods. For the blind outlier detection, we first use an existing algorithm, RANSAC, and then propose a modification called TRANSAC, which provides a further performance improvement.

  14. Second Harmonic Detection of Spin-Dependent Transport in Magnetic Nanostructures

    Yu, Hai-Ming; Granville, S.; Yu, Da-Peng; J-ph., Ansermet

    2010-02-01

    Detection of the second harmonic response of magnetic nanostructures to an ac current is shown to be a very sensitive probe of the magnetization reversal process. A temperature oscillation is obtained by Joule heating instead of using a laser as the heat source, as in thermo-galvanic voltage measurements (TGV). Joule heating is used to produce a large local temperature gradient in asymmetric Co/Cu/Co spin valves. Evidence is found for an effect of a heat current on magnetization.

  15. Comparison of the Ability of Various Imaging Modalities (CT & Plain X- Ray in Detecting Drug Transport in Body Packers

    Morteza Sanei

    2009-01-01

    Full Text Available "ndrugs within the human body. In our country due to vast common border with Afghanistan which is the biggest Opium producer in the world and has the second place in Heroine production, drug smuggling has potential national threat and besides it has a global impact as using our territory as the major smuggling route to the west. Furthermore, in recent years new generations of African smugglers of new types of drugs are using our country as a transit route to transport drugs to Europe or Africa. In this way handmade or automatically produced packets are swallowed, rectally or vaginally inserted, and then transported. The first choice modality is plain x-ray of the abdomen in upright and supine positions. Recently abdominal and pelvic CT without contrast has shown a great success rate in the detection of body packers with changing window modality to detect different types of drugs. "nMaterials and Methods: Plain x-ray and abdominal and pelvic CT without contrast were performed for 12 cases who confessed to drug packet ingestion. The presence, number and location of the packets were evaluated in different modalities and the density of the packets were also measured in Hounsfield units (HU. "nResults: The mean age of our cases was 28.2±5.9 years (range, 17-35 years. Eleven (91.6 % patients were male and only one case was female. All patients had characteristic findings in plain x-ray and also all packets were visualized in all patients "nConclusion: Plain x-ray has a distinctive position in detecting packets in intestines especially when oral contrast materials are used. It is cheaper and more accessible than CT, but using different Hounsfield units in CT windows can even characterize different types of drugs even before extracting them.  

  16. Alteration in cellular acetylcholine influences dauer formation in Caenorhabditis elegans.

    Lee, Jeeyong; Kim, Kwang-Youl; Paik, Young-Ki

    2014-02-01

    Altered acetylcholine (Ach) homeostasis is associated with loss of viability in flies, developmental defects in mice, and cognitive deficits in human. Here, we assessed the importance of Ach in Caenorhabditis elegans development, focusing on the role of Ach during dauer formation. We found that dauer formation was disturbed in choline acetyltransferase (cha-1) and acetylcholinesterase (ace) mutants defective in Ach biosynthesis and degradation, respectively. When examined the potential role of G-proteins in dauer formation, goa-1 and egl-30 mutant worms, expressing mutated versions of mammalian G(o) and G(q) homolog, respectively, showed some abnormalities in dauer formation. Using quantitative mass spectrometry, we also found that dauer larvae had lower Ach content than did reproductively grown larvae. In addition, a proteomic analysis of acetylcholinesterase mutant worms, which have excessive levels of Ach, showed differential expression of metabolic genes. Collectively, these results indicate that alterations in Ach release may influence dauer formation in C. elegans. PMID:24219868

  17. Valence of acetylcholine-receptor-antibody-titers in myasthenia gravis

    In a retrospective study in 47 patients with myasthenia gravis acetylcholine-receptor-antibody-titers (AChR-AB) were correlated with the severity of the disease. In 18 patients the course of titers was studied and two groups of patients could be differentiated: patients with relative constant and patients with fluctuating titers. Age, age of begin of myasthenia and sex did not influence the titers. Also the duration of the disease and the severity of symptoms did not influence the level of AChR-AB-titers. In this retrospective study the influence of immunsuppressive therapy on the intra-individual course of AB-titers and their correlation with the clinical symptoms could not be judged. Measurement of AChR-AB is of value for the diagnosis of myasthenia gravis and important for judging the clinical course and the effect of therapy. (Author)

  18. Structure and superorganization of acetylcholine receptor–rapsyn complexes

    Zuber, Benoît; Unwin, Nigel

    2013-01-01

    The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with insufficient receptor clustering suffer from muscle weakness. However, the detailed organization of the receptor–rapsyn network is poorly understood: it is unclear whether rapsyn first forms a wide meshwork to which receptors can subsequently dock or whether it only forms short bridges linking receptors together to make a large cluster. Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been controversial. Here, we show by cryoelectron tomography and subtomogram averaging of Torpedo postsynaptic membrane that receptors are connected by up to three rapsyn bridges, the minimum number required to form a 2D network. Half of the receptors belong to rapsyn-connected groups comprising between two and fourteen receptors. Our results provide a structural basis for explaining the stability and low diffusion of receptors within clusters. PMID:23754381

  19. Applicability of gamma detection systems for the detection of illicit transports of radioactive substances in cargo containers shielded by the cargo

    measurement conditions on frequently occurring radioactive substances and on minimal detectable activities. The second part is a simulation of portal monitors for gamma radiation. This is done to simulate gamma spectra of radionuclides inside cargo containers transported through a portal monitor. Measurements: Measurements were conducted at the cargo container checkpoint of the German Customs Office at the Hamburg Harbor. At this checkpoint a radiation portal monitor (RTM 910, produced by Mirion Technologies) has been installed. The RTM 910 is capable of neutron and gamma detection with a low energy-resolution. To detect the gamma radiation plastic scintillators are used. The RTM 910 has detectors on both sides of an incoming street to the checkpoint. The distance between the two detector parts is six meters and the cargo containers are transported through this portal monitor with an average velocity of 30 km/h. In the field experiment 30 to 40 cargo containers were controlled per hour. In this time approximately three alarms per hour were given. To examine what kind of radionuclides caused these alarms, it was necessary to analyze spectra of the radiation when such an alarm occurred. For the identification of the radioactive substances two other gamma detectors were used in addition to the RTM 910. One detector (ReGeM, produced by Canberra) was a high-purity-germanium detector with an energy resolution of less than 2 keV (e.g. 0,3% at 662 keV). The other detector (Spir Ident, produced by Mirion Technologies) is based on sodium-iodide and has an energy resolution of about 10 keV. The idea was to take spectra exactly at the time an alarm was given by the RTM 910 and to identify the alarm causing radionuclides by analyzing the spectra. It was possible to identify radionuclides in the spectra that were simultaneously recorded to RTM 910 alarms. All the detected radionuclides are naturally occurring. Simulations: In order to estimate the physical limitations and the remaining

  20. The mechanism of acetylcholine receptor in binding MuSK in myasthenia gravis and the role of HSP90 molecular chaperone

    Chen, Rongbo; Chen, Siqia; Liao, Juan; Chen, Xiaopu; Xu, Xiaoling

    2016-01-01

    As an autoimmune disease, myasthenia gravis is caused by the dysfunction of neural transmission. Acetylcholine is known to exert its function after entering into synaptic cleft through binding onto postsynaptic membrane. The role of acetylcholine in binding MuSK in myasthenia gravis, however, remains unknown. A total of 38 myasthenia gravis patients and 27 healthy controls were included in this study for the detection of the expression of MuSK using immunofluorescent method. Expression of both MuSK and interleukin-6 (IL-6) were measured by Western blot, followed by the correlation analysis between heat shock protein 90 (HSP90) and IL-6 which were measured by enzyme-linked immunosorbent assay (ELISA). In myasthenia gravis patients, MuSK was co-localized with acetylcholine at the postsynaptic membrane. Such accumulation of MuSK, however, did not occur in normal people. Meanwhile we also observed elevated expression of IL-6 in myasthenia gravis patients (pmyasthenia gravis patients, with elevated expression. HSP90 in disease people can activate IL-6 mediated signaling pathways. PMID:27186300

  1. Incrustations detection system for petroleum transport pipes based on gamma transmission

    The scale formed over the inner walls of the ducts conveying the extracted product from offshore oil wheels is a major cause of losses to companies and in some cases even the safety is affected. The consequence of such fouling is the duct's square section reduction that causes extraction flow decrease and can also cause an increase in pressure inside the wheel, with serious consequences for safety. The objective of this work is to propose a mobile inspection system, which can be transported by underwater robots to inspect the lines of ducts in the outputs of the oil wheels. The measurement method to be adopted will be the gamma rays' beam attenuation at a predetermined position of the pipe. This transmission value compared to a clear pipe reading will show if the thickness of the inlay is larger or smaller than an assumed thickness. To carry out the measurements it was designed and built an electronic system comprising power supply, amplifier, single channel analyzer and a counter timer that was connected to a CsI scintillator detector coupled to a PIN photodiode. The system was set up to perform measurements with constant accuracy of ±1%. Tests during the study demonstrated the effectiveness of the proposed method with the obtained results with a carbon steel duct section of 270 mm diameter, removed from the field, with asymmetric BaSO4 inlay. (author)

  2. Electrolyte and protein secretion by the perfused rabbit mandibular gland stimulated with acetylcholine or catecholamines

    Case, R M; Conigrave, A D; Novak, I;

    1980-01-01

    1. A method is described for the isolation and vascular perfusion in vitro of the mandibular gland of the rabbit. The perfusate is a physiological salt solution containing glucose as the only metabolic substrate.2. During perfusion with solutions containing acetylcholine, the gland secretes......) concentrations and the osmolality of acetylcholine evoked saliva exhibited flow-dependency similar to that seen in vivo. The concentrations of Na and Cl, but not K and HCO(3), increased by about 25 mmol l(-1) during periods of prolonged stimulation with acetylcholine even though the salivary secretory rate was...... composition of isoproterenol-evoked saliva was vastly different from that evoked by acetylcholine, being particularly rich in K and HCO(3). The isoproterenol-evoked saliva was also extremely rich in protein so that the total protein secretion evoked by isoproterenol was much greater than that evoked by...

  3. A family of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans.

    Putrenko, Igor; Zakikhani, Mahvash; Dent, Joseph A

    2005-02-25

    The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors. PMID:15579462

  4. Inhibition by substance P of some peripheral actions of acetylcholine in the cat

    Clark, S.L.; Ryall, R. W.

    1982-01-01

    1 The effect of substance P on contractions of the nictitating membrane and pressor responses to acetylcholine (ACh) and dimethylphenyl-piperazinium (DMPP) which were mediated via nocotinic receptors was studied in cats anaesthetized with chloralose.

  5. Notexin preferentially inhibits the release of newly synthesized acetylcholine from rat brain synaptosomal fractions

    An investigation was made of the effects of the snake venom neurotoxin, notexin, on acetylcholine turnover in rat brain P2 fractions using a gas chromatographic mass spectrometric assay for acetylcholine and choline. In contrast to earlier reports, we found a stimulation of the uptake and acetylation of labeled choline by toxin-treated P2 fractions. More significantly, notexin inhibited the release of this newly synthesized transmitter. These effects were found to be dependent on the dose of the toxin and the time of exposure of the P2 fraction to notexin. Longer exposure to notexin or experiments involving resuspension of notexin-treated P2 fractions appeared to result in considerable lysis of the transmitter-containing particles. Thus, notexin may alter acetylcholine compartmentation in the nerve ending and thereby affect acetylcholine synthesis

  6. Effect of spontaneous diffusion in micro/nanoporous chemically crosslinked poly (N-vinyl imidazole) gel on the conformational changes of acetylcholine

    Vaganova, Evgenia; Pierola, Ines F.; Ovadia, Haim; Lyshevski, Sergey E.; Yitzchaik, Shlomo

    2009-02-01

    Interdependent structural properties such as molecular conformation, flexibility and charge redistribution control the intermolecular interactions of acetylcholine (ACh) with adjacent molecules. This paper reports the results of an investigation of the effect of the diffusion of ACh through a nano/microporous poly (N-vinylimidazole) (PVI) gel on its structural properties, namely on changes in its conformation. To investigate the conformational changes of ACh during spontaneous diffusion through the gel, the fluorescence lifetime of the label molecule - fluorescein - was monitored. To clarify the results, analogous experiments were conducted with nicotinic acid and dopamine. In contrast to the nicotinic acid and dopamine, ACh can play the role of a regulator in molecular transport.

  7. NA22 Model Cities Project - LL244T An Intelligent Transportation System-Based Radiation Alert and Detection System

    Peglow, S

    2004-02-24

    The purpose of this project was twofold: first, provide an understanding of the technical foundation and planning required for deployment of Intelligent Transportation System (ITS)-based system architectures for the protection of New York City from a terrorist attack using a vehicle-deployed nuclear device; second, work with stakeholders to develop mutual understanding of the technologies and tactics required for threat detection/identification and establish guidelines for designing operational systems and procedures. During the course of this project we interviewed and coordinated analysis with people from the New Jersey State Attorney General's office, the New Jersey State Police, the Port Authority of New York/New Jersey, the Counterterrorism Division of the New York City Police Department, the New Jersey Transit Authority, the State of New Jersey Department of Transportation, TRANSCOM and a number of contractors involved with state and federal intelligent transportation development and implementation. The basic system architecture is shown in the figure below. In an actual system deployment, radiation sensors would be co-located with existing ITS elements and the data will be sent to the Traffic Operations Center. A key element of successful system operation is the integration of vehicle data, such as license plate, EZ pass ID, vehicle type/color and radiation signature. A threat data base can also be implemented and utilized in cases where there is a suspect vehicle identified from other intelligence sources or a mobile detector system. Another key aspect of an operational architecture is the procedures used to verify the threat and plan interdiction. This was a major focus of our work and discussed later in detail. In support of the operational analysis, we developed a detailed traffic simulation model that is described extensively in the body of the report.

  8. Expression of the α7 nicotinic acetylcholine receptor in human lung cells

    Schuller Hildegard M; Dhar Madhu; Plummer Howard K

    2005-01-01

    Abstract Background We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the α7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a high affinity agonist for the α7 nicotinic acetylcholine receptor. In the present study, our goal was t...

  9. Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

    Matsuda, K; Buckingham, S D; Freeman, J.C.; Squire, M D; Baylis, H. A.; Sattelle, D B

    1998-01-01

    Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine...

  10. Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods

    Collin, Caitlin Alexis; Hauser, Frank; Gonzalez de Valdivia, Ernesto I; Li, Shizhong; Reisenberger, Julia; Carlsen, Eva M.M.; Khan, Zaid; Hansen, Niels Ø.; Puhm, Florian; Søndergaard, Leif; Niemiec, Justyna; Heninger, Magdalena; Ren, Guilin Robin; Grimmelikhuijzen, Cornelis

    2013-01-01

    Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In ...... (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs....

  11. Fusion of waveform events and radionuclide detections with the help of atmospheric transport modelling

    Krysta, Monika; Kushida, Noriyuki; Kotselko, Yuriy; Carter, Jerry

    2016-04-01

    Possibilities of associating information from four pillars constituting CTBT monitoring and verification regime, namely seismic, infrasound, hydracoustic and radionuclide networks, have been explored by the International Data Centre (IDC) of the Comprehensive Nuclear-Test-Ban Treaty Organization (CTBTO) for a long time. Based on a concept of overlying waveform events with the geographical regions constituting possible sources of the detected radionuclides, interactive and non-interactive tools were built in the past. Based on the same concept, a design of a prototype of a Fused Event Bulletin was proposed recently. One of the key design elements of the proposed approach is the ability to access fusion results from either the radionuclide or from the waveform technologies products, which are available on different time scales and through various different automatic and interactive products. To accommodate various time scales a dynamic product evolving while the results of the different technologies are being processed and compiled is envisioned. The product would be available through the Secure Web Portal (SWP). In this presentation we describe implementation of the data fusion functionality in the test framework of the SWP. In addition, we address possible refinements to the already implemented concepts.

  12. Novel aspects of cholinergic regulation of colonic ion transport.

    Bader, Sandra; Diener, Martin

    2015-06-01

    Nicotinic receptors are not only expressed by excitable tissues, but have been identified in various epithelia. One aim of this study was to investigate the expression of nicotinic receptors and their involvement in the regulation of ion transport across colonic epithelium. Ussing chamber experiments with putative nicotinic agonists and antagonists were performed at rat colon combined with reverse transcription polymerase chain reaction (RT-PCR) detection of nicotinic receptor subunits within the epithelium. Dimethylphenylpiperazinium (DMPP) and nicotine induced a tetrodotoxin-resistant anion secretion leading to an increase in short-circuit current (I sc) across colonic mucosa. The response was suppressed by the nicotinic receptor antagonist hexamethonium. RT-PCR experiments revealed the expression of α2, α4, α5, α6, α7, α10, and β4 nicotinic receptor subunits in colonic epithelium. Choline, the product of acetylcholine hydrolysis, is known for its affinity to several nicotinic receptor subtypes. As a strong acetylcholinesterase activity was found in colonic epithelium, the effect of choline on I sc was examined. Choline induced a concentration-dependent, tetrodotoxin-resistant chloride secretion which was, however, resistant against hexamethonium, but was inhibited by atropine. Experiments with inhibitors of muscarinic M1 and M3 receptors revealed that choline-evoked secretion was mainly due to a stimulation of epithelial M3 receptors. Although choline proved to be only a partial agonist, it concentration-dependently desensitized the response to acetylcholine, suggesting that it might act as a modulator of cholinergically induced anion secretion. Thus the cholinergic regulation of colonic ion transport - up to now solely explained by cholinergic submucosal neurons stimulating epithelial muscarinic receptors - is more complex than previously assumed. PMID:26236483

  13. Necessities detected in patients derived to sanitary center in an ambulance of the urgent transport network after being treated by the 061 emergency team

    Sixto Cámara Anguita; Antonio J. Valenzuela Rodríguez

    2007-01-01

    The integral systems of urgencies and sanitary emergencies has, as resources for the urgent transport of patients, with nonwelfare ambulances, ambulances of basic life support and ambulances of advanced life support. In the two first, due to the ambiguity of the norm, the presence of a nurse to give care is not compulsory. Nevertheless, in the present study, it is detected that, the patients of this study, transferred in nonwelfare ambulance of the urgent transport network to a useful hospita...

  14. Characterization of the charge-carrier transport properties of IIa-Tech SC diamond for radiation detection applications

    Single crystal (SC) diamond has since years demonstrated its interest for the fabrication of radiation detectors, especially where the material properties are providing superior interests with respect to the detection application. Among the industrial suppliers able to provide on a commercial basis high-grade single crystal diamond, IIa-Tech has recently appeared in the market as a new player. The aim of this paper is to assess the quality of one SC sample when characterized under α-particles for the measurement of its carrier transport properties. We observed that full charge collection could be observed at biases as low as 0.11 V/μm with no space charge build-up (conventionally typical bias values used are closer to 1 V/μm). Velocity reached values of 38 μm/ns and 53 μm/ns for electrons and holes, respectively (values probed at 0.33 V/μm). Similarly, the α detection spectrum displays a sharp line demonstrating the good uniformity of the material over its surface. By combining the measurements with more conventional optical observations such as birefringence and cathodoluminescence spectroscopy, it comes that the material demonstrates its ability to be used as a detector, with properties that can compare with the highest grade materials today available on the market. (copyright 2015 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  15. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 1010 M-1, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r2>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG

  16. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  17. A radioimmunoassay for the quantitative evaluation of anti-human acetylcholine receptor antibodies in myasthenia gravis

    A radioimmunoassay was developed for the quantitative evaluation of antibodies to the acetylcholine receptor in the serum of myasthenic patients. AcChR was extracted from human muscle. A detailed preparation of the 125I-labelled α-Bgt-AcChR complex used as antigen is reported. Usually, an average of 20 pmol were obtained from 100 g muscle. This preparation is stable for 1 month in presence of an inhibitor of proteolysis and sufficient for performing about fifteen assays. The labelled complex was incubated with increasing amounts of sera and precipitated with anti-human IgG serum. Titres were expressed in pmol 125I-labelled α-Bgt-AcChR complex precipitated per ml serum. Out of thirty-nine sera tested thirty-six had positive titres ranging from 0.1 to 46 pmol/ml. No anti-AcChR were detected in the sera from twenty-seven patients used as controls. (author)

  18. Boosting visual cortex function and plasticity with acetylcholine to enhance visual perception

    Jun-Il eKang

    2014-09-01

    Full Text Available The cholinergic system is a potent neuromodulatory system that plays critical roles in cortical plasticity, attention and learning. In this review, we propose that the cellular effects of acetylcholine in the primary visual cortex during the processing of visual inputs might induce perceptual learning; i.e., long-term changes in visual perception. Specifically, the pairing of cholinergic activation with visual stimulation increases the signal-to-noise ratio, cue detection ability and long-term facilitation in the primary visual cortex. This cholinergic enhancement would increasesthe strength of thalamocortical afferents to facilitate the treatment of a novel stimulus while decreasing the cortico-cortical signaling to reduce recurrent or top-down modulation. This balance would be mediated by different cholinergic receptor subtypes that are located on both glutamatergic and GABAergic neurons of the different cortical layers. The mechanisms of cholinergic enhancement are closely linked to attentional processes, long-term potentiation and modulation of the excitatory/inhibitory balance. Recently, it was found that boosting this system during visual training robustly enhances sensory perception in a long-term manner. Our hypothesis is that repetitive pairing of cholinergic and sensory stimulation over a long period of time induces long-term changes in the processing of trained stimuli that might improve perceptual ability. Various non-invasive approaches to the activation of the cholinergic neurons have strong potential to improve visual perception.

  19. Schizophrenia and the alpha7 nicotinic acetylcholine receptor.

    Martin, Laura F; Freedman, Robert

    2007-01-01

    In addition to the devastating symptoms of psychosis, many people with schizophrenia also suffer from cognitive impairment. These cognitive symptoms lead to marked dysfunction and can impact employability, treatment adherence, and social skills. Deficits in P50 auditory gating are associated with attentional impairment and may contribute to cognitive symptoms and perceptual disturbances. This nicotinic cholinergic-mediated inhibitory process represents a potential new target for therapeutic intervention in schizophrenia. This chapter will review evidence implicating the nicotinic cholinergic, and specifically, the alpha7 nicotinic receptor system in the pathology of schizophrenia. Impaired auditory sensory gating has been linked to the alpha7 nicotinic receptor gene on the chromosome 15q14 locus. A majority of persons with schizophrenia are heavy smokers. Although nicotine can acutely reverse diminished auditory sensory gating in people with schizophrenia, this effect is lost on a chronic basis due to receptor desensitization. The alpha7 nicotinic agonist 3-(2,4 dimethoxy)benzylidene-anabaseine (DMXBA) can also enhance auditory sensory gating in animal models. DMXBA is well tolerated in humans and a new study in persons with schizophrenia has found that DMXBA enhances both P50 auditory gating and cognition. alpha7 Nicotinic acetylcholine receptor agonists appear to be viable candidates for the treatment of cognitive disturbances in schizophrenia. PMID:17349863

  20. Increased expression of the nicotinic acetylcholine receptor in stimulated muscle.

    O'Reilly, Clare; Pette, Dirk; Ohlendieck, Kay

    2003-01-10

    Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts. PMID:12504123

  1. Effects of two oxadiazolidinones on cholinesterases and acetylcholine receptors

    Inhibition of acetylcholinesterase (AChE) and butyryl cholinesterase (BuChE) by 3-(2,3-dihydro-2,2-dimethyl-benzofuran-'7-yl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (DBOX) and 3-(2-methoxyphenyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (MPOX) was measured by the Ellmann spectrophotometric method. Inhibition was quasi first order and irreversible. DBOX was 2-3 orders of magnitude more potent than MPOX. Housefly brain AChE and horse serum BuChE were more sensitive than AChEs of red blood cells or eel and Torpedo electric organs. It is suggested that the nonesteratic oxadiazolidinones are activated to carbanillates on the surface of the enzyme and produce a carbanillated enzyme which ages rapidly. Carbamate anticholinesterases protected AChE against carbanillation as they did against phosphorylation. At higher concentrations, the two oxadiazolidinones also affected binding of [125I] α bungarotoxin and [3H]perhydrohistrionicotoxin to Torpedo nicotinic acetylcholine receptors, but did not affect binding of [3H]quinuclidinyl benzilate to rat brain muscarinic receptors

  2. Therapeutic Potential of α7 Nicotinic Acetylcholine Receptors.

    Bertrand, Daniel; Lee, Chih-Hung L; Flood, Dorothy; Marger, Fabrice; Donnelly-Roberts, Diana

    2015-10-01

    Progress in the fields of neuroscience and molecular biology has identified the forebrain cholinergic system as being important in many higher order brain functions. Further analysis of the genes encoding the nicotinic acetylcholine receptors (nAChRs) has highlighted, in particular, the role of α7 nAChRs in these higher order brain functions as evidenced by their peculiar physiologic and pharmacological properties. As this receptor has gained the attention of scientists from academia and industry, our knowledge of its roles in various brain and bodily functions has increased immensely. We have also seen the development of small molecules that have further refined our understanding of the roles of α7 nAChRs, and these molecules have begun to be tested in clinical trials for several indications. Although a large body of data has confirmed a role of α7 nAChRs in cognition, the translation of small molecules affecting α7 nAChRs into therapeutics has to date only progressed to the stage of testing in clinical trials. Notably, however, most recent human genetic and biochemical studies are further underscoring the crucial role of α7 nAChRs and associated genes in multiple organ systems and disease states. The aim of this review is to discuss our current knowledge of α7 nAChRs and their relevance as a target in specific functional systems and disease states. PMID:26419447

  3. Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors

    Layla AZAM; J Michael MCINTOSH

    2009-01-01

    Cysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the a-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain a-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiol-ogy of Parkinson's disease and nociception. Structure/function characterization of a-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipo-philic moieties has led to improved stability and bioavailability of a-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of a-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.

  4. Acetylcholine esterase activity in mild cognitive impairment and Alzheimer's disease

    Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD), but there is controversy about its relevance at the early stages of the disease and in mild cognitive impairment (MCI). In vivo positron emission tomography imaging of cortical acetylcholine esterase (AChE) activity as a marker of cholinergic innervation that is expressed by cholinergic axons and cholinoceptive neurons has demonstrated a reduction of this enzyme activity in manifest AD. The technique is also useful to measure the inhibition of cerebral AChE induced by cholinesterase inhibitors for treatment of dementia symptoms. A reduction of cortical AchE activity was found consistently in all studies of AD and in few cases of MCI who later concerted to AD. The in vivo findings in MCI and very mild AD are still preliminary, and studies seem to suggest that cholinergic innervation and AChE as the main degrading enzyme are both reduced, which might result in partial compensation of their effect. (orig.)

  5. Bolus injection of acetylcholine terminates atrial fibrillation in rats.

    Fleidervish, Ilya A; Goldberg, Yuri; Ovsyshcher, I Eli

    2008-01-28

    It is well established that a tonic increase in the availability of the atrial muscarinic K(+) channels, either by enhanced vagal tone or by steady infusion of a low-dose of cholinergic or adenosine receptor agonists, promotes the genesis of atrial fibrillation. Here, we aimed to test the hypothesis that bolus administration of a muscarinic receptor agonist would destabilize and terminate atrial arrhythmia by uniformly and transiently activating K(+) channels throughout the atria, and that if the agonist was rapidly hydrolysable, it would dissipate before the more tonic, pro-arrhythmic effects could take hold. The episodes of untreated atrial fibrillation, induced in anesthetized rats by programmed electrical stimulation via trans-esophageal bipolar catheter, lasted on average 8.6+/-2.2 min (n=32). Intravenous injection of a model hydrolysable muscarinic agonist, acetylcholine (0.2 mg/kg body weight), converted atrial fibrillation into sinus rhythm within 8.4+/-1.9 s (n=10, Ppre-atrial fibrillation values within 10-20 s of injection. In conclusion, our evidence indicates that bolus administration of rapidly hydrolysable muscarinic agonist could be an effective way to pharmacologically terminate atrial fibrillation and restore sinus rhythm. PMID:18078927

  6. Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

    Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed

  7. The use of FTA cards for transport and detection of gyrA mutation of Campylobacter jejuni from poultry.

    Sierra-Arguello, Y M; Faulkner, O; Tellez, G; Hargis, B M; Pinheiro do Nascimento, V

    2016-04-01

    The purpose of the present study was to evaluate a technique involving the use of commercially available FTA classic card (Whatman) for transporting and detection of DNA to use in PCR analysis and genetic sequencing of Campylobacter jejuni of poultry origin. Fifty isolates of Campylobacter jejuni were obtained from broiler carcasses in Rio Grande do Sul, Brazil. Antimicrobial susceptibility testing to ciprofloxacin revealed that all 50 isolates were resistant to ciprofloxacin. Each isolate was transferred to Brucella broth tubes and incubated overnight at 41.5°C. Cell cultures were diluted to match a McFarland Turbidity Standard 0.5, and 110 μL of the cell suspension were applied to one circle on Whatman FTA classic cards. The samples were then covered and allowed to dry at room temperature. Cards were identified and stored at room temperature until further use (3 mo after collection). FTA cards were shipped for analysis to the Department of Poultry Science, University of Arkansas. Amplification of the Campylobacter gyrA gene was successful and demonstrated strong bands for a large amplicon for all 50 samples preserved on FTA cards. Mutations present in each gene were confirmed by DNA sequencing. Then, 7 samples were chosen for the sequencing. The detection of a mutation regarding ciprofloxacin-resistant isolates revealed that 7 samples had a mutation in the gyrA gene. In conclusion, the characteristics of the profiles suggest that the DNA has maintained its integrity after 3 mo of storage at room temperature and is a suitable template for PCR and sequencing from Campylobacter samples. The application of this technology has potential in numerous methodologies, especially when working in remote areas and in developing countries where access to laboratory facilities and equipment is limited. PMID:26769268

  8. Comparison of heparin and EDTA transport tubes for detection of cytomegalovirus in leukocytes by shell vial assay, pp65 antigenemia assay, and PCR.

    Storch, G A; Gaudreault-Keener, M; Welby, P C

    1994-01-01

    The anticoagulants heparin and EDTA were compared for inhibitory effects on the detection of cytomegalovirus from washed leukocytes in specimen transport tubes. Evaluation was made by the centrifugation/shell vial culture technique, the pp65 antigenemia assay, and PCR. For each assay, the results with heparin and EDTA were equivalent.

  9. Control of unauthorized transportation of nuclear and radioactive materials across state territories and borders - Detection, response and decision support

    A new solution to control the illicit transportation of nuclear and radioactive materials is proposed and described. This solution consists in the creation of a system of gamma and gamma-neutron radiation monitors and fundamentally new software/hardware package RAVEN (Radiation Alarm and Video Event Notification) which integrates different type monitors in a single network. The main purpose of this system is to analyze and store data coming from radiation monitors, to process these data and to help the user to interpret them. This ensures the user's correct response to all the alarms triggered by radiation monitors and indicating the presence of radioactive and/or nuclear materials in scanned objects. The developed system can integrate fixed radiation monitors that can be installed in different sites within not only one country but in different sites worldwide in a single network. The system can be adapted to the local conditions and allows the user: (i) to detect minimum quantities of special nuclear and radioactive materials (specified by the national and international requirements) by their gamma and/or neutron radiation; (ii) to acquire, store and analyze digital data and video images related to cargos with radioactive materials crossing a state border or an object limits; (iii) to make these data accessible to users at different levels ensuring effective operation of both central alarm stations (state, regional or agency center) and local alarm stations (border cross points or object limits). (author)

  10. Control of unauthorized transportation of nuclear and radioactive materials across state territories and borders: detection, response and decision support

    A new solution to control the illicit transportation of nuclear and radioactive materials is proposed and described. This solution consists in the creation of a system of gamma and gamma-neutron radiation monitors and fundamentally new software/hardware package RAVEN (Radiation Alarm and Video Event Notification) which integrates different type monitors in a single network. The main purpose of this system is to analyze and store data coming from radiation monitors, to process these data and to help the user to interpret them. This ensures the user's correct response to all the alarms triggered by radiation monitors and indicating the presence of radioactive and/or nuclear materials in scanned objects. The developed system can integrate fixed radiation monitors that can be installed in different sites within not only one country but in different sites worldwide in a single network. The system can be adapted to the local conditions and allows the user: (i) To detect minimum quantities of special nuclear and radioactive materials (specified by the national and international requirements) by their gamma and/or neutron radiation; (ii) To acquire, store and analyze digital data and video images related to cargos with radioactive materials crossing a state border or an object limits; (iii) To make these data accessible to users at different levels ensuring effective operation of both central alarm stations (state, regional or agency center) and local alarm stations (border cross points or object limits). (author)

  11. Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene carveol.

    Lozon, Yosra; Sultan, Ahmed; Lansdell, Stuart J; Prytkova, Tatiana; Sadek, Bassem; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Millar, Neil S; Oz, Murat

    2016-04-01

    Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies. PMID:26849939

  12. Loss of Acetylcholine Signaling Reduces Cell Clearance Deficiencies in Caenorhabditis elegans.

    Sérgio M Pinto

    Full Text Available The ability to eliminate undesired cells by apoptosis is a key mechanism to maintain organismal health and homeostasis. Failure to clear apoptotic cells efficiently can cause autoimmune diseases in mammals. Genetic studies in Caenorhabditis elegans have greatly helped to decipher the regulation of apoptotic cell clearance. In this study, we show that the loss of levamisole-sensitive acetylcholine receptor, but not of a typical neuronal acetylcholine receptor causes a reduction in the number of persistent cell corpses in worms suffering from an engulfment deficiency. This reduction is not caused by impaired or delayed cell death but rather by a partial restoration of the cell clearance capacity. Mutants in acetylcholine turn-over elicit a similar phenotype, implying that acetylcholine signaling is the process responsible for these observations. Surprisingly, tissue specific RNAi suggests that UNC-38, a major component of the levamisole-sensitive receptor, functions in the dying germ cell to influence engulfment efficiency. Animals with loss of acetylcholine receptor exhibit a higher fraction of cell corpses positive for the "eat-me" signal phosphatidylserine. Our results suggest that modulation by ion channels of ion flow across plasma membrane in dying cells can influence the dynamics of phosphatidylserine exposure and thus clearance efficiency.

  13. Cognitive effects of dopamine depletion in the context of diminished acetylcholine signaling capacity in mice

    Lilia Zurkovsky

    2013-01-01

    A subset of patients with Parkinson’s disease acquires a debilitating dementia characterized by severe cognitive impairments (i.e. Parkinson’s disease dementia; PDD. Brains from PDD patients show extensive cholinergic loss as well as dopamine (DA depletion. We used a mutant mouse model to directly test whether combined cholinergic and DA depletion leads to a cognitive profile resembling PDD. Mice carrying heterozygous deletion of the high-affinity, hemicholinium-3-sensitive choline transporter (CHTHET show reduced levels of acetylcholine throughout the brain. We achieved bilateral DA depletion in CHTHET and wild-type (WT littermates via intra-striatal infusion of 6-hydroxydopamine (6-OHDA, or used vehicle as control. Executive function and memory were evaluated using rodent versions of cognitive tasks commonly used with human subjects: the set-shifting task and spatial and novel-object recognition paradigms. Our studies revealed impaired acquisition of attentional set in the set-shifting paradigm in WT-6OHDA and CHTHET-vehicle mice that was exacerbated in the CHTHET-6OHDA mice. The object recognition test following a 24-hour delay was also impaired in CHTHET-6OHDA mice compared with all other groups. Treatment with acetylcholinesterase (AChE inhibitors physostigmine (0.05 or 0.1 mg/kg and donepezil (0.1 and 0.3 mg/kg reversed the impaired object recognition of the CHTHET-6OHDA mice. Our data demonstrate an exacerbated cognitive phenotype with dual ACh and DA depletion as compared with either insult alone, with traits analogous to those observed in PDD patients. The results suggest that combined loss of DA and ACh could be sufficient for pathogenesis of specific cognitive deficits in PDD.

  14. Evaluation of PET Radioligands for the neuronal nicotinic acetylcholine receptor

    Full text: A-186253.1, a compound made by Abbott laboratories, was labelled with carbon-11 and evaluated as a PET ligand for the neuronal nicotinic acetylcholine receptor (nAChR). The compound was labelled with C-11 by methylation with 11C-MeI of the desmethyl precursor A-183828.1. The affinity of A-186253.1 for the α4β2 and the α7 subtype of the nAChR was determined in displacement studies. PET-studies were performed in rats and pigs Inhibitory constants (Ki) versus cytsine were 461 ± 99 pM for A-186253.1 and versus α-Bungarotoxin >100 μM. which means a very high selectivity for the α4β2-receptor (>227,000). Highest uptake of [11C]-A-186253.1 was observed in the thalamus where an increase in radiotracer uptake was seen until 45 min p.i.. Thereafter, the radiotracer concentration remained constant until the end of the scan indicating slow washout of [11C]-A-186253.1. Application of cold A-186253.1 (0.5 mg/kg) 40 min p.i. resulted in a decrease in radiotracer concentration in the thalamus and the cortex indicating displacement of [11C]-A-186253.1. Blockade studies with cytisine (0.5 mg/kg), a selective ligand for the α4β2 nicotinic receptor, showed just a slight reduction of the radioligand uptake in the thalamus and in the cortex whereas the blockade with cold A-186253.1 (1 mg/kg) resulted in a 50 % reduction. These results suggest, that 50 % of the [11C]-A-186253.1 in the brain corresponds to specifically bound radioligand, but not to the α4β2 subtype of the nicotinic receptor. (author)

  15. Acetylcholine receptors in dementia and mild cognitive impairment

    Sabri, Osama; Kendziorra, Kai [University of Leipzig, Department of Nuclear Medicine, Leipzig (Germany); Wolf, Henrike; Gertz, Hermann-Josef [University of Leipzig, Department of Psychiatry, Leipzig (Germany); Brust, Peter [Institute of Interdisciplinary Isotope Research, Leipzig (Germany)

    2008-03-15

    To clarify whether changes in the cholinergic transmission occur early in the course of Alzheimer's disease (AD), we carried out positron emission tomography (PET) with the radioligand 2-[{sup 18}F]F-A-85380, which is supposed to be specific for {alpha}4{beta}2 nicotinic acetylcholine receptors (nAChRs). We included patients with moderate to severe AD and patients with amnestic mild cognitive impairment (MCI), presumed to present preclinical AD. Both patients with AD and MCI showed significant reductions in {alpha}4{beta}2 nAChRs in brain regions typically affected by AD pathology. These findings indicate that a reduction in {alpha}4{beta}2 nAChRs occurs during early symptomatic stages of AD. The {alpha}4{beta}2 nAChR availability in these regions correlated with the severity of cognitive impairment, indicating a stage sensitivity of the {alpha}4{beta}2 nAChR status. Together, our results provide evidence for the potential of 2-[{sup 18}]F-A-85380 nAChR PET in the diagnosis of patients at risk for AD. Because of the extraordinary long acquisition time with 2-[{sup 18}F]F-A-85380, we developed the new {alpha}4{beta}2 nAChR-specific radioligands (+)- and (-)-[{sup 18}F]norchloro-fluoro-homoepibatidine (NCFHEB) and evaluated them preclinically. (-)-[{sup 18}F]NCFHEB shows twofold higher brain uptake and significantly shorter acquisition times. Therefore, (-)-[{sup 18}F]NCFHEB should be a suitable radioligand for larger clinical investigations. (orig.)

  16. Acetylcholine receptors in dementia and mild cognitive impairment

    To clarify whether changes in the cholinergic transmission occur early in the course of Alzheimer's disease (AD), we carried out positron emission tomography (PET) with the radioligand 2-[18F]F-A-85380, which is supposed to be specific for α4β2 nicotinic acetylcholine receptors (nAChRs). We included patients with moderate to severe AD and patients with amnestic mild cognitive impairment (MCI), presumed to present preclinical AD. Both patients with AD and MCI showed significant reductions in α4β2 nAChRs in brain regions typically affected by AD pathology. These findings indicate that a reduction in α4β2 nAChRs occurs during early symptomatic stages of AD. The α4β2 nAChR availability in these regions correlated with the severity of cognitive impairment, indicating a stage sensitivity of the α4β2 nAChR status. Together, our results provide evidence for the potential of 2-[18]F-A-85380 nAChR PET in the diagnosis of patients at risk for AD. Because of the extraordinary long acquisition time with 2-[18F]F-A-85380, we developed the new α4β2 nAChR-specific radioligands (+)- and (-)-[18F]norchloro-fluoro-homoepibatidine (NCFHEB) and evaluated them preclinically. (-)-[18F]NCFHEB shows twofold higher brain uptake and significantly shorter acquisition times. Therefore, (-)-[18F]NCFHEB should be a suitable radioligand for larger clinical investigations. (orig.)

  17. A reactive transport model for the geochemical response, detection and potential mitigation of CO2 leakage into a confined aquifer

    Maher, K.; Druhan, J. L.; Vialle, S.; Benson, S. M.; Agarwal, A.

    2013-12-01

    Long-term storage of anthropogenic CO2 in the subsurface generally assumes that caprock formations will serve as physical barriers to upward migration of CO2. Stability and coherence of the caprocks are thus important criteria for site selection, but caprock integritycannot be guaranteed with total certainty over the lifetime of the project. As a result, carbon capture and storage projects require reliable techniques to monitor geologic storage sites for newly formed leaks, and the ability to rapidly deploy mitigation measures should leakage occur. Here, we present two-dimensional reactive transport simulations to evaluate the hydrogeochemical characteristics of a newly formed CO2 leak into an overlying reservoir. Simulations use the ToughReact multi-component reactive transport code and hypothetical reservoir characteristics. We focus on the comparatively short time period of days to months following formation of the leak to consider (1) geochemical shifts in formation water indicative of the leak, (2) hydrodynamics of pumping wells in the vicinity of the leak, and (3) delivery of a sealant to the leak through an adjacent well bore. Our results suggest that characteristic shifts in pH and dissolved inorganic carbon might be detected in down-gradient mentoring wells prior to the breakthrough of CO2, and could offer a potential means of identifying small and newly formed leaks. Injecting water into the aquifer through pumping wells in the vicinity of the leak provides a hydrodynamic control that can prevent CO2 from reaching the top of the reservoir, but this action will likely have only minor influence on the rate of leakage through the caprock defect. Injection of a hypothetical sealant through an adjacent pumping well is considered using an aqueous solute with pH-dependent equilibrium constraints such that the species is soluble in the basic pH range but forms a precipitate at neutral to acidic pH conditions associated with CO2-rich water. Injection of this

  18. Insensitive Acetylcholine Receptor Conferring Resistance of Plutella xylostella to Nereistoxin Insecticides

    CHENG Luo-gen; YU Guang; CHEN Zi-hao; LI Zhong-yin

    2008-01-01

    The combinative rate measurement of (3-[Ⅰ125] iodotyrosyl) α-bungarotoxin was applied in the analysis of the relation between nerve acetylcholine receptor and three types of insecticide resistance in diamondback moth, Plutella xylostella (L.). In the dimehypo-resistant strain and in the cartap-resistant strain, the nerve acetylcholine receptor showed the remarkable insensitivity to dimehypo and cartap, of which the binding rate to ligand was approximately 66 and 60%, respectively, of the susceptible strain. The sensitivity to deltamethrin in the deltamethrin-resistant strain did not show visible change. These results indicated that the decline in the sensitivity of nerve acetylcholine receptor to insecticide might be a potential mechanism to nereistoxin insecticides resistance in the diamondback moth.

  19. Prejunctional inhibition of norepinephrine release caused by acetylcholine in the human saphenous vein

    We performed experiments to determine whether or not acetylcholine exerts a prejunctional inhibitory effect on adrenergic neurotransmission in the human blood vessel wall. Rings of human greater saphenous veins were prepared 2 to 15 hours after death and mounted for isometric tension recording in organ chambers filled with Krebs-Ringer solution. Acetylcholine depressed contractile responses to electric activation of the sympathetic nerve endings significantly more than those to exogenous norepinephrine; the relaxations caused by the cholinergic transmitter were antagonized by atropine. Helical strips were incubated with [/sub 3/H]norepinephrine and mounted for superfusion. Electric stimulation augmented the fractional release of labeled norepinephrine. Acetylcholine caused a depression of the evoked /sub 3/H release which was antagonized by atropine but not by hexamethonium. These experiments demonstrate that, as in animal cutaneous veins, there are prejunctional inhibitory muscarinic receptors on the adrenergic nerve endings in the human saphenous vein. By contrast, the human vein also contains postjunctional inhibitory muscarinic receptors

  20. Reduced acetylcholine synthesis in Alzheimer's disease is a clinically relevant change

    This paper presents a study of patients in the presenium. Psychological assessment was carried out to provide measures of relative severity of dementia, with which pathological and chemical indices of impairment could be compared. Fresh cortical biopsy tissue permitted the assay of ChAT activity, and also the determination of acetylcholine synthesis in a preparation enriched in cortical synaptosomes. Additionally, choline uptake has been measured for comparison. Nuerosurgical samples from all patients were handled and processed for measuring acetylcholine synthesis by incorporation of U-14C-glucoase into C 14-acetylcholine in neocortical tissue prisms. Cortical sections from most of the patients were found on light microscopy to contain the characteristic changes of Alzheimer's disease

  1. Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

    L.F.S. Sampaio

    2005-04-01

    Full Text Available The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5, while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM blocked the response to acetylcholine (3.0 nM-2.0 µM only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

  2. [Intern(euron)al affairs : The role of specific neocortical interneuron classes in the interaction between acetylcholine and GABAergic anesthetics].

    Liebig, L; Grasshoff, C; Hentschke, H

    2016-08-01

    Acetylcholine is a neuromodulator which is released throughout the central nervous system and plays an essential role in consciousness and cognitive processes including attention and learning. Due to its 'activating' effect on the neuronal and behavioral level its interaction with anesthetics has long been of interest to anesthesiologists. It is widely held that a reduction of the release of acetylcholine by general anesthetics constitutes part of the anesthetic effect. This notion is backed by numerous human and animal studies, but is also in seeming contradiction to findings that acetylcholine activates specific classes of inhibitory neurons: if acetylcholine excites elements within the neuronal network responsible for the release of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), its withdrawal should diminish, not enhance, the effect of anesthetics.Focusing on cortical circuits, we present an overview of recent advances in cellular neurophysiology, particularly the interactions between inhibitory neuron classes, which provide insights on the interaction between acetylcholine and GABA. PMID:27380048

  3. Autophagic flux data in differentiated C2C12 myotubes following exposure to acetylcholine and caffeine

    Darin Bloemberg

    2016-06-01

    Full Text Available The C2C12 line of mouse myoblasts is a useful cell culture model in which to conduct in vitro analyses related to skeletal muscle. Here we present data regarding the autophagic response induced by two chemicals known to influence calcium release and contraction in skeletal muscles and C2C12 cells: acetylcholine and caffeine. More specifically, by concurrently administering acetylcholine or caffeine along with chloroquine to differentiated myotubes for various amounts of time and assessing the protein expression of LC3 and p62, we report data on the relative level of autophagic flux induced by these two calcium- and contraction-regulating chemicals.

  4. Enhanced self-administration of alcohol in muscarinic acetylcholine M4 receptor knockout mice

    de la Cour, Cecilie; Sørensen, Gunnar; Wörtwein, Gitta;

    2015-01-01

    Modulation of cholinergic neurotransmission via nicotinic acetylcholine receptors is known to alter alcohol-drinking behavior. It is not known if muscarinic acetylcholine receptor subtypes have similar effects. The muscarinic M4 receptor is highly expressed in the brain reinforcement system and i......4+/+ littermates. The highest alcohol concentration used (10%) did not immediately result in divergent drinking patterns, but after 4 weeks of 10% alcohol self-administration, baseline levels as well as a pattern of M4-/- mice consuming more alcohol than their M4+/+ controls were re...... as a potential target for pharmacological (positive allosteric modulators or future agonists) treatment of alcohol use disorders....

  5. Increases in muscle Ca2+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction.

    Rubin, L L

    1985-01-01

    The synthesis of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) and of acetylcholine receptors (AcChoR) by cultured rat muscle fibers is influenced strongly by the level of muscle contractile activity. If fibers are grown in the presence of tetrodotoxin (TTX) to block spontaneous contraction, the total amount of AcChoE decreases markedly, as does the percentage of AcChoE assembled as the collagen-tailed presumed synaptic form of the enzyme. Under these conditions, ho...

  6. Inhibition of cortical acetylcholine release and cognitive performance by histamine H3 receptor activation in rats.

    Blandina, P.; Giorgetti, M.; L. Bartolini; M.Cecchi; Timmerman, H.; Leurs, R.; Pepeu, G; Giovannini, M. G.

    1996-01-01

    1. The effects of histamine and agents at histamine receptors on spontaneous and 100 mM K(+)-evoked release of acetylcholine, measured by microdialysis from the cortex of freely moving, rats, and on cognitive tests are described. 2. Local administration of histamine (0.1-100 microM) failed to affect spontaneous but inhibited 100 mM K(+)-stimulated release of acetylcholine up to about 50%. The H3 receptor agonists (R)-alpha-methylhistamine (RAMH) (0.1-10 microM), imetit (0.01-10 microM) and im...

  7. Autophagic flux data in differentiated C2C12 myotubes following exposure to acetylcholine and caffeine.

    Bloemberg, Darin; Quadrilatero, Joe

    2016-06-01

    The C2C12 line of mouse myoblasts is a useful cell culture model in which to conduct in vitro analyses related to skeletal muscle. Here we present data regarding the autophagic response induced by two chemicals known to influence calcium release and contraction in skeletal muscles and C2C12 cells: acetylcholine and caffeine. More specifically, by concurrently administering acetylcholine or caffeine along with chloroquine to differentiated myotubes for various amounts of time and assessing the protein expression of LC3 and p62, we report data on the relative level of autophagic flux induced by these two calcium- and contraction-regulating chemicals. PMID:27054179

  8. Interaction of ibogaine with human alpha3beta4-nicotinic acetylcholine receptors in different conformational states.

    Arias, Hugo R; Rosenberg, Avraham; Targowska-Duda, Katarzyna M; Feuerbach, Dominik; Yuan, Xiao Juan; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W

    2010-09-01

    The interaction of ibogaine and phencyclidine (PCP) with human (h) alpha3beta4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca2+ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (+/-)-epibatidine-induced Ca2+ influx in h(alpha)3beta4 AChRs with approximately 9-fold higher potency than that for PCP, (b) [3H]ibogaine binds to a single site in the h(alpha)3beta4 AChR ion channel with relatively high affinity (Kd = 0.46 +/- 0.06 microM), and ibogaine inhibits [3H]ibogaine binding to the desensitized h(alpha)3beta4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [3H]ibogaine binding to the h(alpha)3beta4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6') and valine/phenylalanine (position 13') rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time. PMID:20684041

  9. Interaction of ibogaine with human α3β4-nicotinic acetylcholine receptors in different conformational states

    Arias, Hugo R.; Rosenberg, Avraham; Targowska-Duda, Katarzyna M.; Feuerbach, Dominik; Yuan, Xiao Juan; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W.

    2015-01-01

    The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca2+ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (±)-epibatidine-induced Ca2+ influx in hα3β4 AChRs with ~9-fold higher potency than that for PCP, (b) [3H]ibogaine binds to a single site in the hα3β4 AChR ion channel with relatively high affinity (Kd = 0.46 ± 0.06 µM), and ibogaine inhibits [3H]ibogaine binding to the desensitized hα3β4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [3H]ibogaine binding to the hα3β4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6′) and valine/phenylalanine (position 13′) rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time. PMID:20684041

  10. Transient Receptor Potential Channel Opening Releases Endogenous Acetylcholine, which Contributes to Endothelium-Dependent Relaxation Induced by Mild Hypothermia in Spontaneously Hypertensive Rat but Not Wistar-Kyoto Rat Arteries.

    Zou, Q; Leung, S W S; Vanhoutte, P M

    2015-08-01

    Mild hypothermia causes endothelium-dependent relaxations, which are reduced by the muscarinic receptor antagonist atropine. The present study investigated whether endothelial endogenous acetylcholine contributes to these relaxations. Aortic rings of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats were contracted with prostaglandin F2 α and exposed to progressive mild hypothermia (from 37 to 31°C). Hypothermia induced endothelium-dependent, Nω-nitro-l-arginine methyl ester-sensitive relaxations, which were reduced by atropine, but not by mecamylamine, in SHR but not in WKY rat aortae. The responses in SHR aortae were also reduced by acetylcholinesterase (the enzyme responsible for acetylcholine degradation), bromoacetylcholine (inhibitor of acetylcholine synthesis), hemicholinium-3 (inhibitor of choline uptake), and vesamicol (inhibitor of acetylcholine release). The mild hypothermia-induced relaxations in both SHR and WKY rat aortae were inhibited by AMTB [N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide; the transient receptor potential (TRP) M8 inhibitor]; only those in SHR aortae were inhibited by HC-067047 [2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide; TRPV4 antagonist] while those in WKY rat aortae were reduced by HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; TRPA1 antagonist]. The endothelial uptake of extracellular choline and release of cyclic guanosine monophosphate was enhanced by mild hypothermia and inhibited by HC-067047 in SHR but not in WKY rat aortae. Compared with WKY rats, the SHR preparations expressed similar levels of acetylcholinesterase and choline acetyltransferase, but a lesser amount of vesicular acetylcholine transporter, located mainly in the endothelium. Thus, mild hypothermia causes nitric oxide-dependent relaxations by opening TRPA1 channels in WKY rat aortae

  11. [{sup 123}I]-3-Iodcytisin as possible radiotracer for the imaging of nicotinic acetylcholine receptors using single photon emission computer tomography; [{sup 123}I]-3-Iodcytisin als moeglicher Radiotracer fuer die Darstellung der nikotinergen Acetylcholin Rezeptoren mittels Single-Photon-Emissions-Computertomographie

    Paulik, Dagmar Julia

    2015-03-06

    For the synthesis of [{sup 123}I]-3-Iodcytisin as possible radiotracer for the imaging of nicotinic acetylcholine (nACh) receptors using SPECT two different technologies were used: the radio-iodination with iodogen and the radio-iodination with nitric acid. The latter one showed higher efficiency. The radiotracer will allow to detect degenerative processes and other nACh-depending diseases in the brain (Alzheimer, Parkinson) and to observe the progress. The autoradiography is aimed to the imaging of the nACh receptors in the brain bypassing the brain-blood barrier. The highest activity was measured in the thalamus of mice and rat brains.

  12. A shallow subsurface controlled release facility in Bozeman, Montana, USA, for testing near surface CO2 detection techniques and transport models

    Spangler, Lee H. [Montana State Univ., Bozeman, MT (United States). Dept. of Chemistry and Biochemistry; Dobeck, Laura M. [Montana State Univ., Bozeman, MT (United States). Dept. of Chemistry and Biochemistry; Repasky, Kevin S. [Montana State Univ., Bozeman, MT (United States). Dept. of Electrical and Computer Engineering; Nehrir, Amin R. [Montana State Univ., Bozeman, MT (United States). Dept. of Electrical and Computer Engineering; Humphries, Seth D. [Montana State Univ., Bozeman, MT (United States). Dept. of Electrical and Computer Engineering; Barr, Jamie L. [Montana State Univ., Bozeman, MT (United States). Dept. of Electrical and Computer Engineering; Keith, Charlie J. [Montana State Univ., Bozeman, MT (United States). Dept. of Electrical and Computer Engineering; Shaw, Joseph A. [Montana State Univ., Bozeman, MT (United States). Dept. of Electrical and Computer Engineering; Rouse, Joshua H. [Montana State Univ., Bozeman, MT (United States). Dept. of Electrical and Computer Engineering; Cunningham, Alfred B. [Montana State Univ., Bozeman, MT (United States). Dept. of Civil Engineering; Benson, Sally M. [Stanford Univ., CA (United States). Global Climate and Energy Project; Oldenburg, Curtis M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Div.; Lewicki, Jennifer L. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Div.; Wells, Arthur W. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Div.; Diehl, J. Rodney [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Div.; Strazisar, Brian R. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Div.; Fessenden, Julianna E. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Div. of Earth and Environmental Sciences; Rahn, Thom A. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Div. of Earth and Environmental Sciences; Amonette, James E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Barr, Jon L. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Pickles, William L. [Univ. of California, Santa Cruz, CA (United States). Earth and Planetary Sciences; Jacobson, James D. [Univ. of California, Santa Cruz, CA (United States). Earth and Planetary Sciences; Silver, Eli A. [Univ. of California, Santa Cruz, CA (United States). Earth and Planetary Sciences; Male, Erin J. [Univ. of California, Santa Cruz, CA (United States). Earth and Planetary Sciences; Rauch, Henry W. [Univ. of West Virginia, Morgantown, WV (United States). Dept. of Geology and Geography; Gullickson, Kadie S. [Montana State Univ., Bozeman, MT (United States). Dept. of Chemistry and Biochemistry; Trautz, Robert [Electric Power Research Inst. (EPRI), Palo Alto, CA (United States); Kharaka, Yousif [U.S. Geological Survey, Menlo Park, CA (United States); Birkholzer, Jens [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Div.; Wielopolski, Lucien [Brookhaven National Lab. (BNL), Upton, NY (United States)

    2010-03-01

    A controlled field pilot has been developed in Bozeman, Montana, USA, to study near surface CO2 transport and detection technologies. A slotted horizontal well divided into six zones was installed in the shallow subsurface. The scale and CO2 release rates were chosen to be relevant to developing monitoring strategies for geological carbon storage. The field site was characterized before injection, and CO2 transport and concentrations in saturated soil and the vadose zone were modeled. Controlled releases of CO2 from the horizontal well were performed in the summers of 2007 and 2008, and collaborators from six national labs, three universities, and the U. S. Geological Survey investigated movement of CO2 through the soil, water, plants, and air with a wide range of near surface detection techniques. An overview of these results will be presented.

  13. A shallow subsurface controlled release facility in Bozeman, Montana, USA, for testing near surface CO2 detection techniques and transport models

    Spangler, L.H.; Dobeck, L.M.; Nehrir, A.; Humphries, S.; Barr, J.; Keith, C.; Shaw, J.; Rouse, J.; Cunningham, A.; Benson, S.; Repasky, K.S.; Lewicki, J.; Wells, A.; Diehl, R.; Strazisar, B.; Fessenden, J.; Rahn, T.; Amonette, J.; Barr, J.; Pickles, W.; Jacobson, J.; Silver, E.; Male, E.; Rauch, H.; Gullickson, K.; Trautz, R.; Kharaka, Y.; Birkholzer, J.T.; Wielopolski, L.; Oldenburg, C.M.

    2009-10-20

    A controlled field pilot has been developed in Bozeman, Montana, USA, to study near surface CO2 transport and detection technologies. A slotted horizontal well divided into six zones was installed in the shallow subsurface. The scale and CO2 release rates were chosen to be relevant to developing monitoring strategies for geological carbon storage. The field site was characterized before injection, and CO2 transport and concentrations in saturated soil and the vadose zone were modeled. Controlled releases of CO2 from the horizontal well were performed in the summers of 2007 and 2008, and collaborators from six national labs, three universities, and the U.S. Geological Survey investigated movement of CO2 through the soil, water, plants, and air with a wide range of near surface detection techniques. An overview of these results will be presented.

  14. A shallow subsurface controlled release facility in Bozeman, Montana, USA, for testing near surface CO2 detection techniques and transport models

    Spangler, L.H.; Dobeck, L.M.; Repasky, K.S.; Nehrir, A.R.; Humphries, S.D.; Keith, C.J.; Shaw, J.A.; Rouse, J.H.; Cunningham, A.B.; Benson, S.M.; Oldenburg, C.M.; Lewicki, J.L.; Wells, A.W.; Diehl, J.R.; Strazisar, B.R.; Fessenden, J.E.; Rahn, T.A.; Amonette, J.E.; Barr, J.L.; Pickles, W.L.; Jacobson, J.D.; Silver, E.A.; Male, E.J.; Rauch, H.W.; Gullickson, K.S.; Trautz, R.; Kharaka, Y.; Birkholzer, J.; Wielopolski, L.

    2010-01-01

    A controlled field pilot has been developed in Bozeman, Montana, USA, to study near surface CO2 transport and detection technologies. A slotted horizontal well divided into six zones was installed in the shallow subsurface. The scale and CO2 release rates were chosen to be relevant to developing monitoring strategies for geological carbon storage. The field site was characterized before injection, and CO2 transport and concentrations in saturated soil and the vadose zone were modeled. Controlled releases of CO2 from the horizontal well were performed in the summers of 2007 and 2008, and collaborators from six national labs, three universities, and the U.S. Geological Survey investigated movement of CO2 through the soil, water, plants, and air with a wide range of near surface detection techniques. An overview of these results will be presented. ?? 2009 The Author(s).

  15. Exocrine secretion of epidermal growth factor from Brunner's glands. Stimulation by VIP and acetylcholine

    Poulsen, Steen Seier

    1983-01-01

    Brunner's glands of the duodenum are innervated by cholinergic and VIP-ergic nerves, and the glands have been shown to contain epidermal growth factor (EGF). In this study the effect of VIP and acetylcholine (Ach) on secretion of EGF from Brunner's glands was investigated in the rat. Intravenous ...

  16. Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.

    Elise Courtot

    2015-12-01

    Full Text Available Acetylcholine receptors are pentameric ligand-gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.

  17. Functional Characterization of CCHamide and Muscarinic Acetylcholine Receptor Signalling in Drosophila melanogaster

    Ren, Guilin Robin

    mutants created with the CRISP/Cas9 technique showed thatCCHamide-2 is probly an orexigenic peptide and also that is an important factor for larvaldevelopmental timing.In mammals, muscarinic acetylcholine signalling is involved in the signal transmission of theparasympathetic nervous system. However...

  18. Characterization of the positive and negative inotropic effects of acetylcholine in the human myocardium

    X.Y. Du (Xiaoyi); R.G. Schoemaker (Regien); E. Bos (Egbert); P.R. Saxena (Pramod Ranjan)

    1995-01-01

    textabstractIn the human isolated myocardium, acetylcholine (10−9 to 10−3 M) elicited a biphasic inotropic effect (a decrease in the lower and an increase in the higher concentration range) in atrial and a positive inotropic effect in ventricular trabeculae. However, under conditions of raised contr

  19. MUSCARINIC ACETYLCHOLINE RECEPTOR-EXPRESSION IN ASTROCYTES IN THE CORTEX OF YOUNG AND AGED RATS

    VANDERZEE, EA; DEJONG, GI; STROSBERG, AD; LUITEN, PGM

    1993-01-01

    The present report describes the cellular and subcellular distribution pattern of immunoreactivity to M35, a monoclonal antibody raised against purified muscarinic acetylcholine receptor protein, in astrocytes in the cerebral cortex of young and aged rats. Most M35-positive astrocytes were localized

  20. Synthesis and pharmacological evaluation of DHβE analogs as neuronal nicotinic acetylcholine receptor antagonists

    Jepsen, Tue H.; Jensen, Anders A.; Lund, Mads Henrik; Glibstrup, Emil; Kristensen, Jesper Langgaard

    2014-01-01

    Dihydro-β-erythroidine (DHβE) is a member of the Erythrina family of alkaloids and a potent competitive antagonist of the α4β2-subtype of the nicotinic acetylcholine receptors (nAChRs). Guided by an X-ray structure of DHβE in complex with an ACh binding protein, we detail the design, synthesis, and...

  1. Tying up Nicotine: New Selective Competitive Antagonist of the Neuronal Nicotinic Acetylcholine Receptors

    Petersen, Ida Nymann; Crestey, François; Jensen, Anders A; Indurthi, Dinesh C; Pedersen, Henrik; Andreasen, Jesper T; Balle, Thomas; Kristensen, Jesper L

    2015-01-01

    Conformational restriction of the pyrrolidine nitrogen in nicotine by the introduction of an ethylene bridge provided a potent and selective antagonist of the α4β2-subtype of the nicotinic acetylcholine receptors. Resolution by chiral SFC, pharmacological characterization of the two enantiomers...

  2. Distinct neural pathways mediate alpha7 nicotinic acetylcholine receptor-dependent activation of the forebrain

    Thomsen, Morten S; Hay-Schmidt, Anders; Hansen, Henrik H; Mikkelsen, Jens D

    2010-01-01

    alpha(7) nicotinic acetylcholine receptor (nAChR) agonists are candidates for the treatment of cognitive deficits in schizophrenia. Selective alpha(7) nAChR agonists, such as SSR180711, activate neurons in the medial prefrontal cortex (mPFC) and nucleus accumbens shell (ACCshell) in rats, regions...

  3. alpha(7) Nicotinic acetylcholine receptor activation prevents behavioral and molecular changes induced by repeated phencyclidine treatment

    Thomsen, Morten Skøtt; Christensen, Ditte Z; Hansen, Henrik H; Redrobe, John P; Mikkelsen, Jens D

    determined in a modified Y-maze test. Polymorphisms in the alpha(7) nicotinic acetylcholine receptor (nAChR) gene have been linked to schizophrenia. Here we demonstrate that acute administration of the selective alpha(7) nAChR partial agonist SSR180711 dose-dependently reversed the behavioral impairment...

  4. Apolipoprotein E4 reduces evoked hippocampal acetylcholine release in adult mice

    Dolejší, Eva; Liraz, O.; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; Michaelson, D. M.

    Roč. 136, č. 3 ( 2016 ), s. 503-509. ISSN 0022-3042 R&D Projects: GA MŠk(CZ) LH13269 Institutional support: RVO:67985823 Keywords : acetylcholine release * Alzheimer's disease (AD) * apolipoprotein E4 (apoE4) * hippocampus Subject RIV: FH - Neurology Impact factor: 4.281, year: 2014

  5. Acetylcholine Release in the Hippocampus and Striatum during Place and Response Training

    Pych, Jason C.; Chang, Qing; Colon-Rivera, Cynthia; Haag, Renee; Gold, Paul E.

    2005-01-01

    These experiments examined the release of acetylcholine in the hippocampus and striatum when rats were trained, within single sessions, on place or response versions of food-rewarded mazes. Microdialysis samples of extra-cellular fluid were collected from the hippocampus and striatum at 5-min increments before, during, and after training. These…

  6. Theoretical investigation of interaction between the set of ligands and α7 nicotinic acetylcholine receptor

    Glukhova, O. E.; Prytkova, T. R.; Shmygin, D. S.

    2016-03-01

    Nicotinic acetylcholine receptors (nAChRs) are neuron receptor proteins that provide a transmission of nerve impulse through the synapses. They are composed of a pentametric assembly of five homologous subunits (5 α7 subunits for α7nAChR, for example), oriented around the central pore. These receptors might be found in the chemical synapses of central and peripheral nervous system, and also in the neuromuscular synapses. Transmembrane domain of the one of such receptors constitutes ion channel. The conductive properties of ion channel strongly depend on the receptor conformation changes in the response of binding with some molecule, f.e. acetylcholine. Investigation of interaction between ligands and acetylcholine receptor is important for drug design. In this work we investigate theoretically the interaction between the set of different ligands (such as vanillin, thymoquinone, etc.) and the nicotinic acetylcholine receptor (primarily with subunit of the α7nAChR) by different methods and packages (AutodockVina, GROMACS, KVAZAR, HARLEM, VMD). We calculate interaction energy between different ligands in the subunit using molecular dynamics. On the base of obtained calculation results and using molecular docking we found an optimal location of different ligands in the subunit.

  7. Mechanisms of the inhibition of endplate acetylcholine receptors by antiseptic chlorhexidine (experiments and models)

    Shaihutdinova, A.R.; Nikolsky, E. E.; Vyskočil, František; Skorinkin, A.I.

    2009-01-01

    Roč. 380, č. 6 (2009), s. 551-560. ISSN 0028-1298 R&D Projects: GA AV ČR(CZ) IAA500110905 Institutional research plan: CEZ:AV0Z50110509 Keywords : acetylcholine * endplate currents Subject RIV: ED - Physiology Impact factor: 2.631, year: 2009

  8. VISUALIZATION OF CHOLINOCEPTIVE NEURONS IN THE RAT NEOCORTEX - COLOCALIZATION OF MUSCARINIC AND NICOTINIC ACETYLCHOLINE-RECEPTORS

    VANDERZEE, EA; STREEFLAND, C; STROSBERG, AD; SCHRODER, H; LUITEN, PGM

    1992-01-01

    The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluores

  9. Visualization of cholinoceptive neurons in the rat neocortex : colocalization of muscarinic and nicotinic acetylcholine receptors

    Zee, E.A. van der; Streefland, C.; Strosberg, A.D.; Schröder, H.; Luiten, P.G.M.

    1992-01-01

    The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluores

  10. ATP–Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening

    Iram, Surtaj H.; Gruber, Simon J.; Raguimova, Olga N.; Thomas, David D.; Seth L Robia

    2015-01-01

    Multidrug resistance protein 1 (MRP1) actively transports a wide variety of drugs out of cells. To quantify MRP1 structural dynamics, we engineered a “two-color MRP1” construct by fusing green fluorescent protein (GFP) and TagRFP to MRP1 nucleotide–binding domains NBD1 and NBD2, respectively. The recombinant MRP1 protein expressed and trafficked normally to the plasma membrane. Two-color MRP1 transport activity was normal, as shown by vesicular transport of [3H]17β-estradiol-17-β-(d-glucuroni...

  11. Concomitant release of ventral tegmental acetylcholine and accumbal dopamine by ghrelin in rats.

    Elisabet Jerlhag

    Full Text Available Ghrelin, an orexigenic peptide, regulates energy balance specifically via hypothalamic circuits. Growing evidence suggest that ghrelin increases the incentive value of motivated behaviours via activation of the cholinergic-dopaminergic reward link. It encompasses the cholinergic afferent projection from the laterodorsal tegmental area (LDTg to the dopaminergic cells of the ventral tegmental area (VTA and the mesolimbic dopamine system projecting from the VTA to nucleus accumbens (N.Acc.. Ghrelin receptors (GHS-R1A are expressed in these reward nodes and ghrelin administration into the LDTg increases accumbal dopamine, an effect involving nicotinic acetylcholine receptors in the VTA. The present series of experiments were undertaken directly to test this hypothesis. Here we show that ghrelin, administered peripherally or locally into the LDTg concomitantly increases ventral tegmental acetylcholine as well as accumbal dopamine release. A GHS-R1A antagonist blocks this synchronous neurotransmitter release induced by peripheral ghrelin. In addition, local perfusion of the unselective nicotinic antagonist mecamylamine into the VTA blocks the ability of ghrelin (administered into the LDTg to increase N.Acc.-dopamine, but not VTA-acetylcholine. Collectively our data indicate that ghrelin activates the LDTg causing a release of acetylcholine in the VTA, which in turn activates local nicotinic acetylcholine receptors causing a release of accumbal dopamine. Given that a dysfunction in the cholinergic-dopaminergic reward system is involved in addictive behaviours, including compulsive overeating and alcohol use disorder, and that hyperghrelinemia is associated with such addictive behaviours, ghrelin-responsive circuits may serve as a novel pharmacological target for treatment of alcohol use disorder as well as binge eating.

  12. A shallow subsurface controlled release facility in Bozeman, Montana, USA, for testing near surface CO2 detection techniques and transport models

    Spangler, Lee H.; Dobeck, Laura M.; Repasky, Kevin S.; Nehrir, Amin R.; Humphries, Seth D.; Barr, Jamie L.; Keith, Charlie J.; Shaw, Joseph A.; Rouse, Joshua H.; Cunningham, Alfred B.; Benson, Sally M.; Oldenburg, Curtis M.; Lewicki, Jennifer L.; Wells, Arthur W.; Diehl, J. R.; Strazisar, Brian; Fessenden, Julianna; Rahn, Thom A.; Amonette, James E.; Barr, Jonathan L.; Pickles, William L.; Jacobson, James D.; Silver, Eli A.; Male, Erin J.; Rauch, Henry W.; Gullickson, Kadie; Trautz, Robert; Kharaka, Yousif; Birkholzer, Jens; Wielopolski, Lucien

    2010-03-01

    A facility has been constructed to perform controlled shallow releases of CO2 at flow rates that challenge near surface detection techniques and can be scalable to desired retention rates of large scale CO2 storage projects. Preinjection measurements were made to determine background conditions and characterize natural variability at the site. Modeling of CO2 transport and concentration in saturated soil and the vadose zone was also performed to inform decisions about CO2 release rates and sampling strategies. Four releases of CO2 were carried out over the summer field seasons of 2007 and 2008. Transport of CO2 through soil, water, plants, and air was studied using near surface detection techniques. Soil CO2 flux, soil gas concentration, total carbon in soil, water chemistry, plant health, net CO2 flux, atmospheric CO2 concentration, movement of tracers, and stable isotope ratios were among the quantities measured. Even at relatively low fluxes, most techniques were able to detect elevated levels of CO2 in the soil, atmosphere, or water. Plant stress induced by CO2 was detectable above natural seasonal variations.

  13. Chronic nicotine modifies skeletal muscle Na,K-ATPase activity through its interaction with the nicotinic acetylcholine receptor and phospholemman.

    Alexander V Chibalin

    Full Text Available Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21-31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV while the activity of the α1 isoform decreased (-4.4 mV. Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM, measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser(63 and Ser(68. Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.

  14. Individual response speed is modulated by variants of the gene encoding the alpha 4 sub-unit of the nicotinic acetylcholine receptor (CHRNA4).

    Schneider, Katja Kerstin; Schote, Andrea B; Meyer, Jobst; Markett, Sebastian; Reuter, Martin; Frings, Christian

    2015-05-01

    Acetylcholine (ACh) is a known modulator of several domains of cognition, among them attention, memory and learning. The neurotransmitter also influences the speed of information processing, particularly the detection of targets and the selection of suitable responses. We examined the effect of the rs1044396 (C/T) polymorphism of the gene encoding the nicotinic acetylcholine receptor α4-subunit (CHRNA4) on response speed and selective visual attention. To this end, we administered a Stroop task, a Negative priming task and an exogenous Posner-Cuing task to healthy participants (n = 157). We found that the CHRNA4 rs1044396 polymorphism modulated the average reaction times (RTs) across all three tasks. Dependent on the C allele dosage, the RTs linearly increased. Homozygous T allele carriers were always fastest, while homozygous C allele carriers were always slowest. We did not observe effects of this polymorphism on selective attention. In sum, we conclude that naturally occurring variations within the cholinergic system influence an important factor of information processing. This effect might possibly be produced by the neuromodulator system rather than the deterministic system of cortical ACh. PMID:25639542

  15. Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine

    The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the γ-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist α-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effect on [125I]TID labeling of the AChR. The regions of the AChR α-subunit that incorporate [125]TID were mapped by Staphylococcus aureus V8 protest digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated [125I]TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the α-subunit amino terminus, incorporated no detectable [125I]TID. The mapping results place constraints on suggested models of AChR subunit topology

  16. ELISA detection of multixenobiotic resistance transporter induction in indigenous freshwater Chironomidae larvae (Diptera): A biomarker calibration step for in situ monitoring of xenobiotic exposure

    Moreau, X.; Saez, G.; Thiery, A. [Equipe ' Biomarqueurs and Bioindicateurs Environnementaux' , UMR-CNRS 6116 IMEP, Universite de Provence, 3 Place Victor Hugo, 13331 Marseille cedex 3 (France); Clot-Faybesse, O.; Guiraudie-Capraz, G. [' Neurobiologie Integrative et Adaptative' -UMR 6149, Universite de Provence, 3 Place Victor Hugo, 13331 Marseille cedex 3 (France); Bienboire-Frosini, C. [' Neurobiologie Integrative et Adaptative' -UMR 6149, Universite de Provence, 3 Place Victor Hugo, 13331 Marseille cedex 3 (France); Pherosynthese, Le Rieu Neuf, 84490 St Saturnin d' Apt (France); Martin, C. [Equipe ' Biomarqueurs and Bioindicateurs Environnementaux' , UMR-CNRS 6116 IMEP, UAPV, 33 rue Louis Pasteur, 84000 Avignon (France); De Jong, L. [Equipe ' Biomarqueurs and Bioindicateurs Environnementaux' , UMR-CNRS 6116 IMEP, Universite de Provence, 3 Place Victor Hugo, 13331 Marseille cedex 3 (France)], E-mail: laetitia.moreau@univ-provence.fr

    2008-06-15

    A new simple and sensitive method to distinguish chemically polluted from unpolluted situations in freshwater ecosystems is reported. For this purpose, Chironomus gr thumni larvae were collected from a polluted urban river downstream a sewage treatment plant. For the first time, ELISA assay was used to semi-quantify the multixenobiotic resistance transporters (MXR) in these small pertinent bioindicators. The use of samples immediately fixed in the field gives a delay to isolate larvae and allows multi-sampling along a longitudinal transect in a river at a given time. Results exhibit an induction of MXR proteins in larvae from the polluted river and a deinduction in larvae maintained 11 days in unpolluted water. They show new evidences to use midge larvae in biomonitoring environmental programs. They answer to first biomarker calibration steps for the ongoing development of MXR transporters as a detection tool of xenobiotic impacts on bioindicator invertebrates in their freshwater habitats. - Semi-quantification of midge larval MXR transporters by ELISA is a simple and sensitive method to detect chemically polluted situations in running freshwaters.

  17. ELISA detection of multixenobiotic resistance transporter induction in indigenous freshwater Chironomidae larvae (Diptera): A biomarker calibration step for in situ monitoring of xenobiotic exposure

    A new simple and sensitive method to distinguish chemically polluted from unpolluted situations in freshwater ecosystems is reported. For this purpose, Chironomus gr thumni larvae were collected from a polluted urban river downstream a sewage treatment plant. For the first time, ELISA assay was used to semi-quantify the multixenobiotic resistance transporters (MXR) in these small pertinent bioindicators. The use of samples immediately fixed in the field gives a delay to isolate larvae and allows multi-sampling along a longitudinal transect in a river at a given time. Results exhibit an induction of MXR proteins in larvae from the polluted river and a deinduction in larvae maintained 11 days in unpolluted water. They show new evidences to use midge larvae in biomonitoring environmental programs. They answer to first biomarker calibration steps for the ongoing development of MXR transporters as a detection tool of xenobiotic impacts on bioindicator invertebrates in their freshwater habitats. - Semi-quantification of midge larval MXR transporters by ELISA is a simple and sensitive method to detect chemically polluted situations in running freshwaters

  18. Incrustations detection system for petroleum transport pipes based on gamma transmission; Sistema de deteccao de incrustacoes em dutos de transporte de petroleo pela tecnica de transmissao gama

    Soares, Milton

    2014-07-01

    The scale formed over the inner walls of the ducts conveying the extracted product from offshore oil wheels is a major cause of losses to companies and in some cases even the safety is affected. The consequence of such fouling is the duct's square section reduction that causes extraction flow decrease and can also cause an increase in pressure inside the wheel, with serious consequences for safety. The objective of this work is to propose a mobile inspection system, which can be transported by underwater robots to inspect the lines of ducts in the outputs of the oil wheels. The measurement method to be adopted will be the gamma rays' beam attenuation at a predetermined position of the pipe. This transmission value compared to a clear pipe reading will show if the thickness of the inlay is larger or smaller than an assumed thickness. To carry out the measurements it was designed and built an electronic system comprising power supply, amplifier, single channel analyzer and a counter timer that was connected to a CsI scintillator detector coupled to a PIN photodiode. The system was set up to perform measurements with constant accuracy of ±1%. Tests during the study demonstrated the effectiveness of the proposed method with the obtained results with a carbon steel duct section of 270 mm diameter, removed from the field, with asymmetric BaSO4 inlay. (author)

  19. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  20. Necessities detected in patients derived to sanitary center in an ambulance of the urgent transport network after being treated by the 061 emergency team

    Sixto Cámara Anguita

    2007-03-01

    Full Text Available The integral systems of urgencies and sanitary emergencies has, as resources for the urgent transport of patients, with nonwelfare ambulances, ambulances of basic life support and ambulances of advanced life support. In the two first, due to the ambiguity of the norm, the presence of a nurse to give care is not compulsory. Nevertheless, in the present study, it is detected that, the patients of this study, transferred in nonwelfare ambulance of the urgent transport network to a useful hospitable center with urgency character, once they have been attended by the 061 emergency team, presented/displayed a group of altered basic human necessities, medical levels of dependency as far as autonomy, nurse diagnoses, medical diagnoses, and necessities of pursuit of the restored evolution of the therapeutic technique and that cause the necessity of benefit of nursing care during this transfer.

  1. Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum.

    Emiliane Taillebois

    Full Text Available Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI, thiamethoxam (TMX and clothianidin (CLT. Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 ± 0.04 nM and 41.7 ± 5.9 nM and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008 ± 0.002 nM and 1.135 ± 0.213 nM. Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml and TMX (LC50 = 0.034 µg/ml were more toxic than CLT (LC50 = 0.118 µg/ml. The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.

  2. Anti-acetylcholine receptor antibody titres in the sera of myasthenia patients treated with plasma exchange combined with immunosuppressive therapy.

    Carter, B.; Harrison, R.; Lunt, G G; Behan, P O; Simpson, J. A.

    1980-01-01

    Anti-acetylcholine receptor antibody titres have been monitored in the sera of 19 myasthenic patients treated with plasma exchange combined with a three month period of immunosuppressive therapy. In general the post-exchange titres stabilised at below pre-exchange levels for prolonged periods which were associated with clinical improvement. In seven instances recurrence of symptoms occurred and in six of these cases relapse was shown to be associated with a rise in anti-acetylcholine receptor...

  3. Modulatory effect of neuropeptide Y on acetylcholine-induced oedema and vasoconstriction in isolated perfused lungs of rabbit.

    Delaunois, A; Gustin, P; Dessy-Doize, C; Ansay, M

    1994-01-01

    1. The modulatory role of neuropeptide Y (NPY) on pulmonary oedema induced by acetylcholine and capsaicin was investigated. The effects of NPY on the haemodynamic response to acetylcholine, phenylephrine and substance P were also investigated. 2. Isolated, ventilated, exsanguinated lungs of the rabbit were perfused with a constant flow of recirculating blood-free perfusate. The double/arterial/venous occlusion method was used to partition the total pressure gradient (delta Pt) into four compo...

  4. Combination of Ca2+-activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery

    Stankevičius, E; Lopez-Valverde, V; Rivera, L; Hughes, A D; Mulvany, M J; Simonsen, Ulf

    2006-01-01

    Background and purpose: The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation. Experimental approach: Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+]i) were measured. Key results. A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+-activated K channels abolished acetylcholine (10 μM)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80 mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+]i. Cyclopiazonic acid (CPA, 30 μM), an inhibitor of sarcoplasmic Ca2+-ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+]i, but inhibited the sustained NO release induced by CPA. Conclusions and Implications: Acetylcholine increases endothelial cell [Ca2+]i by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery. PMID:16967048

  5. Quantum Transport Detected by Strong Proximity Interaction at a Graphene-WS2 van der Waals Interface.

    O'Farrell, E C T; Avsar, A; Tan, J Y; Eda, G; Özyilmaz, B

    2015-09-01

    Magnetotransport measurements demonstrate that graphene in a van der Waals heterostructure is a sensitive probe of quantum transport in an adjacent WS2 layer via strong Coulomb interactions. We observe a large low-field magnetoresistance (≫ e(2)/h) and a -ln T temperature dependence of the resistance. In-plane magnetic field resistance indicates the origin is orbital and nonclassical. We demonstrate a strong electron-hole asymmetry in the mobility and coherence length of graphene demonstrating the presence of localized Coulomb interactions with ionized donors in the WS2 substrate, which ultimately leads to screening as the Fermi level of graphene is tuned toward the conduction band of WS2. This leads us to conclude that graphene couples to quantum localization processes in WS2 via the Coulomb interaction and results in the observed signatures of quantum transport. Our results show that theoretical descriptions of the van der Waals interface should not ignore localized strong correlations. PMID:26258760

  6. Acetylcholine produces contraction mediated by cyclooxigenase pathway in arterial vessels in the marine fish (Isacia conceptionis).

    Moraga, F A; Urriola-Urriola, N

    2015-05-01

    Preliminary studies showed that dorsal artery contraction mediated by acetylcholine (ACh) is blocked with indomethacin in intertidal fish (G. laevifrons). Our objective was to characterize the cholinergic pathway in several artery vessels of the I. conceptionis. Afferent and efferent branchial, dorsal and mesenteric arteries were dissected of 6 juvenile specimens, isometric tension studies were done using doses response curves (DRC) for Ach (10(-13) to 10(-3) M), and cholinergic pathways were obtained by blocking with atropine or indomethacin. CRC to ACh showed a pattern of high sensitivity only in efferente branchial artery and low sensibility in all vessels. Furthermore, these contractions were blocked in the presence of atropine and indomethacin in all vessels. Our results corroborate previous results observed in intertidal species that contraction induced by acetylcholine is mediated by receptors that activate a cyclooxygenase contraction pathway. PMID:26132019

  7. Theoretical studies of interaction models of human acetylcholine esterase with different inhibitors

    2009-01-01

    Alzheimer’s disease(AD) is a progressive neurodegenerative disorder and one of the most common causes of dementia in the elderly.Acetylcholine esterase inhibitors(AChEI) are the main drugs used in the treatment of AD.In this work,docking studies have been performed in order to understand the interaction between a number of inhibitors(tacrine,rivastigmine,huperzine A,TV-3326(ladostigil),donepezil and anseculin) and acetylcholine esterase(AChE).The calculated binding affinities between inhibitors and AChE increase in the order tacrine

  8. Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

    Lee, J.H.; El-Fakahany, E.E.

    1985-08-12

    Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent (/sup 3/H)N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37/sup 0/C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system. 20 references, 3 tables.

  9. Changes in acetylcholine content, release and muscarinic receptors in rat hippocampus under cold stress

    The aim was to study the mechanism of the previously established decrease in acetylcholine (ACh) concentration in the rat hippocampus under cold stress. Male rats were exposed for 14 days to cold (5 degree C) or kept (controls) at room temperature (24 degree C). Acetylcholine content, release and muscarinic receptor binding were investigated in the hippocampus. Cold exposure resulted in a decrease of ACh concentration in the dorsal hippocampus. Moreover, the potassium-evoked release of ACh from hippocampal slices was increased and an increase of maximal binding capacity of [3H](-) quinuclidinyl benzilate in the dorsal hippocampus of cold exposed animals was also observed. Thus the decrease of hippocampal ACh concentration under cold exposure is probably due to its increased release. On balance then, our results demonstrate that cold stress in the rat induces significant activation of the hippocampal cholinergic system

  10. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

    Butcher, Adrian J; Bradley, Sophie J; Prihandoko, Rudi; Brooke, Simon M; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M; Bottrill, Andrew R; Challiss, R A John; Broad, Lisa M; Felder, Christian C; Tobin, Andrew B

    2016-04-22

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. PMID:26826123

  11. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory*♦

    Butcher, Adrian J.; Bradley, Sophie J.; Prihandoko, Rudi; Brooke, Simon M.; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M.; Bottrill, Andrew R.; Challiss, R. A. John; Broad, Lisa M.; Felder, Christian C.; Tobin, Andrew B.

    2016-01-01

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo. Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser228) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser228. These data supported the hypothesis that phosphorylation at Ser228 was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser228 on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser228 phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser228 not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. PMID:26826123

  12. Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior

    Picciotto, Marina R.; Higley, Michael J.; Mineur, Yann S.

    2012-01-01

    Acetylcholine in the brain alters neuronal excitability, influences synaptic transmission, induces synaptic plasticity and coordinates the firing of groups of neurons. As a result, it changes the state of neuronal networks throughout the brain and modifies their response to internal and external inputs: the classical role of a neuromodulator. Here we identify actions of cholinergic signaling on cellular and synaptic properties of neurons in several brain areas and discuss the consequences of ...

  13. On homology modeling of the M-2 muscarinic acetylcholine receptor subtype

    Jakubík, Jan; Randáková, Alena; Doležal, Vladimír

    2013-01-01

    Roč. 27, č. 6 (2013), s. 525-538. ISSN 0920-654X R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GBP304/12/G069 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptor * G-protein coupled receptor * homology energy estimation * MM-GBSA Subject RIV: ED - Physiology Impact factor: 2.782, year: 2013

  14. Acetylcholine Elevation Relieves Cognitive Rigidity and Social Deficiency in a Mouse Model of Autism

    Karvat, Golan; Kimchi, Tali

    2013-01-01

    Autism spectrum disorders (ASD) are defined by behavioral deficits in social interaction and communication, repetitive stereotyped behaviors, and restricted interests/cognitive rigidity. Recent studies in humans and animal-models suggest that dysfunction of the cholinergic system may underlie autism-related behavioral symptoms. Here we tested the hypothesis that augmentation of acetylcholine (ACh) in the synaptic cleft by inhibiting acetylcholinesterase may ameliorate autistic phenotypes. We ...

  15. CHRNB2 Is the Second Acetylcholine Receptor Subunit Associated with Autosomal Dominant Nocturnal Frontal Lobe Epilepsy*

    Phillips, Hilary A.; Favre, Isabelle; Kirkpatrick, Martin; Zuberi, Sameer M; Goudie, David; Heron, Sarah E.; Scheffer, Ingrid E.; Sutherland, Grant R.; Berkovic, Samuel F; Bertrand, Daniel; Mulley, John C

    2000-01-01

    Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an uncommon, idiopathic partial epilepsy characterized by clusters of motor seizures occurring in sleep. We describe a mutation of the β2 subunit of the nicotinic acetylcholine receptor, effecting a V287M substitution within the M2 domain. The mutation, in an evolutionary conserved region of CHRNB2, is associated with ADNFLE in a Scottish family. Functional receptors with the V287M mutation are highly expressed in Xenopus oocytes ...

  16. Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms

    Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, E. E.; Jakubík, Jan

    2015-01-01

    Roč. 97, Jul 2015 (2015), s. 27-39. ISSN 1043-6618 R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * atypical agonists * xanomeline * activation mechanism Subject RIV: ED - Physiology Impact factor: 4.408, year: 2014

  17. Evidence for a neurotransmitter function of acetylcholine in rabbit superior colliculus.

    Wichmann, T; Illing, R B; Starke, K

    1987-12-01

    Acetylcholinesterase staining and studies on the uptake of [3H]choline into the subsequent efflux of tritium from collicular slices were carried out in order to provide evidence for a neurotransmitter function of acetylcholine in rabbit superior colliculus. Acetylcholinesterase staining was dense and homogeneous in superficial layers whereas the staining was arranged in patches with slightly higher density caudally than rostrally in the intermediate layers. The accumulation of tritium in slices incubated with [3H]choline depended on time, temperature and concentration, and was inhibited by hemicholinium-3. Accumulation was slightly higher in caudal than in rostral slices. Electrical stimulation enhanced tritium outflow from slices preincubated with [3H]choline. Tetrodotoxin and a low calcium medium inhibited the evoked overflow whereas hemicholinium-3 caused an enhancement. Oxotremorine decreased the evoked overflow; atropine prevented this effect. The opioids [D-Ala2, MePhe4, Glycol5]enkephalin, [D-Ala2, D-Leu5]enkephalin and ethylketocyclazocine caused an inhibition. The effects of the latter two agonists were antagonized by naloxone. The GABAB-receptor-agonist (-)-baclofen decreased the evoked overflow at lower concentrations than GABA, whereas the GABAA-receptor-agonist muscimol was ineffective. Serotonin produced an inhibition which was prevented by metitepin, alpha- and beta-adrenoceptor as well as dopamine-receptor ligands caused no change. It is concluded that in the rabbit superior colliculus the pattern of acetylcholinesterase staining is comparable, but not identical to the distribution in other species. The accumulation of [3H]choline, as well as the tetrodotoxin-sensitive and calcium-dependent overflow of tritium upon electrical stimulation (reflecting presumably release of [3H]acetylcholine) indicate that acetylcholine has a neurotransmitter function in this tissue. The release of [3H]acetylcholine was modulated by various transmitter substances and

  18. Chemical Stimulation of Adherent Cells by Localized Application of Acetylcholine from a Microfluidic System

    Susanne Zibek

    2010-01-01

    Chemical stimulation of cells is inherently cell type selective in contrast to electro-stimulation. The availability of a system for localized application of minute amounts of chemical stimulants could be useful for dose related response studies to test new compounds. It could also bring forward the development of a novel type of neuroprostheses. In an experimental setup micro-droplets of an acetylcholine solution were ejected from a fluidic microsystem and applied to the bottom of a nanop...

  19. M2 Muscarinic acetylcholine receptor modulates rat airway smooth muscle cell proliferation

    Placeres-Uray, Fabiola A; Febres-Aldana, Christopher A; Fernandez-Ruiz, Ruth; Gonzalez de Alfonzo, Ramona; Lippo de Becemberg, Itala A; Alfonzo, Marcelo J

    2013-01-01

    Airways chronic inflammatory conditions in asthma and COPD are characterized by tissue remodeling, being smooth muscle hyperplasia, the most important feature. Non-neuronal and neuronal Acetylcholine acting on muscarinic receptors (MAChRs) has been postulated as determinant of tissue remodeling in asthma and COPD by promoting proliferation and phenotypic changes of airway smooth muscle cells (ASMC). The objective was to evaluate proliferative responses to muscarinic agonist as carbamylcholine...

  20. Acetylcholine release by human colon cancer cells mediates autocrine stimulation of cell proliferation

    Cheng, Kunrong; Samimi, Roxana; Xie, Guofeng; Shant, Jasleen; Drachenberg, Cinthia; Wade, Mark; Davis, Richard J.; Nomikos, George; Raufman, Jean-Pierre

    2008-01-01

    Most colon cancers overexpress M3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor...

  1. INFLUENCE OF ANTIBIOTICS ON THE MECHANICAL RESPONSES OF GUINEA-PIG ILEUM TO ACETYLCHOLINE AND HISTAMINE

    1998-01-01

    The side effects of antibiotics have been extensively described during the last decades, however, their role on digestive motility must be better investigated. Following a line of research, the influence of penicillin, chloranfenicol tetracycline and gentamicine on longitudinal smooth muscle responses to acetylcholine and histamine were studied on guinea-pig ileum. There were no differences between the responses before and after the addition of each antibiotic. Further investigations must be ...

  2. Neonicotinoid Binding, Toxicity and Expression of Nicotinic Acetylcholine Receptor Subunits in the Aphid Acyrthosiphon pisum

    Taillebois, Emiliane; Beloula, Abdelhamid; Quinchard, Sophie; Jaubert-Possamai, Stéphanie; Daguin, Antoine; Servent, Denis; Tagu, Denis; Thany, Steeve H.; Tricoire-Leignel, Helene

    2014-01-01

    Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and ...

  3. Minor structural changes in nicotinoid insecticides confer differential subtype selectivity for mammalian nicotinic acetylcholine receptors

    Tomizawa, Motohiro; Casida, John E.

    1999-01-01

    The major nitroimine insecticide imidacloprid (IMI) and the nicotinic analgesics epibatidine and ABT-594 contain the 6-chloro-3-pyridinyl moiety important for high activity and/or selectivity. ABT-594 has considerable nicotinic acetylcholine receptor (AChR) subtype specificity which might carry over to the chloropyridinyl insecticides. This study considers nine IMI analogues for selectivity in binding to immuno-isolated α1, α3 and α7 containing nicotinic AChRs and to purported α4β2 nicotinic ...

  4. Utilization of Superfused Cerebral Slices in Probing Muscarinic Receptor Autoregulation of Acetylcholine Release

    Alquicer, Glenda; Doležal, Vladimír; El-Fakahany, E. E.

    New York: Springer, 2016 - (Mysliveček, J.; Jakubík, J.), s. 221-233. (Neuromethods. 107). ISBN 978-1-4939-2857-6 R&D Projects: GA ČR(CZ) GA14-05696S; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : muscarinic receptors * acetylcholine release * autoregulation * superfusion Subject RIV: FH - Neurology

  5. Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus

    Sugiyama, Kou-ichi; Tezuka, Takafumi

    2011-01-01

    Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongatio...

  6. Variants in nicotinic acetylcholine receptors α5 and α3 increase risks to nicotine dependence†

    Chen, Xiangning; Chen, Jingchun; Williamson, Vernell S; An, Seon-Sook; Hettema, John M.; Aggen, Steven H.; Neale, Michael C.; Kendler, Kenneth S.

    2009-01-01

    Nicotinic acetylcholine receptors bind to nicotine and initiate the physiological and pharmacological responses to tobacco smoking. In this report, we studied the association of α5 and α3 subunits with nicotine dependence and with the symptoms of alcohol and cannabis abuse and dependence in two independent epidemiological samples (n = 815 and 1,121, respectively). In this study, seven single nucleotide polymorphisms were genotyped in the CHRNA5 and CHRNA3 genes. In both samples, we found that...

  7. Ionomycin-induced acetylcholine release and its inhibition by adenosine at frog motor nerve endings.

    Hunt, J M; Silinsky, E. M.

    1993-01-01

    1. Acetylcholine (ACh) evoked secretion by the calcium ionophore, ionomycin, was studied at frog motor nerve endings. 2. Bath application of ionomycin stimulated an irreversible increase in the rate of spontaneous, quantal ACh release in the presence of extracellular Ca2+. In contrast, local application of ionomycin stimulated a rapid, reversible acceleration of spontaneous ACh release. 3. The magnitude of the secretory response to ionomycin was dependent both upon the concentration of ionoph...

  8. Synthesis of [11C]N-methyl tetrahydroaminoacridine, a potent acetylcholine esterase inhibitor

    Tetrahydroaminoacridine (THA) is a potent central acting acetylcholine esterase (AChE) inhibitor which might be used as therapeutic agent in the treatment of Alzheimer's disease (AZD). In order to study the AChE activity in the brain by PET, the authors selected N-methyl THA, a potent AChE inhibitor, as a potential radioligand. In this paper, they report the synthesis and labelling of N-methyl THA with [11C]methyl iodide

  9. Quantitative Molecular Imaging of Neuronal Nicotinic Acetylcholine Receptors in the Human Brain with A-85380 Radiotracers

    Lotfipour, Shahrdad; Mandelkern, Mark; Brody, Arthur L.

    2011-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated in a spectrum of cognitive functions as well as psychiatric and neurodegenerative disorders, including tobacco addiction and Alzheimer's Disease. The examination of neuronal nAChRs in living humans is a relatively new field. Researchers have developed brain-imaging radiotracers for nAChRs, with radiolabeled A-85380 compounds having the most widespread use. We provide a brief background on nAChRs, followed by a discussion...

  10. Inducibility of human atrial fibrillation in an in silico model reflecting local acetylcholine distribution and concentration

    Hwang, Minki; Lee, Hyun-Seung; Pak, Hui-Nam; Shim, Eun Bo

    2015-01-01

    Vagal nerve activity has been known to play a crucial role in the induction and maintenance of atrial fibrillation (AF). However, it is unclear how the distribution and concentration of local acetylcholine (ACh) promotes AF. In this study, we investigated the effect of the spatial distribution and concentration of ACh on fibrillation patterns in an in silico human atrial model. A human atrial action potential model with an ACh-dependent K+ current (IKAch) was used to examine the effect of vag...

  11. Isolation of acetylcholine receptor clusters in substrate-associated material from cultured rat myotubes using saponin

    1984-01-01

    After exposure of rat myotube cultures to saponin, less than 1% of the cellular protein was found to remain associated with the tissue culture substrate. This substrate-associated material contained approximately 10% of the acetylcholine receptors (AChRs) and greater than 80% of the large, ventral AChR clusters present in the original culture. The domain structure evident in intact cells was maintained in AChR clusters after isolation using saponin. However, vinculin, present at the clusters ...

  12. CaMKIIα, a modulator of M4 muscarinic acetylcholine receptors

    Guo, Ming-Lei; Liu, Zhenguo; Chu, Xiang-Ping; Mao, Li-Min; WANG, John Q.

    2010-01-01

    G protein-coupled receptors (GPCRs) are subject to the regulation by protein kinases. By controlling the phosphorylation-dephosphorylation balance, protein kinases actively modify GPCR expression and function. In a recent study, we have identified a novel phosphorylation-dependent regulation of Gαi/o-coupled muscarinic acetylcholine receptors. A synapse-enriched protein kinase, Ca2+/calmodulin-dependent protein kinase II (CaMKIIα), binds directly and selectively to second intracellular loops ...

  13. Visualization of cholinoceptive neurons in the rat neocortex: colocalization of muscarinic and nicotinic acetylcholine receptors

    Zee, E.A. van der; Streefland, C.; Strosberg, A D; Schröder, H.; Luiten, P.G.M.

    1992-01-01

    The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluorescent double-labeling techniques. For both classes of receptors, pyramidal and nonpyramidal cells were found immunostained and an identical laminar distribution pattern of immunopositive neurons in ...

  14. Hippocampal acetylcholine release during memory testing in rats: augmentation by glucose.

    Ragozzino, M E; Unick, K E; Gold, P. E.

    1996-01-01

    Several lines of evidence indicate that a modest increase in circulating glucose levels enhances memory. One mechanism underlying glucose effects on memory may be an increase in acetylcholine (ACh) release. The present experiment determined whether enhancement of spontaneous alternation performance by systemic glucose treatment is related to an increase in hippocampal ACh output. Samples of extracellular ACh were assessed at 12-min intervals using in vivo microdialysis with HPLC-EC. Twenty-fo...

  15. Blockage of muscle and neuronal nicotinic acetylcholine receptors by fluoxetine (Prozac)

    García-Colunga, J; Awad, J. N.; Miledi, R

    1997-01-01

    Fluoxetine (Prozac), a widely used antidepressant, is said to exert its medicinal effects almost exclusively by blocking the serotonin uptake systems. The present study shows that both muscle and neuronal nicotinic acetylcholine receptors are blocked, in a noncompetitive and voltage-dependent way, by fluoxetine, which also increases the rate of desensitization of the nicotinic receptors. Because these receptors are very widely distributed in the both central and peripheral nervous systems, th...

  16. ACETYLCHOLINE RELEASE IN THE HIPPOCAMPUS AND PRELIMBIC CORTEX DURING ACQUISITION OF A SOCIALLY TRANSMITTED FOOD PREFERENCE

    Gold, P E; Countryman, R.A.; Dukala, D.; Chang, Q.

    2011-01-01

    Interference with cholinergic functions in hippocampus and prefrontal cortex impairs learning and memory for social transmission of food preference, suggesting that acetylcholine (ACh) release in the two brain regions may be important for acquiring the food preference. This experiment examined release of ACh in the hippocampus and prefrontal cortex of rats during training for social transmission of food preference. After demonstrator rats ate a food with novel flavor and odor, a social transm...

  17. The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine

    1988-01-01

    Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The thresho...

  18. Regulation of Synaptic Transmission and Plasticity by Neuronal Nicotinic Acetylcholine Receptors

    McKay, Bruce E.; Placzek, Andon N; Dani, John A.

    2007-01-01

    Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system and participate in a variety of physiological functions. Recent advances have revealed roles of nAChRs in the regulation of synaptic transmission and synaptic plasticity, particularly in the hippocampus and midbrain dopamine centers. In general, activation of nAChRs causes membrane depolarization and directly and indirectly increases the intracellular calcium concentration. Thus, when nAChRs ...

  19. Future perspectives of a cardiac non-neuronal acetylcholine system targeting cardiovascular diseases as an adjunctive tool for metabolic intervention.

    Kakinuma, Yoshihiko

    2015-11-01

    It has been several years since the function of the non-neuronal cholinergic system was independently reported in cardiomyocytes by several research groups. Although these findings initially seemed to be negligible and insignificant, extraordinary findings about cardiomyocytes were subsequently reported in studies involving the knockdown of the non-neuronal cholinergic system. These studies provide the evidence that this system may be indispensable for maintaining principal cardiac functions. Despite the absence of an appropriate and reliable technology to detect cellular ACh in real time in cardiomyocytes, studies of this system have progressed, albeit very slowly, to gradually consolidate the significance of this system. Based on the many significant findings regarding this system, these will be critical to develop adjunctive intervention therapy against cardiovascular diseases, including peripheral artery disease and heart failure. In this study, previous studies focusing on the non-neuronal cholinergic system are reviewed along with our studies, both indicating the biologically significant roles of the cardiac non-neuronal acetylcholine system from a clinical perspective. PMID:26028150

  20. Up-regulated expression of the alpha7 nicotinic acetylcholine receptor subunit on inflammatory infiltrates during Dictyocaulus viviparus infection.

    Lazari, O; Kipar, A; Johnson, D R; Selkirk, M E; Matthews, J B

    2006-09-01

    Cholinergic signalling is known to affect immune cell function, but few studies have addressed its relevance during nematode infection. We therefore analysed the anatomical distribution and expression pattern of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in lungs obtained from Dictyocaulus viviparus-infected and uninfected control cattle. The analysis was performed on trachea and lung parenchyma from uninfected animals and animals necropsied at 15, 22 and 43 days post-infection (DPI). Localization of the alpha7 nAChR was evaluated by immunohistology and mRNA expression analysed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR). In uninfected animals, tracheal, bronchial and bronchiolar epithelium and smooth muscle cells constitutively expressed the alpha7 nAChR, as did type I and II alveolar epithelial cells and alveolar macrophages and a few infiltrating leucocytes. By 15 DPI, immunohistology revealed a massive influx of alpha7 nAChR+ inflammatory cells into the lung parenchyma and tracheal wall. This was reflected in the RT-PCR results. At later time points, both parenchyma and tracheal wall contained large numbers of alpha7 nAChR+ leucocytes, but detection of transcript was restricted to the trachea. Recruitment of nAChR-containing leucocytes to the lungs of D. viviparus-infected cattle suggests that these cells may represent possible downstream targets for parasite-secreted acetylcholinesterases. PMID:16916366

  1. Dopamine modulates acetylcholine release via octopamine and CREB signaling in Caenorhabditis elegans.

    Satoshi Suo

    Full Text Available Animals change their behavior and metabolism in response to external stimuli. cAMP response element binding protein (CREB is a signal-activated transcription factor that enables the coupling of extracellular signals and gene expression to induce adaptive changes. Biogenic amine neurotransmitters regulate CREB and such regulation is important for long-term changes in various nervous system functions, including learning and drug addiction. In Caenorhabditis elegans, the amine neurotransmitter octopamine activates a CREB homolog, CRH-1, in cholinergic SIA neurons, whereas dopamine suppresses CREB activation by inhibiting octopamine signaling in response to food stimuli. However, the physiological role of this activation is unknown. In this study, the effect of dopamine, octopamine, and CREB on acetylcholine signaling was analyzed using the acetylcholinesterase inhibitor aldicarb. Mutants with decreased dopamine signaling exhibited reduced acetylcholine signaling, and octopamine and CREB functioned downstream of dopamine in this regulation. This study demonstrates that the regulation of CREB by amine neurotransmitters modulates acetylcholine release from the neurons of C. elegans.

  2. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists. PMID:27055770

  3. Evidence for the extramembranous location of the putative amphipathic helix of acetylcholine receptor

    Evidence has been obtained demonstrating that the peptides GVKYIAE and AIKYIAE found in the potential amphipathic helices of the α and β subunits, respectively, of acetylcholine receptor are not buried in the membrane. The peptide KYIAE was synthesized, and polyclonal antibodies were prepared against a conjugate of bovine serum albumin and synthetic peptide. An immunoadsorbent capable of binding and subsequently releasing peptides ending with the sequence-YIAE was produced by attaching these specific antibodies to agarose. Native acetylcholine receptor was labeled with pyridoxal phosphate and Na[3H]BH4. The labeled protein was stripped of phospholipid and digested with the protease from Staphylococcus aureus strain V8. The digest was submitted to immunoadsorption to isolate the labeled indigenous peptides. As a control, α and β polypeptides prepared by gel filtration of a solution of acetylcholine receptor in detergent were stripped of detergent and labeled with pyridoxal phosphate and Na[3H]BH4 in the presence of 8 M urea. The labeled α and β polypeptides were digested and submitted to immunoadsorption. The specific radioactivities of the indigenous peptides from the α and β subunits labeled under native and denaturing conditions were nearly equal. In similar experiments using isethionyl (2',4'-dinitrophenyl)-3-aminopropionimidate as the labeling agent, the indigenous peptides from native and denatured receptor were also labeled to the same extent. Since these peptides are labeled to the same extent whether or not the protein is denatured, they cannot be buried in the membrane

  4. A multi-enzyme microreactor-based online electrochemical system for selective and continuous monitoring of acetylcholine.

    Lin, Yuqing; Yu, Ping; Mao, Lanqun

    2015-06-01

    This study demonstrates an online electrochemical system (OECS) for selective and continuous measurements of acetylcholine (ACh) through efficiently integrating in vivo microdialysis, a multi-enzyme microreactor and an electrochemical detector. A multi-enzyme microreactor was prepared first by co-immobilizing two kinds of enzymes, i.e. choline oxidase (ChOx) and catalase (Cat), onto magnetite nanoparticles and then confining the as-formed nanoparticles into a fused-silica capillary with the assistance of an external magnet. The multi-enzyme microreactor was settled between an in vivo microdialysis sampling system and an electrochemical detector to suppress the interference from choline toward ACh detection. Selective detection of ACh was accomplished using the electrochemical detector with ACh esterase (AChE) and ChOx as the recognition units for ACh and Prussian blue (PB) as the electrocatalyst for the reduction of hydrogen peroxide (H2O2). The current recorded with the OECS was linear with the concentration of ACh (I/nA = -3.90CACh/μM + 1.21, γ = 0.998) within a concentration range of 5 μM to 100 μM. The detection limit, based on a signal-to-noise ratio of 3, was calculated to be 1 μM. Interference investigation demonstrates that the OECS did not produce an observable current response toward physiological levels of common electroactive species, such as ascorbic acid (AA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and uric acid (UA). The high selectivity and the good linearity in combination with the high stability may enable the OECS developed here as a potential system for continuous monitoring of cerebral ACh release in some physiological and pathological processes. PMID:25529471

  5. Evaluation of [1-11C]-α-aminoisobutyric acid for tumor detection and amino acid transport measurement: Spontaneous canine tumor studies

    Alpha-aminoisobutyric acid (AIB) or α-methyl alanine, is a nonmetabolized amino acid treansported into cells particularly malignant cells, predominantly by the ''A'' amino acid transport system. Since it is not metabolized, [1-11C]-AIB can be used to quantify A-type amino acid transport into cells using a relatively simple compartmental model and quantitative imaging procedures (e.g. positron tomography). The tissue distribution of [1-11C]-AIB was determined in six dogs bearing spontaneous tumors, including lymphosarcoma, osteogenic sarcoma, mammary carcinoma, and adenocarcinoma. Quantitative imaging with tissue radioassay confirmation at necropsy showed poor to excellent tumor localization. However, in all cases the concentrations achieved appear adequate for amino acid transport measurement at known tumor locations. The observed low normal brain (due to blood-brain barrier exclusion) and high (relative to brain) tumor concentrations of [1-11C]-AIB suggest that this agent may prove effective for the early detection of human brain tumors. (orig.)

  6. 13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1

    CLC transporters catalyze the exchange of Cl− for H+ across cellular membranes. To do so, they must couple Cl− and H+ binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state 13C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H+) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H+-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl−-permeation pathway, to the extracellular solution. The H+-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H+ binding is mechanistically coupled to closing of the intracellular access-pathway for Cl−

  7. {sup 13}C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1

    Abraham, Sherwin J.; Cheng, Ricky C.; Chew, Thomas A.; Khantwal, Chandra M. [Stanford University School of Medicine, Department of Molecular & Cellular Physiology (United States); Liu, Corey W. [Stanford University School of Medicine, Stanford Magnetic Resonance Laboratory (United States); Gong, Shimei; Nakamoto, Robert K. [University of Virginia, Department of Molecular Physiology and Biological Physics (United States); Maduke, Merritt, E-mail: maduke@stanford.edu [Stanford University School of Medicine, Department of Molecular & Cellular Physiology (United States)

    2015-04-15

    CLC transporters catalyze the exchange of Cl{sup −} for H{sup +} across cellular membranes. To do so, they must couple Cl{sup −} and H{sup +} binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state {sup 13}C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H{sup +}) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H{sup +}-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl{sup −}-permeation pathway, to the extracellular solution. The H{sup +}-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H{sup +} binding is mechanistically coupled to closing of the intracellular access-pathway for Cl{sup −}.

  8. Effects of alpha-7 nicotinic acetylcholine receptor positive allosteric modulator on lipopolysaccharide-induced neuroinflammatory pain in mice.

    Abbas, Muzaffar; Rahman, Shafiqur

    2016-07-15

    Evidence indicates that microglial activation contributes to the pathophysiology and maintenance of neuroinflammatory pain involving central nervous system alpha-7 nicotinic acetylcholine receptors. The objective of the present study was to determine the effects of 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS), an alpha-7 nicotinic acetylcholine receptor positive allosteric modulator (PAM), on tactile allodynia and thermal hyperalgesia following lipopolysaccharide (LPS)-induced microglial activation in hippocampus, a neuroinflammatory pain model in mice. In addition, we examined the effects of TQS on microglial activation marker, an ionized calcium-binding adapter molecule 1 (Iba-1), in the hippocampus may be associated with neuroinflammatory pain. Pretreatment of TQS (4mg/kg) significantly reduced LPS (1mg/kg)-induced tactile allodynia and thermal hyperalgesia. Moreover, pretreatment of methyllycaconitine (3mg/kg) significantly reversed TQS-induced antiallodynic and antihyperalgesic responses indicating the involvement of alpha-7 nicotinic acetylcholine receptor. Pretreatment of TQS significantly decreased LPS-induced increased in hippocampal Iba-1 expression. Overall, these results suggest that TQS reduces LPS-induced neuroinflammatory pain like symptoms via modulating microglial activation likely in the hippocampus and/or other brain region by targeting alpha-7 nicotinic acetylcholine receptor. Therefore, alpha-7 nicotinic acetylcholine receptor PAM such as TQS could be a potential drug candidate for the treatment of neuroinflammatory pain. PMID:27154173

  9. Acetylcholine Attenuates Hypoxia/ Reoxygenation-Induced Mitochondrial and Cytosolic ROS Formation in H9c2 Cells via M2 Acetylcholine Receptor

    Yi Miao

    2013-02-01

    Full Text Available Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS by measuring mitochondrial ROS (mtROS, mitochondrial DNA (mtDNA copy number, xanthine oxidase (XO and NADPH oxidase (NOX activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

  10. Growth of high quality mercurous halide single crystals by physical vapor transport method for AOM and radiation detection applications

    Amarasinghe, Priyanthi M.; Kim, Joo-Soo; Chen, Henry; Trivedi, Sudhir; Qadri, Syed B.; Soos, Jolanta; Diestler, Mark; Zhang, Dajie; Gupta, Neelam; Jensen, Janet L.; Jensen, James

    2016-09-01

    Single crystals of mercurous halide were grown by physical vapor transport method (PVT). The orientation and the crystalline quality of the grown crystals were determined using high resolution x-ray diffraction (HRXRD) technique. The full width at half maximum (FWHM) of the grown mercurous bromide crystals was measured to be 0.13 degrees for (004) reflection, which is the best that has been achieved so far for PVT grown mercurous halide single crystals. The extended defects of the crystals were also analyzed using high resolution x-ray diffraction topography. Preliminary studies were carried out to evaluate the performance of the crystals on acousto-optic modulator (AOM) and gamma-ray detector applications. The results indicate the grown mercurous halide crystals are excellent materials for acousto-optic modulator device fabrication. The diffraction efficiencies of the fabricated AOM device with 1152 and 1523 nm wavelength lasers polarizing parallel to the acoustic wave were found to be 35% and 28%, respectively. The results also indicate the grown crystals are a promising material for gamma-ray detector application with a very high energy resolution of 1.86% FWHM.

  11. Spectroscopic investigation of sulfonate phthalocyanine to probe enzyme reactions for heavy metals detection

    Optical absorption and Raman spectra of the sulfonated copper phthalocyanine (CuTsPc) layer were exploited for detection of cadmium (Cd) contaminants in water. Acetylcholine esterase was immobilized by freely suspending them in calcium alginate microbeads and this gel was then spincoated on the drop cast sulfonated copper phthalocyanine film on a glass substrate to form a bilayer. The inhibition of catalytic reaction between acetylcholine chloride and enzyme due to Cd contaminants was monitored by recording changes in spectra of drop cast CuTsPc as an indicator. The detection limit of cadmium content in water was found to be 1 ppm.

  12. Introducing Thermal Wave Transport Analysis (TWTA): A Thermal Technique for Dopamine Detection by Screen-Printed Electrodes Functionalized with Molecularly Imprinted Polymer (MIP) Particles.

    Peeters, Marloes M; van Grinsven, Bart; Foster, Christopher W; Cleij, Thomas J; Banks, Craig E

    2016-01-01

    A novel procedure is developed for producing bulk modified Molecularly Imprinted Polymer (MIP) screen-printed electrodes (SPEs), which involves the direct mixing of the polymer particles within the screen-printed ink. This allowed reduction of the sample preparation time from 45 min to 1 min, and resulted in higher reproducibility of the electrodes. The samples are measured with a novel detection method, namely, thermal wave transport analysis (TWTA), relying on the analysis of thermal waves through a functional interface. As a first proof-of-principle, MIPs for dopamine are developed and successfully incorporated within a bulk modified MIP SPE. The detection limits of dopamine within buffer solutions for the MIP SPEs are determined via three independent techniques. With cyclic voltammetry this was determined to be 4.7 × 10(-6) M, whereas by using the heat-transfer method (HTM) 0.35 × 10(-6) M was obtained, and with the novel TWTA concept 0.26 × 10(-6) M is possible. This TWTA technique is measured simultaneously with HTM and has the benefits of reducing measurement time to less than 5 min and increasing effect size by nearly a factor of two. The two thermal methods are able to enhance dopamine detection by one order of magnitude compared to the electrochemical method. In previous research, it was not possible to measure neurotransmitters in complex samples with HTM, but with the improved signal-to-noise of TWTA for the first time, spiked dopamine concentrations were determined in a relevant food sample. In summary, novel concepts are presented for both the sensor functionalization side by employing screen-printing technology, and on the sensing side, the novel TWTA thermal technique is reported. The developed bio-sensing platform is cost-effective and suitable for mass-production due to the nature of screen-printing technology, which makes it very interesting for neurotransmitter detection in clinical diagnostic applications. PMID:27128891

  13. Introducing Thermal Wave Transport Analysis (TWTA: A Thermal Technique for Dopamine Detection by Screen-Printed Electrodes Functionalized with Molecularly Imprinted Polymer (MIP Particles

    Marloes M. Peeters

    2016-04-01

    Full Text Available A novel procedure is developed for producing bulk modified Molecularly Imprinted Polymer (MIP screen-printed electrodes (SPEs, which involves the direct mixing of the polymer particles within the screen-printed ink. This allowed reduction of the sample preparation time from 45 min to 1 min, and resulted in higher reproducibility of the electrodes. The samples are measured with a novel detection method, namely, thermal wave transport analysis (TWTA, relying on the analysis of thermal waves through a functional interface. As a first proof-of-principle, MIPs for dopamine are developed and successfully incorporated within a bulk modified MIP SPE. The detection limits of dopamine within buffer solutions for the MIP SPEs are determined via three independent techniques. With cyclic voltammetry this was determined to be 4.7 × 10−6 M, whereas by using the heat-transfer method (HTM 0.35 × 10−6 M was obtained, and with the novel TWTA concept 0.26 × 10−6 M is possible. This TWTA technique is measured simultaneously with HTM and has the benefits of reducing measurement time to less than 5 min and increasing effect size by nearly a factor of two. The two thermal methods are able to enhance dopamine detection by one order of magnitude compared to the electrochemical method. In previous research, it was not possible to measure neurotransmitters in complex samples with HTM, but with the improved signal-to-noise of TWTA for the first time, spiked dopamine concentrations were determined in a relevant food sample. In summary, novel concepts are presented for both the sensor functionalization side by employing screen-printing technology, and on the sensing side, the novel TWTA thermal technique is reported. The developed bio-sensing platform is cost-effective and suitable for mass-production due to the nature of screen-printing technology, which makes it very interesting for neurotransmitter detection in clinical diagnostic applications.

  14. Refinements in the use of equivalent latitude for assimilating sporadic inhomogeneous stratospheric tracer observations, 1: Detecting transport of Pinatubo aerosol across a strong vortex edge

    P. Good

    2004-01-01

    Full Text Available The use of PV equivalent latitude for assimilating stratospheric tracer observations is discussed - with particular regard to the errors in the equivalent latitude coordinate, and to the assimilation of sparse data. Some example measurements are assimilated: they sample the stratosphere sporadically and inhomogeneously. The aim was to obtain precise information about the isentropic tracer distribution and evolution as a function of equivalent latitude. Precision is important, if transport across barriers like the vortex edge are to be detected directly. The main challenges addressed are the errors in modelled equivalent latitude, and the non-ideal observational sampling. The methods presented allow first some assessment of equivalent latitude errors and a picture of how good or poor the observational coverage is. This information determines choices in the approach for estimating as precisely as possible the true equivalent latitude distribution of the tracer, in periods of good and poor observational coverage. This is in practice an optimisation process, since better understanding of the equivalent latitude distribution of the tracer feeds back into a clearer picture of the errors in the modelled equivalent latitude coordinate. Error estimates constrain the reliability of using equivalent latitude to make statements like 'this observation samples air poleward of the vortex edge' or that of more general model-measurement comparisons. The approach is demonstrated for ground-based lidar soundings of the Mount Pinatubo aerosol cloud, focusing on the 1991-92 arctic vortex edge between 475-520K. Equivalent latitude is estimated at the observation times and locations from Eulerian model tracers initialised with PV and forced by UK Meteorological Office analyses. With the model formulation chosen, it is shown that tracer transport of a few days resulted in an error distribution that was much closer to Gaussian form, although the mean error was not

  15. Selective effects of carbamate pesticides on rat neuronal nicotinic acetylcholine receptors and rat brain acetylcholinesterase

    Effects of commonly used carbamate pesticides on rat neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. The potencies of these effects have been compared to the potencies of the carbamates to inhibit rat brain acetylcholinesterase. The potency order of six carbamates to inhibit α4β4 nicotinic receptors is fenoxycarb > EPTC > carbaryl, bendiocarb > propoxur > aldicarb with IC50 values ranging from 3 μM for fenoxycarb to 165 μM for propoxur and >1 mM for aldicarb. Conversely, the potency order of these carbamates to inhibit rat brain acetylcholinesterase is bendiocarb > propoxur, aldicarb > carbaryl >> EPTC, fenoxycarb with IC50 values ranging from 1 μM for bendiocarb to 17 μM for carbaryl and >>1 mM for EPTC and fenoxycarb. The α4β2, α3β4, and α3β2 nicotinic acetylcholine receptors are inhibited by fenoxycarb, EPTC, and carbaryl with potency orders similar to that for α4β4 receptors. Comparing the potencies of inhibition of the distinct subtypes of nicotinic acetylcholine receptors shows that the α3β2 receptor is less sensitive to inhibition by fenoxycarb and EPTC. The potency of inhibition depends on the carbamate as well as on a combination of α and β subunit properties. It is concluded that carbamate pesticides affect different subtypes of neuronal nicotinic receptors independently of acetylcholinesterase inhibition. This implicates that neuronal nicotinic receptors are additional targets for some carbamate pesticides and that these receptors may contribute to carbamate pesticide toxicology, especially after long-term exposure

  16. A neuronal acetylcholine receptor regulates the balance of muscle excitation and inhibition in Caenorhabditis elegans.

    Maelle Jospin

    2009-12-01

    Full Text Available In the nematode Caenorhabditis elegans, cholinergic motor neurons stimulate muscle contraction as well as activate GABAergic motor neurons that inhibit contraction of the contralateral muscles. Here, we describe the composition of an ionotropic acetylcholine receptor that is required to maintain excitation of the cholinergic motor neurons. We identified a gain-of-function mutation that leads to spontaneous muscle convulsions. The mutation is in the pore domain of the ACR-2 acetylcholine receptor subunit and is identical to a hyperactivating mutation in the muscle receptor of patients with myasthenia gravis. Screens for suppressors of the convulsion phenotype led to the identification of other receptor subunits. Cell-specific rescue experiments indicate that these subunits function in the cholinergic motor neurons. Expression of these subunits in Xenopus oocytes demonstrates that the functional receptor is comprised of three alpha-subunits, UNC-38, UNC-63 and ACR-12, and two non-alpha-subunits, ACR-2 and ACR-3. Although this receptor exhibits a partially overlapping subunit composition with the C. elegans muscle acetylcholine receptor, it shows distinct pharmacology. Recordings from intact animals demonstrate that loss-of-function mutations in acr-2 reduce the excitability of the cholinergic motor neurons. By contrast, the acr-2(gf mutation leads to a hyperactivation of cholinergic motor neurons and an inactivation of downstream GABAergic motor neurons in a calcium dependent manner. Presumably, this imbalance between excitatory and inhibitory input into muscles leads to convulsions. These data indicate that the ACR-2 receptor is important for the coordinated excitation and inhibition of body muscles underlying sinusoidal movement.

  17. Partial nicotinic acetylcholine (α4β2 agonists as promising new medications for smoking cessation

    Singh J

    2008-01-01

    Full Text Available Objective: To review the pharmacology, clinical efficacy and safety of partial agonists of a4β 2 nicotinic acetylcholine receptor. Data Sources: Primary literature and review articles were obtained via a PUBMED search (1988-August 2006 using the key terms smoking cessation, partial agonist alpha4beta2 nicotinic acetylcholine receptor, varenicline, cytisine and SSR591813. Additional studies and abstracts were identified from the bibliographies of reviewed literature. Study Selection and Data Extraction: Studies and review articles related to varenicline, cytisine and the partial agonist alpha4beta2 nicotinic acetylcholine receptor were reviewed. Data Synthesis: Smoking is widely recognized as a serious health problem. Smoking cessation has major health benefits. According to the US Public Health Services, all patients attempting to quit smoking should be encouraged to use one or more effective pharmacotherapy. Currently, along with nicotine replacement therapy, bupropion, nortriptyline and clonidine, are the mainstay of pharmacotherapy. More than ¾ of patients receiving treatment for smoking cessation return to smoking within the first year. Nicotine, through stimulating α4β 2 nAChR, releases dopamine in the reward pathway. Partial agonist of α4β 2 nAChR elicits moderate and sustained release of dopamine, which is countered during the cessation attempts; it simultaneously blocks the effects of nicotine by binding with α4β 2 receptors during smoking. Recently, varenicline, a partial agonist at α4β 2 nAChR, has been approved by the FDA (Food and Drug Administration for smoking cessation. Conclusion: Partial agonist α4β 2 nAChR appears to be a promising target in smoking cessation. Varenicline of this group is approved for treatment of smoking cessation by the FDA in May 2006.

  18. Temperature effect on proximal to distal gradient of quantal release of acetylcholine at frog endplate

    Samigullin, D.; Bukharaeva, E.; Nikolsky, E.; Vyskočil, František

    2003-01-01

    Roč. 28, 3-4 (2003), s. 507-514. ISSN 0364-3190 R&D Projects: GA AV ČR IAA7011902; GA ČR GA305/02/1333; GA ČR GA202/02/1213 Grant ostatní: RFBR(RU) 02/04/48901 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100003 Keywords : neuromuscular junction * acetylcholine release * temperature Subject RIV: ED - Physiology Impact factor: 1.511, year: 2003

  19. INFLUENCE OF ANTIBIOTICS ON THE MECHANICAL RESPONSES OF GUINEA-PIG ILEUM TO ACETYLCHOLINE AND HISTAMINE

    Petroianu Andy

    1998-01-01

    Full Text Available The side effects of antibiotics have been extensively described during the last decades, however, their role on digestive motility must be better investigated. Following a line of research, the influence of penicillin, chloranfenicol tetracycline and gentamicine on longitudinal smooth muscle responses to acetylcholine and histamine were studied on guinea-pig ileum. There were no differences between the responses before and after the addition of each antibiotic. Further investigations must be performed in order to find a possible influence of antibiotics on digestive motility.

  20. Catecholamines and acetylcholine are key regulators of the interaction between microbes and the immune system.

    Weinstein, Leon Islas; Revuelta, Alberto; Pando, Rogelio Hernandez

    2015-09-01

    Recent studies suggest that catecholamines (CAs) and acetylcholine (ACh) play essential roles in the crosstalk between microbes and the immune system. Host cholinergic afferent fibers sense pathogen-associated molecular patterns and trigger efferent cholinergic and catecholaminergic pathways that alter immune cell proliferation, differentiation, and cytokine production. On the other hand, microbes have the ability to produce and degrade ACh and also regulate autogenous functions in response to CAs. Understanding the role played by these neurotransmitters in host-microbe interactions may provide valuable information for the development of novel therapies. PMID:26378438

  1. Prostate stem cell antigen interacts with nicotinic acetylcholine receptors and is affected in Alzheimer's disease

    Jensen, Majbrit Myrup; Mikkelsen, Jens D.; Arvaniti, Maria; Pinborg, Lars Hageman; Thomsen, Morten Skøtt

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder involving impaired cholinergic neurotransmission and dysregulation of nicotinic acetylcholine receptors (nAChRs). Ly-6/neurotoxin (Lynx) proteins have been shown to modulate cognition and neural plasticity by binding to nAChR subtypes and...... are present in the human brain. We further showed that PSCA forms stable complexes with the α4 nAChR subunit and decreases nicotine-induced extracellular-signal regulated kinase phosphorylation in PC12 cells. In addition, we analyzed protein levels of PSCA and Lypd6 in postmortem tissue of medial...

  2. alpha4beta2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief

    McGranahan, Tresa M.; Patzlaff, Natalie E.; Grady, Sharon R; Heinemann, Stephen F.; Booker, T.K.

    2011-01-01

    Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbra...

  3. The role of alpha4 containing nicotinic acetylcholine receptors in dopamine neurons

    McGranahan, Tresa Michelle

    2011-01-01

    Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and, thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4- containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midb...

  4. The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori

    Zhang Chuan-Xi; Dong Ke; Shao Ya-Ming

    2007-01-01

    Abstract Background Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic transmission in the insect central nervous system. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Like mammalian nAChRs, insect nAChRs are considered to be made up of five subunits, coded by homologous genes belonging to the same family. The nAChR subunit genes of Drosophila melanogaster, Apis mellifera and Anopheles gambiae have been cloned previously based o...

  5. Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states

    Arias, Hugo R.; Rosenberg, Avraham; Feuerbach, Dominik; Targowska-Duda, Katarzyna M.; Maciejewski, Ryszard; Jozwiak, Krzysztof; Moaddel, Ruin; Glick, Stanley D.; Wainer, Irving W.

    2010-01-01

    The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (±)-epibatidine-induced AChR Ca2+ influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [3H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in...

  6. Changes in acetylcholine release from the chick retina are not associated with myopia development

    Full text: The effectiveness of muscarinic receptor antagonists in inhibiting myopia progression in animal models and humans implicates cholinergic signalling in ocular growth regulation. Therefore to determine if changes in the release of acetylcholine from the retina are involved in myopia development, the efflux of acetylcholine from the in vitro retina of normal and myopic chick eyes was investigated. Chicks were monocularly deprived (MD) of pattern vision with translucent occluders for 2 or 7 days and refractive error of MD groups and age matched normals was monitored using retinoscopy (n=6 each group). 3H-choline-Cl (1 Ci in 7μL) was injected into the vitreous of each eye under 2.5% halothane anaesthesia. After 1hr, the eyes were enucleated, under terminal anaesthesia (sodium pentobarbital, 120 mg/kg, im). Retinas were flat-mounted on acetate filter discs and superfused with oxygenated physiological saline solution (PSS) for 30min at 0.4mL/min. Five baseline fractions were collected (B1-B5), then three stimulated fractions were collected in the presence of PSS containing 50mM KCl (K1-K3) at 2min intervals. 3H-acetylcholine ( 3H-ACh) in each fraction was quantified by liquid scintillation counting. Significant amounts of myopia were induced in MD eyes after 2 (-5.1±0.8D) and 7 days (-18.8±2.4D) relative to control eyes (paired t-test p3H-ACh release was 146±15% above basal levels (K2/B1%) from retinas of normal animals. After 2 days MD, there was no significant difference between KCl-evoked release of 3H-ACh from deprived eyes (147 39%) compared to control eyes (198±61%, paired t-test, p=0.27) or the eyes of normal animals (ANOVA, p>0.5). Similar results were obtained following 7 days MD. The results demonstrate that evoked acetylcholine release from the chick retina of myopic eyes is unaltered relative to control or normal eyes using an in vitro approach. Copyright (2002) Australian Neuroscience Society

  7. Block by acetylcholine of mouse muscle nicotinic receptors, stably expressed in fibroblasts

    1995-01-01

    We have measured the concentration and voltage dependence of block by acetylcholine (ACh) of fetal- and adult-type mouse muscle nicotinic receptors, expressed in a fibroblast cell line. Data, obtained at a transmembrane potential of -60 mV and with ACh concentrations of 1 mM and above, are broadly consistent with the occlusion of an open channel with a single ACh+ ion (simple open channel block). The rate of recovery from block is approximately 40,000s-1 and has only a weak voltage dependence...

  8. Synthesis, Nicotinic Acetylcholine Receptor Binding, and Pharmacological Properties of 3’- (Substituted phenyl) Deschloroepibatidine Analogs

    F. Ivy Carroll; Yokota, Yasuno; Ma, Wei; Lee, Jeffrey R.; Brieaddy, Lawrence E.; Burgess, Jason P.; Navarro, Hernán A.; Damaj, M. I.; Martin, Billy R.

    2007-01-01

    A series of 3’-(substituted phenyl)deschloroepibatidine analogs (5a–j) were synthesized. The α4β2* and α7 nicotinic acetylcholine receptor (nAChR) binding properties and functional activity in the tail-flick, hot-plate, locomotor, and body temperature tests in mice of 5a–j were compared to those of the nAChR agonist, nicotine (1), epibatidine (4), and deschloroepibatidine (13) the partial agonist, varenicline (3) and the antagonist 2’-fluoro-3’-(substituted phenyl)deschloroepibatidine analogs...

  9. Spontaneous quantal and non-quantal release of acetylcholine at mouse endplate during onset of hypoxia

    Bukharaeva, E.A.; Salakhutdinov, R.I.; Vyskočil, František; Nikolsky, E.E.

    2005-01-01

    Roč. 54, č. 2 (2005), s. 251-255. ISSN 0862-8408 R&D Projects: GA AV ČR(CZ) IAA5011411; GA ČR(CZ) GA305/02/1333 Grant ostatní: RFBR(RU) 05-04-49723; Scientific Schools of Russia(RU) 1063.2003.4 Institutional research plan: CEZ:AV0Z50110509 Keywords : hypoxia * non-quantal * acetylcholine Subject RIV: ED - Physiology Impact factor: 1.806, year: 2005

  10. Regional distribution of muscarinic acetylcholine receptors in the telencephalon of the pigeon (Columba livia f. domestica)

    The distribution of muscarinic acetylcholine receptors was studied autoradiographically in croystat sections of the pigeon telencephalon using 3H-quinuclidinylbenzylate as a ligand. Highest receptor density was observed in the hyperstriatum ventrale, palaeostriatum augmentatum, septum, and parts of the archistriatum. In sites of known sensory input of neostriatum (field L) and ectostriatum low receptor binding was observed. Acetylcholinesterase distribution is in good agreement with the receptor picture only in the basal telencephalon. In the pallium differences in the pattern of these two components can be seen. (author)

  11. Decreased cerebral α4β2* nicotinic acetylcholine receptor availability in patients with mild cognitive impairment and Alzheimer's disease assessed with positron emission tomography

    Postmortem studies indicate a loss of nicotinic acetylcholine receptor (nAChRs) in Alzheimer's disease (AD). In order to establish whether these changes in the cholinergic system occur at an early stage of AD, we carried out positron emission tomography (PET) with a specific radioligand for the α4β2* nicotinic acetylcholine receptor (α4β2* nAChR) in patients with mild to moderate AD and in patients with amnestic mild cognitive impairment (MCI), who have a high risk to progress to AD. Nine patients with moderate AD, eight patients with MCI and seven age-matched healthy controls underwent 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[18F]FA-85380) PET. After coregistration with individual magnetic resonance imaging the binding potential (BPND) of 2-[18F]FA-85380 was calculated using either the corpus callosum or the cerebellum as reference regions. PET data were analysed by region of interest analysis and by voxel-based analysis. Both patients with AD and MCI showed a significant reduction in 2-[18F]FA-85380 BPND in typical AD-affected brain regions. Thereby, the corpus callosum was identified as the most suitable reference region. The 2-[18F]FA-85380 BPND correlated with the severity of cognitive impairment. Only MCI patients that converted to AD in the later course (n = 5) had a reduction in 2-[18F]FA-85380 BPND. 2-[18F]FA-85380 PET appears to be a sensitive and feasible tool for the detection of a reduction in α4β2* nAChRs which seems to be an early event in AD. In addition, 2-[18F]FA-85380 PET might give prognostic information about a conversion from MCI to AD. (orig.)

  12. Decreased cerebral {alpha}4{beta}2* nicotinic acetylcholine receptor availability in patients with mild cognitive impairment and Alzheimer's disease assessed with positron emission tomography

    Kendziorra, Kai; Meyer, Philipp Mael; Barthel, Henryk; Hesse, Swen; Becker, Georg Alexander; Luthardt, Julia; Schildan, Andreas; Patt, Marianne; Sorger, Dietlind; Seese, Anita; Sabri, Osama [University of Leipzig, Department of Nuclear Medicine, Leipzig (Germany); Wolf, Henrike [University of Leipzig, Department of Psychiatry, Leipzig (Germany); University of Zurich, Department of Old Age Psychiatry and Psychiatry Research, Psychiatric University Hospital (PUK) Zurich, Zurich (Switzerland); Gertz, Herman-Josef [University of Leipzig, Department of Psychiatry, Leipzig (Germany)

    2011-03-15

    Postmortem studies indicate a loss of nicotinic acetylcholine receptor (nAChRs) in Alzheimer's disease (AD). In order to establish whether these changes in the cholinergic system occur at an early stage of AD, we carried out positron emission tomography (PET) with a specific radioligand for the {alpha}4{beta}2* nicotinic acetylcholine receptor ({alpha}4{beta}2* nAChR) in patients with mild to moderate AD and in patients with amnestic mild cognitive impairment (MCI), who have a high risk to progress to AD. Nine patients with moderate AD, eight patients with MCI and seven age-matched healthy controls underwent 2-[{sup 18}F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[{sup 18}F]FA-85380) PET. After coregistration with individual magnetic resonance imaging the binding potential (BP{sub ND}) of 2-[{sup 18}F]FA-85380 was calculated using either the corpus callosum or the cerebellum as reference regions. PET data were analysed by region of interest analysis and by voxel-based analysis. Both patients with AD and MCI showed a significant reduction in 2-[{sup 18}F]FA-85380 BP{sub ND} in typical AD-affected brain regions. Thereby, the corpus callosum was identified as the most suitable reference region. The 2-[{sup 18}F]FA-85380 BP{sub ND} correlated with the severity of cognitive impairment. Only MCI patients that converted to AD in the later course (n = 5) had a reduction in 2-[{sup 18}F]FA-85380 BP{sub ND}. 2-[{sup 18}F]FA-85380 PET appears to be a sensitive and feasible tool for the detection of a reduction in {alpha}4{beta}2* nAChRs which seems to be an early event in AD. In addition, 2-[{sup 18}F]FA-85380 PET might give prognostic information about a conversion from MCI to AD. (orig.)

  13. An extract of lionfish (Pterois volitans) spine tissue contains acetylcholine and a toxin that affects neuromuscular transmission.

    Cohen, A S; Olek, A J

    1989-01-01

    A soluble toxic extract derived from spine tissue of the lionfish (Pterois volitans) decreased heart rate and force of contraction in isolated clam and frog hearts. These actions were due to the presence of micromolar concentrations of acetylcholine in the extract. Toxicity was retained after hydrolysis of acetylcholine by exogenous acetylcholinesterase, but heart function was no longer affected. Toxin treated in this way induced muscle fibrillation in an isolated nerve-muscle preparation, followed by blockade of neuromuscular transmission. Bursts of transient depolarizations were recorded at the muscle endplate shortly after toxin addition that correlated in time with the duration of toxin-induced muscle fibrillation. These effects are thought to be due to the increased release and then depletion of acetylcholine from the nerve terminal. PMID:2560846

  14. Topological dispositions of lysine α380 and lysine γ486 in the acetylcholine receptor from Torpedo californica

    The locations have been determined, with respect to the plasma membrane, of lysine α380 and lysine γ486 in the α subunit and the γ subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the α subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the γ subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [3H]-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine α380 and lysine γ486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine α380 is on the inside surface of a vesicle and lysine γ486 is on the outside surface. Because a majority (85%) of the total binding sites for α-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine α380 and lysine γ486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor

  15. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    Dwyer, B.P. (Univ. of California, San Diego, La Jolla (USA))

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  16. Noninvasive detection by ATR and NIR-DR methods for skin-care ionic materials transported into the skin by iontophoresis

    Ueda, Toyotoshi; Watanabe, Yukio; Akao, Ken-ichi; Suzuki, Harue

    2003-12-01

    Two analytical methods without damage to the skin were proposed in order to detect and measure the quantity of the medication transported into the skin by the iontophoresis. The infrared attenuated total reflection (ATR) method was proven to be able to evaluate the content of such a substance as sodium all- trans-retinoate or magnesium ℓ-ascorbyl-2-phosphate in the top (horny) layer of epidermis (about 1 μm under the skin surface), using characteristic bands to the above ion. Another method of near-infrared diffusive-reflection (NIR-DR) technique was shown probably to detect it in the dermis (1 mm under the surface), based on the shift of frequency and the change in intensity for the vibrational combination band of water molecules hydrating the ion. The quantity of the above material decreased monotonically in the horny layer for several hours after the treatment, while in the dermis it increased at first and then decreased via the maximum value.

  17. Nicotinic acetylcholine receptor polymorphism, smoking behavior, and tobacco-related cancer and lung and cardiovascular diseases: a cohort study

    Kaur-Knudsen, Diljit; Bojesen, Stig E; Tybjærg-Hansen, Anne; Nordestgaard, Børge G

    2011-01-01

    We examined the associations between the nicotinic acetylcholine receptor polymorphism (rs1051730) on chromosome 15q25 marking the gene cluster CHRNA3-CHRNB4-CHRNA5, smoking behavior, and tobacco-related cancer and lung and cardiovascular diseases in the general population.......We examined the associations between the nicotinic acetylcholine receptor polymorphism (rs1051730) on chromosome 15q25 marking the gene cluster CHRNA3-CHRNB4-CHRNA5, smoking behavior, and tobacco-related cancer and lung and cardiovascular diseases in the general population....

  18. Contrasting Effects of Allosteric and Orthosteric Agonists on M1 Muscarinic Acetylcholine Receptor Internalization and Down-regulation

    Thomas, Rachel L.; Christopher J Langmead; Wood, Martyn D; Challiss, R.A. John

    2009-01-01

    A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allosteric [4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine (AC-42); 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1)] agonists on M1 mAC...

  19. Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway

    AMONYINGCHAROEN, SUMET; SURIYO, TAWIT; THIANTANAWAT, APINYA; WATCHARASIT, PIYAJIT; SATAYAVIVAD, JUTAMAAD

    2015-01-01

    Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1–40 μM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth. PMID:25815516

  20. Interfacial Recognition of Acetylcholine by an Amphiphilic p-Sulfonatocalix[8]arene Derivative Incorporated into Dimyristoyl Phosphatidylcholine Vesicles

    Yasuhiro Ooi

    2008-10-01

    Full Text Available Dodecyl ether derivatives 1-3 of p-sulfonatocalix[n]arene were incorporated into dimyristoyl phosphatidylcholine (DMPC vesicles, and their binding abilities for acetylcholine (ACh were examined by using steady-state fluorescence/fluorescence anisotropy and fluorescence correlation spectroscopy (FCS. For the detection of ACh binding to the DMPC vesicles containing 5 mol % of 1-3, competitive fluorophore displacement experiments were performed, where rhodamine 6G (Rh6G was used as a fluorescent guest. The addition of Rh6G to the DMPC vesicles containing 3 resulted in a decrease in the fluorescence intensity of Rh6G with an increase of its fluorescence anisotropy, indicating that Rh6G binds to the DMPC-3 vesicles. In the case of DMPC-1 and DMPC-2 vesicles, significant changes in the fluorescence spectra of Rh6G were not observed. When ACh was added to the DMPC-3 vesicles in the presence of Rh6G ([3]/[Rh6G]=100, the fluorescence intensity of Rh6G increased with a decrease in its fluorescence anisotropy. From the analysis of fluorescence titration data, the association constants were determined to be 7.1×105 M-1 for Rh6G-3 complex and 1.1×102 M-1 for ACh-3 complex at the DMPC-3 vesicles. To get a direct evidence for the binding of Rh6G and its displacement by ACh at the DMPC-3 vesicles, diffusion times of the Rh6G were measured by using FCS. Binding selectivity of the DMPC-3 vesicles for ACh, choline, GABA, L-aspartic acid, L-glutamic acid, L-arginine, L-lysine, L-histamine and ammonium chloride was also evaluated using FCS.

  1. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    Miles, K.; Anthony, D.T.; Rubin, L.L.; Greengard, P.; Huganir, R.L.

    1987-09-01

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with (/sup 32/P)orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the ..beta.. and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the ..cap alpha.. subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 ..mu..M in the presence of 35 ..mu..M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of ..cap alpha.. subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.

  2. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with [32P]orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the β and δ subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the δ subunit 20-fold over basal phosphorylation and induced phosphorylation of the α subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 μM in the presence of 35 μM Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of δ subunit phosphorylation was almost maximal within 5 min, whereas stimulation of α subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the δ subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the δ subunit increases the rate of AcChoR desensitization in rat myotubes

  3. Characterization of a putative acetylcholine receptor in chick ciliary ganglion neurons

    Monoclonal antibodies to the main immunogenic region on the alpha subunit of acetylcholine receptors in muscle and electric organ recognize membrane components in chick brain and ciliary ganglia that are candidates for the neuronal receptor. The component in chick brain has been purified by immunoaffinity chromatography. It specifically binds nicotine but not alpha-bungarotoxin, and can be affinity labeled with (3H)bromoacetylcholine. The cross-reacting component in ciliary ganglion neurons is concentrated in synaptic membrane, and can be modulated by exposure of the cells to cholinergic ligands in culture. The cross-reacting component in ciliary ganglion neurons is an integral membrane component that binds concanavalin A, and it is distinct from the alpha-bungarotoxin binding component. The acetylcholine receptor function in these neurons can be locked by affinity alkylation with bromoacetylcholine, indicating similarity in this respect to receptors from muscle and electric organ. Antisera raised against the partially purified component from chick brain also block receptor function on ciliary ganglion neurons. The subcellular distribution of the ganglion component in culture is assessed, and it is shown that approximately 2/3 of the cross-reacting components are intracellular; the majority of these seem not to be destined for insertion into the plasma membrane

  4. Regulation of phosphorylation of nicotinic acetylcholine receptors in mouse BC3H1 myocytes

    Smith, M.M.; Merlie, J.P.; Lawrence, J.C. Jr.

    1987-09-01

    By using /sup 32/P-labeling methods and performing immunoprecipitations with specific antibodies, the authors have found that three subunits of the nicotinic acetylcholine receptor and phosphorylated in mouse skeletal muscle cells. In nonstimulated cells, the molar ratios of phosphate estimated in ..cap alpha.., ..beta.., and delta subunits were 0.02, 0.05, and 0.5, respectively. All three subunits contained predominantly phosphoserine with some phosphothreonine; the ..beta.., subunit also contained phosphotyrosine. Incubating cells with agents that stimulate cAMP-dependent pathways (isoproterenol, forskolin, 8-Br-cAMP) increased the phosphorylation of the delta subunit by 50%, but phosphate labeling of the ..beta.. subunit was depressed by a third. In contrast, when cells were incubated with the divalent cation ionophores A-23187 or ionomycin, phosphorylation of both the delta and ..beta.. subunits increased. The results indicate that acetylcholine receptors are phosphorylated to significant levels in skeletal muscle cells and that cAMP-dependent and Ca/sup 2 +/-dependent pathways exist for controlling the phosphorylation state of the receptor subunits.

  5. The Anti-Acetylcholine Receptor Antibody Test in Suspected Ocular Myasthenia Gravis

    Jung Jin Lee

    2014-01-01

    Full Text Available Aim. To estimate the clinical significance of anti-acetylcholine receptor antibody (anti-AChR-Ab levels in suspected ocular myasthenia gravis. Methods. In total, 144 patients complaining of fluctuating diplopia and ptosis were evaluated for serum levels of anti-acetylcholine receptor antibody and their medical charts were retrospectively reviewed. Subjects were classified into three groups: variable diplopia only, ptosis only, and both variable diplopia and ptosis. We investigated serum anti-AChR-Ab titer levels and performed thyroid autoantibody tests. Results. Patients’ chief complaints were diplopia (N=103, ptosis (N=12, and their concurrence (N=29. Abnormal anti-AChR-Ab was observed in 21 of 144 patients (14.1%. Between the three groups, mean age, number of seropositive patients, and mean anti-AChR-Ab level were not significantly different (P=0.224, 0.073, and 0.062, resp.. Overall, 27.5% of patients had abnormal thyroid autoantibodies. Conclusion. The sensitivity of anti-AChR-Ab was 14.1% in suspected ocular myasthenia gravis and seropositivity in myasthenia gravis patients showed a high correlation with the presence of thyroid autoantibodies.

  6. Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

    Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+]i) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of Gαq/11-coupled M1, M3 and M5 receptors and intracellular calcium stores, whereas Gαi/o-protein coupled M2 receptor activity mediated neuronal differentiation

  7. Relation between Pro-inflammatory Cytokines and Acetylcholine Levels in Relapsing-Remitting Multiple Sclerosis Patients

    Ada Maria Tata

    2012-10-01

    Full Text Available Multiple sclerosis (MS is a chronic inflammatory, demyelinating and neurodegenerative disorder. Since acetylcholine (ACh is known to participate in the inflammatory response, we investigated the possible relationship between pro-inflammatory cytokines and acetylcholine levels in relapsing-remitting multiple sclerosis (RR-MS patients. Levels of ACh and pro-inflammatory cytokines IL1-β and IL-17 were measured both in cerebrospinal fluid (CSF and sera of 22 RR-MS patients in the relapsing phase and in 17 control subjects affected by other non-neurological diseases (OND. We observed higher levels of pro-inflammatory cytokines such as IL-1β and IL-17 in both CSF and serum of RR-MS patients compared to control subjects. Moreover, ACh levels were lower in CSF and serum of RR-MS patients compared to levels of control subjects. Although the relationship between high inflammatory cytokine levels and low ACh levels need to be further investigated in the future, our data suggest that IL-1β, and cytokines induced by it, such as IL-17 and ACh, may be involved in the pathogenesis of MS.

  8. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors.

    Thal, David M; Sun, Bingfa; Feng, Dan; Nawaratne, Vindhya; Leach, Katie; Felder, Christian C; Bures, Mark G; Evans, David A; Weis, William I; Bachhawat, Priti; Kobilka, Tong Sun; Sexton, Patrick M; Kobilka, Brian K; Christopoulos, Arthur

    2016-03-17

    Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains. PMID:26958838

  9. Luminescent silica nanoparticles for sensing acetylcholinesterase-catalyzed hydrolysis of acetylcholine.

    Mukhametshina, Alsu R; Fedorenko, Svetlana V; Zueva, Irina V; Petrov, Konstantin A; Masson, Patrick; Nizameev, Irek R; Mustafina, Asiya R; Sinyashin, Oleg G

    2016-03-15

    This work highlights the H-function of Tb(III)-doped silica nanoparticles in aqueous solutions of acetic acid as a route to sense acetylcholinesterase-catalyzed hydrolysis of acetylcholine (ACh). The H-function results from H(+)-induced quenching of Tb(III)-centered luminescence due to protonation of Tb(III) complexes located close to silica/water interface. The H-function can be turned on/switched off by the concentration of complexes within core or nanoparticle shell zones, by the silica surface decoration and adsorption of both organic and inorganic cations on silica surface. Results indicate the optimal synthetic procedure for making nanoparticles capable of sensing acetic acid produced by enzymatic hydrolysis of acetylcholine. The H-function of nanoparticles was determined at various concentrations of ACh and AChE. The measurements show experimental conditions for fitting the H-function to Michaelis-Menten kinetics. Results confirm that reliable fluorescent monitoring AChE-catalyzed hydrolysis of ACh is possible through the H-function properties of Tb(III)-doped silica nanoparticles. PMID:26516688

  10. Auto-transporter A protein of Neisseria meningitidis: a potent CD4+ T-cell and B-cell stimulating antigen detected by expression cloning.

    Ait-Tahar, K; Wooldridge, K G; Turner, D P; Atta, M; Todd, I; Ala'Aldeen, D A

    2000-09-01

    A meningococcal genomic expression library was screened for potent CD4+ T-cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto-transporter A) as its peptide sequence shares molecular characteristics of the auto-transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T-cell responses in PBLs of patients and some healthy donors, and induced strong primary T-cell responses in healthy donors. The human B-cell immunogenicity and cross-reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross-reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB-specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross-react with AutA. Thus, AutA, being a potent CD4+ T-cell and B-cell-stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate. PMID:10972828

  11. Hippocampal α7 nicotinic acetylcholine receptor levels in patients with schizophrenia, bipolar disorder, or major depressive disorder

    Thomsen, Morten Skøtt; Weyn, Annelies; Mikkelsen, Jens D

    The α7 nicotinic acetylcholine receptor (nAChR) is involved in cognitive function and synaptic plasticity. Consequently, changes in α7 nAChR function have been implicated in a variety of mental disorders, especially schizophrenia. However, there is little knowledge regarding the levels of the α7 n...

  12. Changes in Temperature Have Opposing Effects on Current Amplitude in alpha 7 and alpha 4 beta 2 Nicotinic Acetylcholine Receptors

    Jindřichová, Marie; Lansdell, S. J.; Millar, N. S.

    2012-01-01

    Roč. 7, č. 2 (2012), e32073. E-ISSN 1932-6203 Institutional research plan: CEZ:AV0Z50110509 Keywords : effect of temperature * nicotinic acetylcholine receptor * voltage - clamp recording Subject RIV: ED - Physiology Impact factor: 3.730, year: 2012

  13. An allosteric enhancer of M4muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine

    Dencker, Ditte; Weikop, Pia; Sørensen, Gunnar;

    2012-01-01

    The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M4 acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M4 receptors could be...

  14. Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor

    Grant, M.K.O.; Noetzel, M.J.; De Lorme, K.C.; Jakubík, Jan; Doležal, Vladimír; El-Fakahany, E. E.

    2010-01-01

    Roč. 5, č. 12 (2010), e15722-16. E-ISSN 1932-6203 R&D Projects: GA ČR GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : Xanomeline * muscarinic acetylcholine receptor Subject RIV: CE - Biochemistry Impact factor: 4.411, year: 2010

  15. Utrophin abundance is reduced at neuromuscular junctions of patients with both inherited and acquired acetylcholine receptor deficiencies

    Slater, CR; Young, C; Wood, SJ; Bewick, GS; Anderson, LVB; Baxter, P; Fawcett, PRW; Roberts, M; Jacobson, L; Kuks, J; Vincent, A; NewsomDavis, J

    1997-01-01

    Congenital myasthenic syndromes are a heterogenous group of conditions in which muscle weakness resulting from impaired neuromuscular transmission is often present from infancy. One form of congenital myasthenic syndrome is due to a reduction of the number of acetylcholine receptors (AChRs) at the n

  16. Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor

    Absalom, Nathan L; Quek, Gracia; Lewis, Trevor M;

    2013-01-01

    The α4β2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, with different sensitivities to acetylcholine (ACh), but their pharmacologi......The α4β2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, with different sensitivities to acetylcholine (ACh), but their...... competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4β2 receptor...... containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(β2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(β2)2 and...

  17. A subpopulation of neuronal M4 muscarinic acetylcholine receptors plays a critical role in modulating dopamine-dependent behaviors

    Jeon, Jongrye; Nielsen, Ditte Dencker; Wörtwein, Gitta;

    2010-01-01

    Acetylcholine (ACh) regulates many key functions of the CNS by activating cell surface receptors referred to as muscarinic ACh receptors (M(1)-M(5) mAChRs). Like other mAChR subtypes, the M(4) mAChR is widely expressed in different regions of the forebrain. Interestingly, M(4) mAChRs are coexpres...

  18. Determination of anti-acetylcholine receptor antibodies in myasthenic patients by use of time-resolved fluorescence

    Říčný, Jan; Šimková, L.; Vincent, A.

    2002-01-01

    Roč. 48, č. 3 (2002), s. 549-554. ISSN 0009-9147 R&D Projects: GA MZd NF4646 Institutional research plan: CEZ:AV0Z5011922 Keywords : nicotinic acetylcholine receptor * time-resolved fluorescence method * myasthenia gravis Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.788, year: 2002

  19. Gamma-lactams--a novel scaffold for highly potent and selective alpha 7 nicotinic acetylcholine receptor agonists.

    Enz, Albert; Feuerbach, Dominik; Frederiksen, Mathias U; Gentsch, Conrad; Hurth, Konstanze; Müller, Werner; Nozulak, Joachim; Roy, Bernard L

    2009-03-01

    A novel class of alpha7 nicotinic acetylcholine receptor (nAChR) agonists has been discovered through high-throughput screening. The cis gamma-lactam scaffold has been optimized to reveal highly potent and selective alpha7 nAChR agonists with in vitro activity and selectivity and with good brain penetration in mice. PMID:19208472

  20. Maximal acetylcholine dose of 200 μg into the left coronary artery as a spasm provocation test: comparison with 100 μg of acetylcholine.

    Sueda, Shozo; Kohno, Hiroaki; Miyoshi, Toru; Sakaue, Tomoki; Sasaki, Yasuhiro; Habara, Hirokazu

    2015-11-01

    As a spasm provocation test of acetylcholine (ACH), incremental dose up (20/50/100 μg) into the left coronary artery (LCA) is recommended in the guidelines established by Japanese Circulation Society. Recently, Ong et al. reported the ACOVA study which maximal ACH dose was 200 μg in the LCA. We compared the angiographic findings between ACH 100 μg and ACH 200 μg in the LCA and also examined the usefulness and safety of ACH 200 μg in Japanese patients without variant angina. As a spasm provocation test, we performed intracoronary injection of ACH 200 μg after ACH 100 μg in 88 patients (55 males, 68.4 ± 11.7 years old) including 59 ischemic heart disease (IHD) patients and 29 non-IHD patients. Positive spasm was defined as >99 % transient stenosis (focal spasm) or 90 % severe diffuse vasoconstriction (diffuse spasm). Positive spasm by ACH 200 μg was significantly higher than that by ACH 100 μg (36 pts: 40.9 % vs. 17 pts: 19.3 %, p LCA was safe and useful. We may reexamine the maximal ACH dose into the LCA. PMID:25179297

  1. Activation of the Macrophage α7 Nicotinic Acetylcholine Receptor and Control of Inflammation.

    Báez-Pagán, Carlos A; Delgado-Vélez, Manuel; Lasalde-Dominicci, José A

    2015-09-01

    Inflammatory responses to stimuli are essential body defenses against foreign threats. However, uncontrolled inflammation may result in serious health problems, which can be life-threatening. The α7 nicotinic acetylcholine receptor, a ligand-gated ion channel expressed in the nervous and immune systems, has an essential role in the control of inflammation. Activation of the macrophage α7 receptor by acetylcholine, nicotine, or other agonists, selectively inhibits production of pro-inflammatory cytokines while leaving anti-inflammatory cytokines undisturbed. The neural control of this regulation pathway was discovered recently and it was named the cholinergic anti-inflammatory pathway (CAP). When afferent vagus nerve terminals are activated by cytokines or other pro-inflammatory stimuli, the message travels through the afferent vagus nerve, resulting in action potentials traveling down efferent vagus nerve fibers in a process that eventually leads to macrophage α7 activation by acetylcholine and inhibition of pro-inflammatory cytokines production. The mechanism by which activation of α7 in macrophages regulates pro-inflammatory responses is subject of intense research, and important insights have thus been made. The results suggest that activation of the macrophage α7 controls inflammation by inhibiting NF-κB nuclear translocation, and activating the JAK2/STAT3 pathway among other suggested pathways. While the α7 is well characterized as a ligand-gated ion channel in neurons, whole-cell patch clamp experiments suggest that α7's ion channel activity, defined as the translocation of ions across the membrane in response to ligands, is absent in leukocytes, and therefore, ion channel activity is generally assumed not to be required for the operation of the CAP. In this perspective, we briefly review macrophage α7 activation as it relates to the control of inflammation, and broaden the current view by providing single-channel currents as evidence that the α7

  2. Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein

    Israel López-Reyes

    2010-01-01

    Full Text Available Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT. Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.

  3. Voltammetric detection of phytochelatin transported\

    Navrátil, Tomáš; Nováková, Kateřina; Josypčuk, Bohdan; Sokolová, Romana; Šestáková, Ivana

    2016-01-01

    Roč. 147, č. 1 (2016), s. 165-171. ISSN 0026-9247 R&D Projects: GA ČR(CZ) GAP208/12/1645 Grant ostatní: Rada Programu interní podpory projektů mezinárodní spolupráce AV ČR M200401201 Institutional support: RVO:61388955 Keywords : Barley protoplasts * electrochemical impendance spectroscopy * mercury electrode Subject RIV: CG - Electrochemistry Impact factor: 1.222, year: 2014

  4. Analogues of neuroactive polyamine wasp toxins that lack inner basic sites exhibit enhanced antagonism toward a muscle-type mammalian nicotinic acetylcholine receptor

    Stromgaard, K; Brierley, M J; Andersen, K; Sløk, F A; Mellor, I R; Usherwood, P N; Krogsgaard-Larsen, P; Jaroszewski, J W

    1999-01-01

    noncompetitively antagonized the nicotinic acetylcholine receptor (nAChR) in a concentration-, time-, and voltage-dependent manner. The amplitudes of acetylcholine-induced currents were compared at their peaks and at the end of a 1 s application in the presence or absence of the analogues. Most of the analogues...... properties (stepwise macroscopic pK(a) values) were determined by (13)C NMR titrations. All analogues are fully protonated at physiological pH. The effects of these compounds on acetylcholine-induced currents in TE671 cells clamped at various holding potentials were determined. All of the analogues...

  5. [3H]imidacloprid: synthesis of a candidate radioligand for the nicotinic acetylcholine receptor

    Imidacloprid is an exceptionally potent insecticide known from physiological studies to act at the nicotinic acetylcholine receptor. To prepare [3H]imidacloprid as a candidate radioligand, 6-chloronicotinoyl chloride was reduced with NaB2H4 (in model studies) or NaB3H4 in absolute ethanol to 2-chloro-5-pyridinylmethanol which was transformed to 2-chloro-5-chloromethylpyridine on refluxing with thionyl chloride. Coupling with 4,5-dihydro-N-nitro-1H-imidazol-2-amine then gave [2H2]imidacloprid incorporating about 95% of the deuterium or [3H2]imidacloprid (25 Ci/mmol) in 80% radiochemical yield. In studies not detailed here [3H] imidacloprid was found to undergo high affinity, specific and saturable binding to a site in insect brain. (author)

  6. Dynamic heterogeneity and non-Gaussian statistics for acetylcholine receptors on live cell membrane

    He, W.; Song, H.; Su, Y.; Geng, L.; Ackerson, B. J.; Peng, H. B.; Tong, P.

    2016-05-01

    The Brownian motion of molecules at thermal equilibrium usually has a finite correlation time and will eventually be randomized after a long delay time, so that their displacement follows the Gaussian statistics. This is true even when the molecules have experienced a complex environment with a finite correlation time. Here, we report that the lateral motion of the acetylcholine receptors on live muscle cell membranes does not follow the Gaussian statistics for normal Brownian diffusion. From a careful analysis of a large volume of the protein trajectories obtained over a wide range of sampling rates and long durations, we find that the normalized histogram of the protein displacements shows an exponential tail, which is robust and universal for cells under different conditions. The experiment indicates that the observed non-Gaussian statistics and dynamic heterogeneity are inherently linked to the slow-active remodelling of the underlying cortical actin network.

  7. Nicotinic Acetylcholine Receptor Gene Family of the Pea Aphid, Acyrthosiphon pisum

    LIU Yi-peng; LIN Ke-jian; LIU Yang; GUI Fu-rong; WANG Gui-rong

    2013-01-01

    The nicotinic acetylcholine receptors (nAchRs) are cholinergic receptors that form ligand-gated ion channels by ifve subunits in insect and vertebrate nervous systems. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Here, we identiifed and cloned 11 candidate nAChR subunit genes in Acyrthosiphon pisum using genome-based bioinformatics combined modern molecular techniques. Most A. pisum nAChRs including α1, α2, α3, α4, α6, α8, and β1 show highly sequence identities with the counterparts of other insects examined. Expression proifles analysis showed that all subunit genes were expressed in adult head. At least two subunits have alternative splicing that obviously increase A. pisum nicotinic receptor diversity. This study will be invaluable for exploring the molecular mechanisms of neonicotinoid-like insecticides in sucking pests, and for ultimately establishing the screening platform of novel insecticides.

  8. The effect of cooling on the acetylcholine-induced current of identified Helix pomatia Br neuron.

    Nedeljkovic, Miodrag; Kartelija, Gordana; Radenovic, Lidija; Todorovic, Natasa

    2005-05-01

    The Br neuron of the snail Helix pomatia, involved in neuronal regulation of various homeostatic and adaptive mechanisms, represents an interesting model for studying effects of temperature changes on neuronal activity of poikilotherms. The acetylcholine (ACh) induces a transient, inward dose-dependent current in the identified Br neuron. In the work presented, we analyses the effects of cooling on the ACh-induced inward current. The amplitude of ACh-induced inward current was markedly decreased after cooling and the speed of the decay of ACh response was decreased. Sensitivity to cooling of Ach-activated current on the Br neuron is mediated by a mechanism that does not involve change in the apparent receptor affinity or the cooperativity of binding. PMID:15759140

  9. Steroids induce acetylcholine receptors on cultured human muscle: Implications for myasthenia gravis

    Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. The authors show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. The results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also a direct effect on muscle. They propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications

  10. Neuronal nicotinic acetylcholine receptors serve as sensitive targets that mediate β-amyloid neurotoxicity

    Qiang LIU; Jie WU

    2006-01-01

    Alzheimer's disease (AD) is the most common form of brain dementia characterized by the accumulation of β-amyloid peptides (Aβ) and loss of forebrain cholinergic neurons. Aβ accumulation and aggregation are thought to contribute to cholinergic neuronal degeneration, in turn causing learning and memory deficits, but the specific targets that mediate Aβ neurotoxicity remain elusive. Recently, accumlating lines of evidence have demonstrated that Aβ directly modulates the function of neuronal nicotinic acetylcholine receptors (nAChRs), which leads to the new hypothesis that neuronal nAChRs may serve as important targets that mediate Aβ neurotoxicity. In this review, we summarize current studies performed in our laboratory and in others to address the question of how Aβ modulates neuronal nAChRs, especially nAChR subunit function.

  11. Steroids induce acetylcholine receptors on cultured human muscle: Implications for myasthenia gravis

    Kaplan, I.; Blakely, B.T.; Pavlath, G.K.; Travis, M.; Blau, H.M. (Stanford Univ. School of Medicine, CA (USA))

    1990-10-01

    Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. The authors show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. The results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also a direct effect on muscle. They propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.

  12. Neuronal nicotinic acetylcholine receptors: Common molecular substrates of nicotine and alcohol dependence

    AndrewR.Tapper

    2013-04-01

    Full Text Available Alcohol and nicotine are often co-abused. As many as 80-95% of alcoholics are also smokers, suggesting that ethanol and nicotine, the primary addictive component of tobacco smoke, may functionally interact in the central nervous system and/or share a common mechanism of action. While nicotine initiates dependence by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs, ligand-gated cation channels normally activated by endogenous acetylcholine (ACh, ethanol is much less specific with the ability to modulate multiple gene products including those encoding voltage-gated ion channels, and excitatory/inhibitory neurotransmitter receptors. However, emerging data indicate that ethanol interacts with nAChRs, both directly and indirectly, in the mesocorticolimbic dopaminergic (DAergic reward circuitry to affect brain reward systems. Like nicotine, ethanol activates DAergic neurons of the ventral tegmental area (VTA which project to the nucleus accumbens (NAc. Blockade of VTA nAChRs reduces ethanol-mediated activation of DAergic neurons, NAc DA release, consumption, and operant responding for ethanol in rodents. Thus, ethanol may increase ACh release into the VTA driving activation of DAergic neurons through nAChRs. In addition, ethanol potentiates distinct nAChR subtype responses to ACh and nicotine in vitro and in DAergic neurons. The smoking cessation therapeutic and nAChR partial agonist, varenicline, reduces alcohol consumption in heavy drinking smokers and rodent models of alcohol consumption. Finally, single nucleotide polymorphisms in nAChR subunit genes are associated with alcohol dependence phenotypes and smoking behaviors in human populations. Together, results from preclinical, clinical, and genetic studies indicate that nAChRs may have an inherent role in the abusive properties of ethanol, as well as in nicotine and alcohol co-dependence.

  13. Design and synthesis of new agents for neuronal nicotinic acetylcholine receptor (nAChRs) imaging

    Introduction: The most abundant subtype of cerebral nicotinic acetylcholine receptors (nAChR), α4β2, plays a critical role in various brain functions and pathological states. Due to rapid technological progress in chemistry, bioinformatics, structural biology and computer technology, computer aided drug design (CADD) plays a more and more important role in today's drug discovery. Methods: Two novel 3-pyridyl ether nicotinic ligands-3-((pyridine-2-yl)methoxy)-5-iodopyridine, and 3-(((S)-pyrrolidin-2-yl)methoxy)-5-((4-iodobenzyloxy)-methyl)pyridine were designed and synthesized and radiolabeled with I-125 based on our 3D-QSAR models reported previously. Their ability to label high-affinity brain nicotinic acetylcholine receptors (nAChRs) was evaluated. Results: [125I]3-((pyridin-2-yl)methoxy)-5-iodopyridine shows rapid accumulation and elimination with peak (1.86%ID/g) at 5 min post injection, but has high blood uptake. [125I]3-(((S)-pyrrolidin-2-yl)methoxy)-5-((4-iodobenzyloxy)methyl)pyridine entered the brain with maximal uptake value 3.01%ID/g at 15 min after injection, and showed approximately 27% inhibition of radioactivity uptake in thalamus in mice pretreated with nicotine. Conclusions: The results of this preliminary study show that [125I]3-(((S)-pyrrolidin-2-yl)methoxy)-5-((4-iodobenzyloxy)methyl)pyridine shows relatively high uptake to the brain, however, since the in vivo selectivity for α4β2 nAChRs was not enough, [125I]3-(((S)-pyrrolidin-2-yl)methoxy)-5-((4-iodobenzyloxy)methyl)pyridine does not have the required properties for imaging nAChRs using SPECT. Structure optimization is needed for specific visualization of brain α4β2 nAChRs in vivo.

  14. Carbon monoxide-induced delayed amnesia, delayed neuronal death and change in acetylcholine concentration in mice

    Nabeshima, T.; Katoh, A.; Ishimaru, H.; Yoneda, Y.; Ogita, K.; Murase, K.; Ohtsuka, H.; Inari, K.; Fukuta, T.; Kameyama, T. (Meijo Univ., Nagoya (Japan))

    1991-01-01

    We investigated the interrelationship of delayed amnesia, delayed neuronal death and changes in acetylcholine concentration induced by carbon monoxide (CO)-exposure in mice. In the test for retention of the passive avoidance task, amnesia was observed 5 and 7 days after CO-exposure when the mice were exposed to CO 1 day after training; in the case when the mice were exposed to CO 5 and 7 days before training, amnesia was also observed in a retention test given 1 day after training. The number of pyramidal cells in the hippocampal CA1 subfield was lower than that of the control 3, 5 and 7 days after CO-exposure. But the neurodegeneration in the parietal cortex, area 1, was not observed until 7 days after CO-exposure. The findings indicated that the amnesia and the neuronal death were produced after a delay when the mice were exposed to CO. In addition, the delayed amnesia was closely related to the delayed neuronal death in the hippocampal CA1 subfield. Moreover, (3H)glutamate and (3H)glycine binding sites did not change after CO-exposure but, 7 days after CO-exposure, the concentration of acetylcholine and the binding of (3H)quinuclidinyl benzilate in the frontal cortex and the striatum were found to have significantly changed, but those in the hippocampus did not show significant change. Therefore, we suggest that delayed amnesia induced by CO-exposure may result from delayed neuronal death in the hippocampal CA1 subfield and dysfunction in the acetylcholinergic neurons, in the frontal cortex, the striatum and/or the hippocampus.

  15. Acetylcholine modulates human working memory and subsequent familiarity based recognition via alpha oscillations.

    Eckart, Cindy; Woźniak-Kwaśniewska, Agata; Herweg, Nora A; Fuentemilla, Lluis; Bunzeck, Nico

    2016-08-15

    Working memory (WM) can be defined as the ability to maintain and process physically absent information for a short period of time. This vital cognitive function has been related to cholinergic neuromodulation and, in independent work, to theta (4-8Hz) and alpha (9-14Hz) band oscillations. However, the relationship between both aspects remains unclear. To fill this apparent gap, we used electroencephalography (EEG) and a within-subject design in healthy humans who either received the acetylcholinesterase inhibitor galantamine (8mg) or a placebo before they performed a Sternberg WM paradigm. Here, sequences of sample images were memorized for a delay of 5s in three different load conditions (two, four or six items). On the next day, long-term memory (LTM) for the images was tested according to a remember/know paradigm. As a main finding, we can show that both theta and alpha oscillations scale during WM maintenance as a function of WM load; this resembles the typical performance decrease. Importantly, cholinergic stimulation via galantamine administration slowed down retrieval speed during WM and reduced associated alpha but not theta power, suggesting a functional relationship between alpha oscillations and WM performance. At LTM, this pattern was accompanied by impaired familiarity based recognition. These findings show that stimulating the healthy cholinergic system impairs WM and subsequent recognition, which is in line with the notion of a quadratic relationship between acetylcholine levels and cognitive functions. Moreover, our data provide empirical evidence for a specific role of alpha oscillations in acetylcholine dependent WM and associated LTM formation. PMID:27222217

  16. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  17. The role of visual cortex acetylcholine in learning to discriminate temporally modulated visual stimuli

    Victor H Minces

    2013-03-01

    Full Text Available Cholinergic neurons in the basal forebrain innervate discrete regions of the cortical mantle, bestowing the cholinergic system with the potential to dynamically modulate sub-regions of the cortex according to behavioral demands. Cortical cholinergic activity has been shown to facilitate learning and modulate attention. Experiments addressing these issues have primarily focused on widespread cholinergic depletions, extending to areas involved in general cognitive processes and sleep cycle regulation, making a definitive interpretation of the behavioral role of cholinergic projections difficult. Furthermore, a review of the electrophysiological literature suggests that cholinergic modulation is particularly important in representing the fine temporal details of stimuli, an issue rarely addressed in behavioral experimentation. The goal of this work is to understand the role cholinergic projections, specific to the sensory cortex, in learning to discriminate fine differences in the temporal structure of stimuli. A novel visual Go/No-Go task was developed to assess the ability of rats to learn and discriminate fine differences in the temporal structure of visual stimuli (lights flashing at various frequencies. The cholinergic contribution to this task was examined by selectively eliminating acetylcholine projections to visual cortex (using 192 IgG-saporin, either before or after discrimination training.We find that in the face of compromised cholinergic input to the visual cortex, the rats’ ability to learn to perform fine discriminations is impaired, whereas their ability to perform discriminations remains unaffected.These results suggest that acetylcholine serves the role of facilitating plastic changes in the sensory cortices that are needed for an animal to refine their sensitivity to the temporal characteristics of relevant stimuli.

  18. The role of visual cortex acetylcholine in learning to discriminate temporally modulated visual stimuli.

    Minces, V H; Alexander, A S; Datlow, M; Alfonso, S I; Chiba, A A

    2013-01-01

    Cholinergic neurons in the basal forebrain innervate discrete regions of the cortical mantle, bestowing the cholinergic system with the potential to dynamically modulate sub-regions of the cortex according to behavioral demands. Cortical cholinergic activity has been shown to facilitate learning and modulate attention. Experiments addressing these issues have primarily focused on widespread cholinergic depletions, extending to areas involved in general cognitive processes and sleep cycle regulation, making a definitive interpretation of the behavioral role of cholinergic projections difficult. Furthermore, a review of the electrophysiological literature suggests that cholinergic modulation is particularly important in representing the fine temporal details of stimuli, an issue rarely addressed in behavioral experimentation. The goal of this work is to understand the role of cholinergic projections, specific to the sensory cortices, in learning to discriminate fine differences in the temporal structure of stimuli. A novel visual Go/No-Go task was developed to assess the ability of rats to learn to discriminate fine differences in the temporal structure of visual stimuli (lights flashing at various frequencies). The cholinergic contribution to this task was examined by selective reduction of acetylcholine projections to visual cortex (VCx) (using 192 IgG-saporin), either before or after discrimination training. We find that in the face of compromised cholinergic input to the VCx, the rats' ability to learn to perform fine discriminations is impaired, whereas their ability to perform previously learned discriminations remains unaffected. These results suggest that acetylcholine serves the role of facilitating plastic changes in the sensory cortices that are necessary for an animal to refine its sensitivity to the temporal characteristics of relevant stimuli. PMID:23519084

  19. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

    Crittenden Elizabeth L

    2004-07-01

    Full Text Available Abstract Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

  20. Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines.

    Avi Ring

    Full Text Available Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase, but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21 and neuronal (SH-SY5Y cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.

  1. Carbon monoxide-induced delayed amnesia, delayed neuronal death and change in acetylcholine concentration in mice

    We investigated the interrelationship of delayed amnesia, delayed neuronal death and changes in acetylcholine concentration induced by carbon monoxide (CO)-exposure in mice. In the test for retention of the passive avoidance task, amnesia was observed 5 and 7 days after CO-exposure when the mice were exposed to CO 1 day after training; in the case when the mice were exposed to CO 5 and 7 days before training, amnesia was also observed in a retention test given 1 day after training. The number of pyramidal cells in the hippocampal CA1 subfield was lower than that of the control 3, 5 and 7 days after CO-exposure. But the neurodegeneration in the parietal cortex, area 1, was not observed until 7 days after CO-exposure. The findings indicated that the amnesia and the neuronal death were produced after a delay when the mice were exposed to CO. In addition, the delayed amnesia was closely related to the delayed neuronal death in the hippocampal CA1 subfield. Moreover, [3H]glutamate and [3H]glycine binding sites did not change after CO-exposure but, 7 days after CO-exposure, the concentration of acetylcholine and the binding of [3H]quinuclidinyl benzilate in the frontal cortex and the striatum were found to have significantly changed, but those in the hippocampus did not show significant change. Therefore, we suggest that delayed amnesia induced by CO-exposure may result from delayed neuronal death in the hippocampal CA1 subfield and dysfunction in the acetylcholinergic neurons, in the frontal cortex, the striatum and/or the hippocampus

  2. Acetylcholine muscarinic receptors and response to anti-cholinesterase therapy in patients with Alzheimer's disease

    An acetylcholine deficit remains the most consistent neurotransmitter abnormality found in Alzheimer's disease and various therapeutic agents have been targeted at this. In this study we investigated the action of Donepezil, a cholinesterase inhibitor that has few side-effects. In particular we set out to investigate whether muscarinic acetylcholine receptor (mAChR) availability influences the response to this therapy. We used the novel single-photon emission tomography (SPET) tracer (R,R)[123I]I-quinuclidinyl benzilate (R,R[123I]I-QNB), which has high affinity for the M1 subtype of mAChR. Regional cerebral perfusion was also assessed using technetium-99m hexamethylpropylene amine oxime. We investigated 20 patients on Donepezil treatment and ten age-matched controls. The results showed a reduction in (R,R)[123I]I-QNB binding in the caudal anterior cingulate in patients compared with controls and relatively high binding in the putamen and rostral anterior cingulate, suggesting a relative sparing of mAChR in these regions. The main finding of the study was that mAChR availability as assessed by (R,R)[123I]I-QNB binding did not distinguish responders from non-responders. Interestingly, we found that the extent of cognitive improvement showed no positive correlation with (R,R)[123I]I-QNB binding in any brain region but was inversely related to binding in the insular cortex. This suggests that, within the advised cognitive performance band for use of Donepezil, response is greater in those patients with evidence of a more marked cholinergic deficit. A larger study should investigate this. (orig.)

  3. Regional selectivity of a gamma-aminobutyric acid-induced (/sup 3/H)acetylcholine release sensitive to inhibitors of gamma-aminobutyric acid uptake

    Bonanno, G.; Raiteri, M.

    1987-05-01

    The effects of gamma-aminobutyric acid (GABA) on the release of (/sup 3/H)acetylcholine ((/sup 3/H)ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with (/sup 3/H)choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized (/sup 3/H)ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of (/sup 3/H)ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of (/sup 3/H)ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of (/sup 3/H)ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of (/sup 3/H)ACh following penetration into cholinergic nerve terminals through a GABA transport system.

  4. Role of nitric oxide and carbon monoxide in N(omega)-Nitro-L-arginine methyl ester-resistant acetylcholine-induced relaxation in chicken carotid artery.

    Leo, Marie Dennis Marcus; Siddegowda, Yeshavanth K B; Kumar, Dinesh; Tandan, Surendra K; Sastry, Kochiganti V H; Prakash, Vellanki Ravi; Mishra, Santosh K

    2008-10-31

    The current study examined the hypothesis that acetylcholine-induced N(omega)-Nitro-L-arginine methyl ester (L-NAME)-resistant endothelium-dependent relaxations in the chicken carotid artery are mediated by nitric oxide and carbon monoxide. Acetylcholine (1 nM-3 microM) caused a concentration-dependent relaxation (pD(2) 6.81+/-0.05, R(max) 115+/-3%) of the artery segments precontracted with phenylephrine (3 microM). L-NAME (1 mM) decreased the sensitivity (pD(2) 6.44+/-0.06), but not the efficacy (R(max) 108+/-3%) of acetylcholine. It also partially decreased the acetylcholine (3 microM)-stimulated nitrite release. While treatment with N(omega)-Nitro-L-arginine (l-NNA; 1 mM) plus L-NAME (1 mM) decreased the acetylcholine-stimulated nitrite release to the basal level, it moderately inhibited (R(max) 77+/-3%) the maximal relaxation elicited with the muscarinic agonist. 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM) a specific scavenger of nitric oxide (NO) plus the two NOS inhibitors further decreased the acetylcholine-evoked relaxation (R(max) 34+/-2%). Although soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM) markedly inhibited the acetylcholine-stimulated increase in tissue cGMP to less than the basal levels, it only decreased the sensitivity, but not the efficacy of the agonist either in the presence or absence of L-NAME (1 mM). Zinc Protoporphyrin-IX (ZnPP; 10 microM), a hemeoxygenase (HO) inhibitor, partially inhibited (R(max) 72+/-3%) the L-NAME-resistant acetylcholine-induced relaxations. A combined treatment of the arterial rings with L-NAME, l-NNA, PTIO and ZnPP nearly abolished (R(max) 7+/-0.9%) the vasodilator responses to acetylcholine. Endothelium removal abolished the relaxation response to acetylcholine. In conclusion, it is suggested that the acetylcholine-induced L-NAME-resistant relaxation is primarily, mediated by NO with a small but significant contribution from

  5. Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

    Teresa A MURRAY; Qiang LIU; Paul WHITEAKER; Jie WU; Ronald J LUKAS

    2009-01-01

    Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR a.7 subunit without compromising formation of functional receptors.Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR al sub-unit (a7Y). SH-EP1 cells were transfected with mouse nAChR wild type a.7 subunits (a.7) or with a7Y subunits, alone or with the chaperone protein, hRJC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled a-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Results: Whole-cell currents revealed that a7Y nAChRs and al nAChRs were functional with comparable EC50 values for the a7 nAChR-selective agonist, choline, and IC50 values for the a.7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that a7Y had primarily intracel-lular rather than surface expression. TIRF microscopy confirmed that little a7Y localized to the plasma membrane, typical of a7 nAChRs.Conclusion: nAChRs composed as homooligomers of a7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of a.7 nAChRs. a7Y nAChRs may be used to elucidate properties of a.7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

  6. Supplementing female rats with DHA-lysophosphatidylcholine increases docosahexaenoic acid and acetylcholine contents in the brain and improves the memory and learning capabilities of the pups.

    Rojas, I.; Zañartu, P.; Nieto, S.; Sanhueza, J.; Morgado, N.; A. Valenzuela

    2010-01-01

    Docosahexaenoic acid (DHA) is supplied to the foetus and newborn through the mother from their own reserves and their diet. No consensus about the best form to supplement DHA has been established. We propose that DHAcontaining lysophosphatidylcholine (DHA-LPC), obtained from DHA-rich eggs may be a suitable form of DHA and choline (the precursor of acetylcholine) supplementation. We evaluated the effectiveness of DHA-LPC to increase DHA and acetylcholine concentration in the brain of pups born...

  7. Dopamine transporter distribution in patients with Parkinson disease of different stages detected using single-photon emission computed tomography brain imaging

    Jiwu Zhang; Lijuan Zhu; Jianqiang Du; Bo Liu

    2007-01-01

    BACKGROUND: Literatures have reported that the density changes of dopamine transporter is negatively correlated with the severity degree and grading of disease condition of Parkinson disease (PD). However, the distribution of dopamine transporter in each nucleus of corpora striatum at each period is still unclear.OBJECTIVE: To observe the radioactive uptake distribution of dopamine transporter in bilateral corpora striata of patients with different stages of PD using single photon emission computed tomography (SPECT),and make a comparison with healthy controls.DESIGN: Case-control analysis.SETTING: Department of Imageology, Second Hospital Affiliated to Guangzhou University of Chinese Medicine.PARTICIPANTS: Thirty patients with PD admitted to Second Hospital Affiliated to Guangzhou University of Traditional Chinese Medicine between January and December 2005 were recruited. The involved patients,19 male and 11 female, were aged from 36 to 80 years and with disease course of 2.5 months to 10 years.They all met the clinical diagnosis criteria of Britain Parkinson's disease Association Think Tank; Following Hoehn-Yahr grading: grade Ⅰ: unilateral morbidity; grade Ⅱ: bilateral morbidity, but without balance disorder; grade Ⅲ: bilateral morbidity, accompanied with early posture balance disorder; grade Ⅳ: severe morbidity, needs more help; grade Ⅴ: without help, only in bed or wheelchair. There were 11 patients with mild PD (grade Ⅰ - Ⅱ ), 9 patients with moderate PD (grade Ⅲ) and 10 patients with advanced PD (grade Ⅳ -V). Meanwhile, 6 healthy persons were selected as normal controls. Informed consents were obtained from all the subjects.METHODS: Twenty-four hours after withdrawal of PD drugs, 30 patients with PD and 6 healthy controls took kalium perchloricum 400 mg orally. After lying down for 30 minutes, all the subjects were intravenously injected with 740 MBq 99Tc m-TRODAT-1 (Jiangsu Institute of Atomic Medicine, Batch No.20040310) at elbow part

  8. Muscarinic Acetylcholine Receptor Subtypes as Potential Drug Targets for the Treatment of Schizophrenia, Drug Abuse and Parkinson's Disease

    Dencker, Ditte; Thomsen, Morgane; Wörtwein, Gitta;

    2011-01-01

    's disease and drug abuse. Dopaminergic systems are regulated by cholinergic, especially muscarinic, input. Not surprisingly, increasing evidence implicates muscarinic acetylcholine receptor-mediated pathways as potential targets for the treatment of these disorders classically viewed as "dopamine based......) acetylcholine binding site. Such agents may lead to the development of novel classes of drugs useful for the treatment of psychosis, drug abuse and Parkinson's disease. The present review highlights recent studies carried out using muscarinic receptor knock-out mice and new subtype-selective allosteric ligands...... to assess the roles of M(1), M(4), and M(5) receptors in various central processes that are under strong dopaminergic control. The outcome of these studies opens new perspectives for the use of novel muscarinic drugs for several severe disorders of the CNS....

  9. Increased efflux of amyloid-β peptides through the blood-brain barrier by muscarinic acetylcholine receptor inhibition reduces pathological phenotypes in mouse models of brain amyloidosis.

    Paganetti, Paolo; Antoniello, Katia; Devraj, Kavi; Toni, Nicolas; Kieran, Dairin; Madani, Rime; Pihlgren, Maria; Adolfsson, Oskar; Froestl, Wolfgang; Schrattenholz, André; Liebner, Stefan; Havas, Daniel; Windisch, Manfred; Cirrito, John R; Pfeifer, Andrea; Muhs, Andreas

    2014-01-01

    The formation and accumulation of toxic amyloid-β peptides (Aβ) in the brain may drive the pathogenesis of Alzheimer's disease. Accordingly, disease-modifying therapies for Alzheimer's disease and related disorders could result from treatments regulating Aβ homeostasis. Examples are the inhibition of production, misfolding, and accumulation of Aβ or the enhancement of its clearance. Here we show that oral treatment with ACI-91 (Pirenzepine) dose-dependently reduced brain Aβ burden in AβPPPS1, hAβPPSL, and AβPP/PS1 transgenic mice. A possible mechanism of action of ACI-91 may occur through selective inhibition of muscarinic acetylcholine receptors (AChR) on endothelial cells of brain microvessels and enhanced Aβ peptide clearance across the blood-brain barrier. One month treatment with ACI-91 increased the clearance of intrathecally-injected Aβ in plaque-bearing mice. ACI-91 also accelerated the clearance of brain-injected Aβ in blood and peripheral tissues by favoring its urinal excretion. A single oral dose of ACI-91 reduced the half-life of interstitial Aβ peptide in pre-plaque mhAβPP/PS1d mice. By extending our studies to an in vitro model, we showed that muscarinic AChR inhibition by ACI-91 and Darifenacin augmented the capacity of differentiated endothelial monolayers for active transport of Aβ peptide. Finally, ACI-91 was found to consistently affect, in vitro and in vivo, the expression of endothelial cell genes involved in Aβ transport across the Blood Brain Brain (BBB). Thus increased Aβ clearance through the BBB may contribute to reduced Aβ burden and associated phenotypes. Inhibition of muscarinic AChR restricted to the periphery may present a therapeutic advantage as it avoids adverse central cholinergic effects. PMID:24072071

  10. Kinetics of Carbamylcholine Binding to Membrane-Bound Acetylcholine Receptor Monitored by Fluorescence Changes of a Covalently Bound Probe

    Dunn, Susan M.J.; Blanchard, Steven G.; Raftery, Michael A.

    1980-01-01

    The fluorescent probe 5-(iodoacetamido)salicylic acid has been used to alkylate acetylcholine receptor enriched membrane fragments from Torpedo californica following their reduction with low concentrations of dithiothreitol. This modification did not affect the equilibrium binding of carbamylcholine to the receptor. The fluorescence of bound 5-(iodoacetamido)salicylic acid was enhanced when the labeled membrane fragments were mixed with carbamylcholine. This increase in fluorescence was ab...

  11. 6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

    Kasheverov, Igor E.; Irina V. Shelukhina; Kudryavtsev, Denis S.; Tatyana N. Makarieva; Spirova, Ekaterina N.; Alla G. Guzii; Stonik, Valentin A.; Tsetlin, Victor I.

    2015-01-01

    6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of ch...

  12. Acetylcholine release in the mesocorticolimbic dopamine system during cocaine-seeking: Conditioned and unconditioned contributions to reward and motivation

    You, Zhi-Bing; Wang, Bin; Zitzman, Dawnya; Wise, Roy A.

    2008-01-01

    Microdialysis was used to assess the contribution to cocaine-seeking of cholinergic input to the mesocorticolimbic dopamine system in ventral tegmental area (VTA). VTA acetylcholine (ACh) was elevated in animals lever-pressing for IV cocaine and in cocaine-experienced and cocaine-naïve animals passively receiving similar “yoked” injections. In cocaine-trained animals, the elevations comprised an initial (first hour) peak to about 160% of baseline and a subsequent plateau of 140% of baseline f...

  13. Effects of extracts from Cordyceps sinensis on M1 muscarinic acetylcholine receptor in vitro and in vivo

    Chiba, Tomohiro

    2010-01-01

    Tomohiro Chiba1, Marina Yamada1, Kosuke Torii2, Masataka Suzuki1, Jumpei Sasabe1, Minoru Ito2, Kenzo Terashita1, Sadakazu Aiso11Department of Anatomy, Keio University, School of Medicine, Tokyo, Japan; 2Department of Research and Development, Noevir Co. Ltd., Tokyo, JapanAbstract: Cholinergic dysfunction is implicated in the pathogenesis of memory impairment related to Alzheimer’s disease (AD). Accordingly, regulation of M1 muscarinic acetylcholine receptor (M1 mAChR) has been one o...

  14. Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

    Detjen, K.; Yang, J; Logsdon, C D

    1995-01-01

    Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in t...

  15. The M4 muscarinic acetylcholine receptor play a key role in the control of murine hair follicle cycling and pigmentation

    Hasse, Sybille; Chernyavsky, Alex I; Grando, Sergei A.; Paus, Ralf

    2007-01-01

    Cholinergic receptors of the muscarinic class (M1-M5) are expressed in epidermal keratinocytes and melanocytes as well as in the hair follicle. Knockout (KO) mice of all five receptors have been created and resulted in different phenotypes. KO mice with a deletion of the M4 muscarinic acetylcholine receptor (M4R) present a striking hair phenotype, which we have analyzed here in greater detail by quantitative histomorphometry. Earlier studies revealed a retarded hair follicle morphogenesis in ...

  16. Nicotinic Acetylcholine Receptor Agonists Attenuate Septic Acute Kidney Injury in Mice by Suppressing Inflammation and Proteasome Activity

    Chatterjee, Prodyot K.; Yeboah, Michael M.; Oonagh Dowling; Xiangying Xue; Powell, Saul R.; Yousef Al-Abed; Metz, Christine N

    2012-01-01

    Sepsis is one of the leading causes of acute kidney injury (AKI). Septic patients who develop acute kidney injury (AKI) are at increased risk of death. To date there is no effective treatment for AKI or septic AKI. Based on their anti-inflammatory properties, we examined the effects of nicotinic acetylcholine receptor agonists on renal damage using a mouse model of lipopolysaccharide (LPS)-induced AKI where localized LPS promotes inflammation-mediated kidney damage. Administration of nicotine...

  17. Microvascular involvement in systemic sclerosis: laser Doppler evaluation of reactivity to acetylcholine and sodium nitroprusside by iontophoresis

    Civita, L; Rossi, M.; Vagheggini, G; F. Storino; Credidio, L; Pasero, G; C. Giusti(INFN, Pavia); Ferri, C

    1998-01-01

    OBJECTIVES—To investigate the skin vasodilatory response to iontophoretically applied acetylcholine (Ach), an endothelium dependent vasodilator, and to sodium nitroprusside (SNP), an endothelium independent vasodilator, in patients with systemic sclerosis (SSc).
METHODS—Eleven SSc patients were preliminarily studied (10 females, mean age 40.5; mean disease duration 6.5 years), and 16 age and sex matched control subjects. By means of laser Doppler flowmetry skin blood flow was evaluated at thi...

  18. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-08-01

    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 α3β2 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 β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations 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 α3β2-nAChRs, while it inhibited cell growth via α7-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 α7 and α3β2-nAChRs.

  19. Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer β-amyloid peptide in rodent

    Greig, Nigel H.; Utsuki, Tadanobu; Ingram, Donald K.; Wang, Yue; Pepeu, Giancarlo; Scali, Carla; Yu, Qian-sheng; Mamczarz, Jacek; Holloway, Harold W.; Giordano, Tony; Chen, DeMao; Furukawa, Katsutoshi; Sambamurti, Kumar; Brossi, Arnold; Lahiri, Debomoy K.

    2005-01-01

    Like acetylcholinesterase, butyrylcholinesterase (BChE) inactivates the neurotransmitter acetylcholine (ACh) and is hence a viable therapeutic target in Alzheimer's disease, which is characterized by a cholinergic deficit. Potent, reversible, and brain-targeted BChE inhibitors (cymserine analogs) were developed based on binding domain structures to help elucidate the role of this enzyme in the central nervous system. In rats, cymserine analogs caused long-term inhibition of brain BChE and ele...

  20. Requirement of the nicotinic acetylcholine receptor β2 subunit for the anatomical and functional development of the visual system

    Rossi, Francesco Mattia; Pizzorusso, Tommaso; Porciatti, Vittorio; Marubio, Lisa M.; Maffei, Lamberto; Changeux, Jean-Pierre

    2001-01-01

    In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal nicotinic receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. ...

  1. Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments

    Asmar-Rovira, Guillermo A.; Asseo-García, Aloysha M.; Quesada, Orestes; Hanson, Michael A.; Nogueras, Carlos; Lasalde-Dominicci, José A.; Stevens, Raymond C.

    2008-01-01

    The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, the molecular mechanisms by which detergents influence membrane protein stability and function remain poorly understood, and elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examined nine detergents upon nAChR solubilization and purification, to assess re...

  2. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-01-01

    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 α3β2 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 β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM 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 α3β2-nAChRs, while it inhibited cell growth via α7-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 α7 and α3β2-nAChRs. PMID:27485575

  3. Synthesis, Nicotinic Acetylcholine Receptor Binding, and Antinociceptive Properties of 3′-(Substituted Phenyl)epibatidine Analogues. Nicotinic Partial Agonists⊥

    Carroll, F. Ivy; Ma, Wei; Deng, Liu; Navarro, Hernán A.; Damaj, M. Imad; Martin, Billy R.

    2010-01-01

    In 1992, John Daly et al. reported the isolation and structure determination of epibatidine. Epibatidine’s unique structure and its potent nicotinic agonist activity have had a tremendous impact on nicotine receptor research. This research has led to a better understanding of the nicotinic acetylcholine receptor (nAChR) pharmacophore and to epibatidine analogues with potential as pharmacotherapies for treating various CNS disorders. In this study, we report the synthesis, receptor binding ([3...

  4. Naloxone-insensitive inhibition of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by the novel opioid, nociceptin

    Patel, Hema J; Giembycz, Mark A; Spicuzza, Lucia; Barnes, Peter J; Belvisi, Maria G

    1997-01-01

    The novel peptide, nociceptin and the μ-opioid agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) produced a concentration-dependent inhibition of electrical field stimulation (EFS)-evoked release of acetylcholine (ACh) from cholinergic nerves innervating guinea-pig trachea. The non-selective opioid receptor antagonist, naloxone, did not antagonize the inhibitory action of nociceptin under conditions where the inhibition of ACh release evoked by DAMGO was completely reversed. It is sugge...

  5. Calcium-dependent effect of the thymic polypeptide thymopoietin on the desensitization of the nicotinic acetylcholine receptor

    The effects of the thymic polypeptide thymopoietin (Tpo) on the properties of the nicotinic acetylcholine receptor (AcChoR) were investigated by patch clamp techniques on mouse C2 myotubes and by biochemical assays on AcChoR-rich membrane fragments purified from the Torpedo marmorata electric organ. At high concentrations (> 100 nM), Tpo inhibits the binding of cholinergic agonists to the AcChoR in a Ca2+-insensitive manner. At lower concentrations (2 nM), Tpo applied on C2 myotubes simultaneously with nondesensitizing concentrations of acetylcholine results in the appearance of long closed times separating groups of openings. This effect depends on the presence of Ca2+ in the external medium. Outside-out recordings, performed with various concentrations of EGTA in the intracellular medium, suggest that Ca2+ acts on the cytoplasmic face of the membrane after entry through acetylcholine-activated channels. Parallel studies with T. marmorata AcChoR-rich membranes show that in the presence of Ca2+ Tpo causes a decrease in the apparent equilibrium dissociation constant of the noncompetitive blocker [3H]phencyclidine, enhances, at low concentrations, the binding of [3H]acetylcholine, and also alters the binding kinetics of the fluorescent agonist 6-(5-dimethylamino-1-naphthalenesulfonamido)-n-hexanoic acid β-(N-trimethylammonium bromide) ethyl ester to the AcChoR. It was concluded that, in the presence of Ca2+, Tpo displaces the conformational equilibrium of the AcChoR towards a high-affinity desensitized state and increases the transition rate towards the same state

  6. Mis-spliced transcripts of nicotinic acetylcholine receptor alpha6 are associated with field evolved spinosad resistance in Plutella xylostella (L..

    Simon W Baxter

    2010-01-01

    Full Text Available The evolution of insecticide resistance is a global constraint to agricultural production. Spinosad is a new, low-environmental-risk insecticide that primarily targets nicotinic acetylcholine receptors (nAChR and is effective against a wide range of pest species. However, after only a few years of application, field evolved resistance emerged in the diamondback moth, Plutella xylostella, an important pest of brassica crops worldwide. Spinosad resistance in a Hawaiian population results from a single incompletely recessive and autosomal gene, and here we use AFLP linkage mapping to identify the chromosome controlling resistance in a backcross family. Recombinational mapping with more than 700 backcross progeny positioned a putative spinosad target, nAChR alpha 6 (Pxalpha6, at the resistance locus, PxSpinR. A mutation within the ninth intron splice junction of Pxalpha6 results in mis-splicing of transcripts, which produce a predicted protein truncated between the third and fourth transmembrane domains. Additional resistance-associated Pxalpha6 transcripts that excluded the mutation containing exon were detected, and these were also predicted to produce truncated proteins. Identification of the locus of resistance in this important crop pest will facilitate field monitoring of the spread of resistance and offer insights into the genetic basis of spinosad resistance in other species.

  7. Influence of Y151 F mutation in loop B on the agonist potency in insect nicotinic acetylcholine receptor

    Feng Song; Yi-Xi Zhang; Xiang-Mei Yao; Ze-Wen Liu

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels,which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems.The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A-C present in α subunits together with loops D-F present in either non-α subunits or homomer-forrning α subunits.Although Y151 in loop B has been identified as important in agonist binding,various residues at the 151-site are found among vertebrate and invertebrate nAChR ot subunits,such as F151.In Xenopus oocytes expressing N1α1 or N1α1~(Y151F) plus rat β2,Y151F mutation was found to significantly change the rate of receptor desensitization and altered the pharmacological properties of acetylcholine,but not imidacloprid,including the decrease of I_(max),the increase of EC_(50)(the concentration causing 50% of the maximum response) and the fast time-constant of decay (τ_f).By comparisons of residue structure,the hydroxyl group in the side chain of Y151 was thought to be important in the interaction between N1α1/β2 nAChRs and acetylcholine,and the phenyl group to be important between N1α1/β2 nAChRs and imidacloprid.

  8. Effect of elevated potassium ion concentrations on electrically evoked release of [3H]acetylcholine in slices of rat hippocampus

    To establish the effect of raising the concentration of extracellular potassium ions on axonal conduction and transmitter release in a mammalian central pathway, the septohippocampal cholinergic tract, the rate of [3H] acetylcholine release evoked by electrical stimulation was measured in rat hippocampal slices superfused with Krebs' solution containing 3-15 mM K+ The evoked release of [3H] acetylcholine was abolished by the presence of tetrodotoxin or by the omission of Ca2+ in the superfusion medium, indicating that it originated from terminals depolarized by conducted action potentials. Potassium concentrations between 3 and 8 mM had no effect but 10-15 mM K+ reduced the rate of evoked release and decreased the size of the releasable pool of [3H] acetylcholine. Decreasing the sodium content of the Krebs' solution to 97 mM or less had effects similar to those of elevated [K+]. Elevated K+ (10-15 mM) reversibly reduced the size of compound action potentials in the fimbria and the alveus. The results suggest that extracellular potassium concentrations occurring under physiological conditions do not affect axonal conduction and transmitter release but that both are reduced in pathological states when extracellular [K+] above 8 mM occur. (author)

  9. Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations.

    Mathé-Allainmat, Monique; Swale, Daniel; Leray, Xavier; Benzidane, Yassine; Lebreton, Jacques; Bloomquist, Jeffrey R; Thany, Steeve H

    2013-12-01

    We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors. PMID:23884575

  10. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor α subunit

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the α subunit of the receptor, with little or no change in the levels of γ- and δ-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of α-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of α subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of α-subunit mRNA, with little change in the amount of γ- and δ-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the α-subunit nuclear precursor

  11. Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies

    A high density of galanin binding sites was found by using 125I-labeled galanin, iodinated by chloramine-T, followed by autoradiography in the ventral, but not in the dorsal, hippocampus of the rat. Lesions of the fimbria and of the septum caused disappearance of a major population of these binding sites, suggesting that a large proportion of them is localized on cholinergic nerve terminals of septal afferents. As a functional correlate to these putative galanin receptor sites, it was shown, both in vivo and in vitro, that galanin, in a concentration-dependent manner, inhibited the evoked release of acetylcholine in the ventral, but not in the dorsal, hippocampus. Intracerebroventricularly applied galanin fully inhibited the scopolamine stimulated release of acetylcholine in the ventral, but not in the dorsal, hippocampus, as measured by the microdialysis technique. In vitro, galanin inhibited the 25 mM K+-evoked release of [3H]acetylcholine from slices of the ventral hippocampus, with an IC50 value of ≅ 50 nM. These results are discussed with respect to the colocalization of galanin- and choline acetyltransferase-like immunoreactivity in septal somata projecting to the hippocampus

  12. Studies on the effects of acetylcholine and antiepileptic drugs on 32P incorporation into phospholipids of rat brain synaptosomes

    Studies were conducted on the effects of antiepileptic drugs on the acetylcholine-stimulated 32P labeling of phospholipids in rat brain synaptosomes. Of the four antiepileptic drugs investigated in the present study, namely phenytoin, carbamazepine, phenobarbital, and valproate, only phenytoin blocked the acetylcholine-stimulated 32P labeling of phosphatidylinositol and phosphatidic acid, and the acetylcholine-stimulated breakdown of polyphosphoinositides. Phenytoin alone, like atropine alone, had no effect on the 32P labeling of phospholipids nor on the specific radioactivity of [32P]ATP. Omission of Na+ drastically reduced both the 32P labeling of synaptosomal phospholipids and the specific radioactivity of [32P]ATP and furthermore it significantly decreased the phosphoinositide effect. It was concluded that certain antiepileptic drugs, such as phenytoin, could exert their pharmacological actions through their antimuscarinic effects. In addition the finding that phenytoin, which acts to regulate NA+ and Ca2+ permeability of neuronal membranes, also inhibited the phosphoinositide effects in synaptosomes, support the conclusions that Ca2+ and Na+ are probably involved in the molecular mechanism underlying this phenomenon in excitable tissues

  13. Methanol extract of Tephrosia vogelii leaves potentiates the contractile action of acetylcholine on isolated rabbit jejunum

    Tavershima Dzenda; Joseph Olusegun Ayo; Alexander Babatunde Adelaiye; Ambrose Osemattah Adaudi

    2015-01-01

    To investigate the modulating role of methanol extract of Tephrosia vogelii leaves on acetylcholine (ACh)-induced contraction of isolated rabbit jejunum. Methods: Rabbit jejunum segment was removed and placed in an organ bath containing Tyrode’s solution, and its contractions were recorded isometrically. Results: ACh (2.0 × 10-10 g/mL) and the extract (2.0 × 10-4 g/mL) individually increased the frequency of contraction (mean ± SEM) of the isolated smooth muscle tissue by 47.6% ± 9.5%and 77.8% ± 66.5%, respectively. When ACh and the extract were combined, the frequency of contraction of the tissue was increased by 222.2% ± 25.9%, representing a 366.7% increase (P < 0.001) over the effect of ACh alone. Similarly, ACh (2.0 × 10-9 g/mL) and the extract individually increased significantly (P < 0.001) the amplitude of contraction of the tissue by 685.7% ± 61.1% and 455.2% ± 38.1%, respectively. When ACh and the extract were combined, the amplitude of contraction of the tissue rose by 1263.8% ± 69.0%, representing 84.3% increase over the effect of ACh alone. Conclusions: The findings demonstrate that methanol extract of Tephrosia vogelii leaves potentiates the contractile effect of ACh on intestinal smooth muscle, supporting the traditional claim that the plant is purgative.

  14. α7-Nicotinic acetylcholine receptor: role in early odor learning preference in mice.

    Jennifer L Hellier

    Full Text Available Recently, we have shown that mice with decreased expression of α7-nicotinic acetylcholine receptors (α7 in the olfactory bulb were associated with a deficit in odor discrimination compared to wild-type mice. However, it is unknown if mice with decreased α7-receptor expression also show a deficit in early odor learning preference (ELP, an enhanced behavioral response to odors with attractive value observed in rats. In this study, we modified ELP methods performed in rats and implemented similar conditions in mice. From post-natal days 5-18, wild-type mice were stroked simultaneously with an odor presentation (conditioned odor for 90 s daily. Control mice were only stroked, exposed to odor, or neither. On the day of testing (P21, mice that were stroked in concert with a conditioned odor significantly investigated the conditioned odor compared to a novel odor, as observed similarly in rats. However, mice with a decrease in α7-receptor expression that were stroked during a conditioned odor did not show a behavioral response to that odorant. These results suggest that decreased α7-receptor expression has a role in associative learning, olfactory preference, and/or sensory processing deficits.

  15. Thrombin action decreases acetylcholine receptor aggregate number and stability in cultured mouse myotubes.

    Davenport, R W; Lanuza, M; Kim, S; Jia, M; Snyder, E; Nelson, P G

    2000-08-30

    Neurons develop and make very stable, long-term synaptic connections with other nerve cells and with muscle. Synaptic stability at the neuromuscular junction changes over development in that a proliferation of synaptic input are made to individual myotubes and synapses from all but one neuron are lost during development. In an established co-culture paradigm in which spinal motoneurons synaptically contact myotubes, thrombin and associated protease inhibitors have been shown to affect the loss of functional synaptic contacts [6]. Evidence has not been provided which clearly demonstrate whether protease/protease inhibitors affect either the pre- or postsynaptic terminal, or both. In an effort to determine whether these reagents directly affect postsynaptic receptors on myotubes, myotubes were cultured in the absence of neurons and the spontaneous presence and stability of aggregates of acetylcholine receptors (AChR) in control and thrombin-containing media were evaluated. In dishes fixed after treatment and in dishes in which individual aggregates were observed live, thrombin action appeared to increase loss of AChR aggregates over time. Hirudin, a specific inhibitor of the thrombin protease, diminished this loss. Neither reagent affected the overall incorporation or degradation of AChR; therefore, it appears these protease/protease inhibitors affect the state of AChR aggregation. PMID:10960680

  16. Nucleus accumbens core acetylcholine is preferentially activated during acquisition of drug- vs food-reinforced behavior.

    Crespo, Jose A; Stöckl, Petra; Zorn, Katja; Saria, Alois; Zernig, Gerald

    2008-12-01

    Acquisition of drug-reinforced behavior is accompanied by a systematic increase of release of the neurotransmitter acetylcholine (ACh) rather than dopamine, the expected prime reward neurotransmitter candidate, in the nucleus accumbens core (AcbC), with activation of both muscarinic and nicotinic ACh receptors in the AcbC by ACh volume transmission being necessary for the drug conditioning. The present findings suggest that the AcbC ACh system is preferentially activated by drug reinforcers, because (1) acquisition of food-reinforced behavior was not paralleled by activation of ACh release in the AcbC whereas acquisition of morphine-reinforced behavior, like that of cocaine or remifentanil (tested previously), was, and because (2) local intra-AcbC administration of muscarinic or nicotinic ACh receptor antagonists (atropine or mecamylamine, respectively) did not block the acquisition of food-reinforced behavior whereas acquisition of drug-reinforced behavior had been blocked. Interestingly, the speed with which a drug of abuse distributed into the AcbC and was eliminated from the AcbC determined the size of the AcbC ACh signal, with the temporally more sharply delineated drug stimulus producing a more pronounced AcbC ACh signal. The present findings suggest that muscarinic and nicotinic ACh receptors in the AcbC are preferentially involved during reward conditioning for drugs of abuse vs sweetened condensed milk as a food reinforcer. PMID:18418362

  17. Captopril augments acetylcholine-induced bronchial smooth muscle contractions in vitro via kinin-dependent mechanisms.

    Agrawal, Naman; Akella, Aparna; Deshpande, Shripad B

    2016-06-01

    Angiotensin converting enzyme (ACE) inhibitors therapy is aassociated with bothersome dry cough as an adverse effect. The mechanisms underlying this adverse effect are not clear. Therefore, influence of captopril (an ACE inhibitor) on acetylcholine (ACh)-induced bronchial smooth muscle contractions was investigated. Further, the mechanisms underlying the captopril-induced changes were also explored. In vitro contractions of rat bronchial smooth muscle to cumulative concentrations of ACh were recorded before and after exposure to captopril. Further, the involvement of kinin and inositol triphosphate (IP₃) pathways for captopril-induced alterations were explored. ACh produced concentration-dependent (5-500 µM) increase in bronchial smooth muscle contractions. Pre-treatment with captopril augmented the ACh-induced contractions at each concentration significantly. Pre-treatment with aprotinin (kinin synthesis inhibitor) or heparin (inositol triphosphate, IP₃-inhibitor), blocked the captopril-induced augmentation of bronchial smooth muscle contractions evoked by ACh. Further, captopril-induced augmentation was absent in calcium-free medium. These results suggest that captopril sensitizes bronchial smooth muscles to ACh-induced contractions. This sensitization may be responsible for dry cough associated with captopril therapy. PMID:27468462

  18. Acetylcholine esterase activity in mild cognitive impairment and Alzheimer's disease

    Herholz, Karl [University of Manchester, Wolfson Molecular Imaging Centre, Clinical Neuroscience, Manchester (United Kingdom); University of Cologne, Cologne (Germany)

    2008-03-15

    Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD), but there is controversy about its relevance at the early stages of the disease and in mild cognitive impairment (MCI). In vivo positron emission tomography imaging of cortical acetylcholine esterase (AChE) activity as a marker of cholinergic innervation that is expressed by cholinergic axons and cholinoceptive neurons has demonstrated a reduction of this enzyme activity in manifest AD. The technique is also useful to measure the inhibition of cerebral AChE induced by cholinesterase inhibitors for treatment of dementia symptoms. A reduction of cortical AchE activity was found consistently in all studies of AD and in few cases of MCI who later concerted to AD. The in vivo findings in MCI and very mild AD are still preliminary, and studies seem to suggest that cholinergic innervation and AChE as the main degrading enzyme are both reduced, which might result in partial compensation of their effect. (orig.)

  19. Synthesis of carbon-11 labeled dexetimide and levetimide for studying muscarinic acetylcholine receptors

    The localization and quantitation of the muscarinic acetylcholine receptor (m-AChR) in the living human brain using a non-invasive method, such as positron emission tomography (PET), may provide valuable information about receptor changes which have been observed post mortem in patients with Huntington's chorea and Alzheimer's dementia, as well as normal brain mechanisms mediated by the m-AChR. Although quinuclidinyl benzilate has been radioiodinated and radiomethylatd, the former is not useful with PET and the latter does not penetrate the blood-brain barrier; therefore, the authors chose to radiolabel dexetimide, a ligand which labels m-AChR in vitro and in vivo, and levetimide, its inactive enantiomer. Carbon-11 labeled carbon dioxide is bubbled through a tetrahydrofuran (THF) solution of phenylmagnesium chloride (1 M, l ml) after which 2 mg of lithium aluminium hydride is added in THF (500 μl). After evaporation of the solvent, 48% hydriodic acid (l ml) is added and the solution is heated for 1 minute. Carbon-11 labeled benzyl iodide is extracted into methylene chloride, added to a solution of nor-benzyl dexetimide or levetimide, and heated for several minutes. Purification is accomplished using semi-preparative reverse phase high performance liquid chromatography (HPLC). Analytical HPLC is used to determine the radiochemical purity and specific activity

  20. Studies of two naturally occurring compounds which effect release of acetylcholine from synaptosomes

    Two naturally occurring compounds which effect the release of neurotransmitter from synaptosomes have been purified to apparent homogeneity. Iotrochotin (IOT) isolated from wound exudate of the Caribbean purple bleeder sponge promotes release in a manner that is independent of the extracellular Ca2+ ion concentration. Leptinotarsin (LPT-d), a protein taken from hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, stimulates Ca2+-dependent release. IOT is slightly acidic and has a molecular weight of approximately 18 kD. [3H]acetylcholine which has been introduced into synaptosomes as [3H]choline can be released by IOT. The toxin releasable pool of labelled neurotransmitter is not depleted by depolarization of the synaptosomes with high potassium, and therefore seems to be primarily extravesicular. LPT-d is a larger protein (molecular weight = 45 kD) than IOT, and seems to effect primarily vesicular release by opening at least one type of presynaptic Ca2+ channel. The facilitatory effects of the toxin on synaptosomal release can be inhibited by inorganic Ca2+ channel antagonists, but are not generally affected by organic antagonists

  1. Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

    Supavilai, P.; Karobath, M.

    1985-02-04

    GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with /sup 3/H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 ..mu..M. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BR agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro. 24 references, 3 figures, 5 table.

  2. Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

    GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with 3H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 μM. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BR agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro. 24 references, 3 figures, 5 table

  3. Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

    Dacosta, Corrie J. B.; Free, Chris R.; Sine, Steven M.

    2015-08-01

    α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homopentameric α7-acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state.

  4. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees

    Moffat, Christopher; Buckland, Stephen T.; Samson, Andrew J.; McArthur, Robin; Chamosa Pino, Victor; Bollan, Karen A.; Huang, Jeffrey T.-J.; Connolly, Christopher N.

    2016-04-01

    There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species.

  5. Dopamine regulation of [3H]acetylcholine release from guinea-pig stomach

    The involvement of dopamine receptors in cholinergic transmission of guinea-pig stomach was investigated by analyzing the effects of dopamine receptor agonists and antagonists on acetylcholine (ACh) release from this organ. Electrical stimulation (1-20 Hz) of strips of guinea-pig stomach preloaded with [3H] choline induced a [3H]ACh release that was calcium dependent and tetrodotoxin sensitive. Dopamine inhibited this transmural stimulation-induced [3H]ACh release in a concentration-dependent manner (10(-8)-10(-4) M). This effect of dopamine was not altered by 10(-5) M hexamethonium, thereby suggesting that the major dopamine receptors are located on the postganglionic cholinergic neurons. Concentration-response curves for dopamine on [3H]ACh release were inhibited by haloperidol, sulpiride and domperidone but not by prazosin, yohimbine, propranolol and ketanserin. LY 171555, an agonist for the D2 dopamine receptor, but not SKF 38-393, an agonist for the D1 dopamine receptor, to some extent decreased the release of [3H]ACh induced by transmural stimulation. In view of the results, the release of ACh from postganglionic cholinergic neurons is probably required through dopamine receptors antagonized by D2 antagonists but not by adrenergic or serotonin receptor antagonists

  6. Neuronal Acetylcholine Nicotinic Receptors as New Targets for Lung Cancer Treatment.

    Mucchietto, Vanessa; Crespi, Arianna; Fasoli, Francesca; Clementi, Francesco; Gotti, Cecilia

    2016-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Smoking accounts for approximately 70% of the cases of non- small cell lung cancer (NSCLC) and 90% of the cases of small-cell lung cancer (SCLC), although some patients develop lung cancer without a history of smoking. Nicotine is the most active addictive component of tobacco smoke. It does not initiate tumorigenesis in humans and rodents, but it alters the pathophysiology of lung cells by inducing the secretion of growth factors, neurotransmitters and cytokines, and promotes tumour growth and metastases by inducing cell cycle progression, migration, invasion, angiogenesis and the evasion of apoptosis. Most of these effects are a result of nicotine binding and activation of cell-surface neuronal nicotinic acetylcholine receptors (nAChRs) and downstream intracellular signalling cascades, and many are blocked by nAChR subtype-selective antagonists. Recent genome-wide association studies have revealed single nucleotide polymorphisms of nAChR subunits that influence nicotine dependence and lung cancer. This review describes the molecular basis of nAChR structural and functional diversity in normal and cancer lung cells, and the genetic alterations facilitating smoking-induced lung cancers. It also summarises current knowledge concerning the intracellular pathways activated by nicotine and other compounds present in tobacco smoke. PMID:26845123

  7. Different patterns of nicotinic acetylcholine receptor subunit transcription in human thymus.

    Bruno, Roxana; Sabater, Lidia; Tolosa, Eva; Sospedra, Mireia; Ferrer-Francesch, Xavier; Coll, Jaume; Foz, Marius; Melms, Arthur; Pujol-Borrell, Ricardo

    2004-04-01

    Clinical observations suggest that the thymus is strongly implicated in the pathogenesis of myasthenia gravis (MG), but questions such as the level and location of nicotinic acetylcholine receptor (AChR) subunit expression that are fundamental to postulate any pathogenic mechanism, remain controversial. We have re-examined this question by combining calibrated RT-PCR and real-time PCR to study nicotinic AChR subunit mRNA expression in a panel of normal and myasthenic thymi. The results suggest that the expression of the different AChR subunits follows three distinct patterns: constitutive for, neonatal for gamma and individually variable for alpha1, beta1 and delta. Experiments using confocal laser microdissection suggest that AChR is mainly expressed in the medullary compartment of the thymus but there is not a clear compartmentalization of subunit expression. The different patterns of subunit expression may influence decisively the level of central tolerance to the subunits and explain the focusing of the T cell response to the alpha and gamma subunits. PMID:15020075

  8. Catharanthine alkaloids are noncompetitive antagonists of muscle-type nicotinic acetylcholine receptors.

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof

    2010-09-01

    We compared the interaction of several catharanthine alkaloids including, ibogaine, vincristine, and vinblastine, with that for the noncompetitive antagonist phencyclidine (PCP) at muscle nicotinic acetylcholine receptors (AChRs) in different conformational states. The results established that catharanthine alkaloids: (a) inhibit, in a noncompetitive manner, (+/-)-epibatidine-induced Ca(2+) influx in TE671-halpha1beta1gammadelta cells with similar potencies (IC(50)=17-25microM), (b) inhibit [(3)H]TCP binding to the desensitized Torpedo AChR with higher affinity compared to the resting AChR, and (c) enhance [(3)H]cytisine binding to resting but activatable Torpedo AChRs, suggesting desensitizing properties. Interestingly, PCP inhibits [(3)H]ibogaine binding to the AChR in a steric fashion. This is corroborated by additional docking experiments indicating that the amino groups of neutral ibogaine form hydrogen bonds with the serine ring (position 6'), a location shared with PCP. Since protonated ibogaine forms a salt bridge with one of the acidic residues at the outer ring (position 20'), this ligand could be first attracted to the entrance of the channel by electrostatic interactions. Our data indicate that the catharanthine moiety is a minimum structural requirement for AChR inhibition including, ion channel blocking and desensitization, and that ibogaine and PCP bind to overlapping sites in the desensitized AChR ion channel. PMID:20493225

  9. Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states.

    Arias, Hugo R; Rosenberg, Avraham; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Maciejewski, Ryszard; Jozwiak, Krzysztof; Moaddel, Ruin; Glick, Stanley D; Wainer, Irving W

    2010-06-01

    The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (+/-)-epibatidine-induced AChR Ca(2+) influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [(3)H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6') and valine (position 13') rings, and (c) inhibits [(3)H]TCP, [(3)H]ibogaine, and [(3)H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization. PMID:20303928

  10. Tetrodotoxin effects in the stimulated acetylcholine release by agonist of glutamate in mice striatum tissue

    The toxins of animal venoms have been used as important tools for biochemical studies of physiological and pathological processes of diverse systems. In this work we used the action of tetrodotoxin on sodium channels to map the localization of glutamate receptors in cholinergic neurons from striatum tissue of rats. All glutamate receptors are exciting, so they promote the release of other neurotransmitters. In this work we focus on acetylcholine. The localization of glutamate receptor, on the soma or on the excitatory terminal, may contribute for a better understanding of its function. For this work we applied the in vitro method of tritiated neurotransmitter release. The agonists of glutamate receptors chosen were glutamic acid 500μM, NMDA 100μM, kainic acid 300μM, quisqualic acid 300μM and AMPA 1mM. In the first part of the assay the basal and stimulated releases were measured and in the second, the same protocol was performed in the presence of tetrodotoxin 1μM. The reductions observed in basal and stimulated release in the presence of tetrodotoxin suggested that the receptors type AMPA and NMDA were located in soma of cholinergic cell preferentially and the other ones presented a more equilibrate distribution among the axons and the soma. (author)

  11. Nicotinic acetylcholine receptor-based blockade: applications of molecular targets for cancer therapy.

    Wu, Chih-Hsiung; Lee, Chia-Hwa; Ho, Yuan-Soon

    2011-06-01

    The nicotinic acetylcholine receptor (nAChR) was first characterized in 1970 as a membrane receptor of a neurotransmitter and an ion channel. nAChRs have been shown to be involved in smoking-induced cancer formation in multiple types of human cancer cells. In vitro and in vivo animal studies have shown that homopentameric nAChR inhibitors, such as methyllycaconitine and α-Bgtx, can attenuate nicotine-induced proliferative, angiogenic, and metastatic effects in lung, colon, and bladder cancer cells. Recent publications have shown that α9-nAChR is important for breast cancer formation, and in many in vivo studies, α9-nAChR-specific antagonists (e.g., α-ImI, α-ImI, Vc1.1, RgIA, and It14a) produced an analgesic effect. Vc1.1 functions in a variety of animal pain models and currently has entered phase II clinical trials. For cancer therapy, natural compounds such as garcinol and EGCG have been found to block nicotine- and estrogen-induced breast cancer cell proliferation through inhibition of the α9-nAChR signaling pathway. A detailed investigation of the carcinogenic effects of nAChRs and their specific antagonists would enhance our understanding of their value as targets for clinical translation. PMID:21444681

  12. Nicotinic Acetylcholine Receptor α4 Subunit Gene Variation Associated with Attention Deficit Hyperactivity Disorder

    HUANG Xuezhu; XU Yong; LI Qianqian; LIU Pozi; YANG Yuan; ZHANG Fuquan; GUO Tianyou; YANG Chuang; GUO Lanting

    2009-01-01

    Previous pharmacological, human genetics, and animal models have implicated the nicotinic ace-tylcholine receptor a4 subunit (CHRNA4) gene in the pathogenesis of attention deficit/hyperactivity disorder (ADHD). The objective of this study is to examine the genetic association between single nucleotide poly-morphisms in the CHRNA4 gene (rs2273502, rs1044396, rs1044397, and rs3827020 loci) and ADHD. Both case-control and family-based designs are used. Children aged 6 to 16 years were interviewed and as-sessed with the children behavior checklist and the revised conner' parent rating scale to identify probands. No significant differences in the frequency distribution of genotypes or alleles were found between the case and control groups. However, further haplotype analyses showed the CCGG haplotype on dsk for ADHD in 164 case-control samples and the standard transmission disequilibrium test analyses suggest that the allele C of rs2273502 was over-transferred in 98 ADHD parent-offspring tdos. These findings suggest that the CHRNA4 gene may play a role in the pathogenesis of ADHD.

  13. Functional nicotinic acetylcholine receptor reconstitution in Au(111)-supported thiolipid monolayers

    Pissinis, Diego E.; Diaz, Carolina; Maza, Eliana; Bonini, Ida C.; Barrantes, Francisco J.; Salvarezza, Roberto C.; Schilardi, Patricia L.

    2015-09-01

    The insertion and function of the muscle-type nicotinic acetylcholine receptor (nAChR) in Au(111)-supported thiolipid self-assembled monolayers have been studied by atomic force microscopy (AFM), surface plasmon resonance (SPR), and electrochemical techniques. It was possible for the first time to resolve the supramolecular arrangement of the protein spontaneously inserted in a thiolipid monolayer in an aqueous solution. Geometric supramolecular arrays of nAChRs were observed, most commonly in a triangular form compatible with three nAChR dimers of ~20 nm each. Addition of the full agonist carbamoylcholine activated and opened the nAChR ion channel, as revealed by the increase in capacitance relative to that of the nAChR-thiolipid system under basal conditions. Thus, the self-assembled system appears to be a viable biomimetic model to measure ionic conductance mediated by ion-gated ion channels under different experimental conditions, with potential applications in biotechnology and pharmacology.

  14. Synthesis and 125I labelling of a precursor for imaging nicotinic acetylcholine receptors

    Nicotinic Acetylcholine Receptors (nAChRs) are involved in various pharmacological effects or diseases, such as Alzheimer's Disease, Parkinson's Disease and tobacco addiction. It will be very appealing to image nAChRs in vivo, diagnose and treat the above diseases, and probe the mechanism of nAChRs in tobacco addiction if the suitable radioactive labeled compound can be synthesized. In this study, (s)-5-(tri-butylstannyl)-3{[1-(tert-butoxycarbonyl)-2-azetidinyl]methoxy} pyridine, a precursor for imaging nAChRs, was synthesized with commercial 2-furfurylamine and (s)-2-azetidinecarboxylic acid as starting materials, and was further labeled with 125/123I. The whole procedure for radiosynthesis needs 50-55 min and more than 30% of the 125I are found in the purified 5-[125I]-A-85380. Even staying for 3 days at room temperature in vitro, the purified 5-[125I]-I-85380 can maintain its stability, with a radiochemical purity of more than 95%. (authors)

  15. Segregation of acetylcholine and GABA in the rat superior cervical ganglia: functional correlation.

    Diana eElinos

    2016-04-01

    Full Text Available Sympathetic neurons have the capability to segregate their neurotransmitters (NTs and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh and other classical NTs such as gamma aminobutyric acid (GABA. Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX. We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region show larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons.

  16. Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

    Gu, Y.

    1989-01-01

    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.

  17. Neuronal Nicotinic Acetylcholine Receptors: Neuroplastic Changes underlying Alcohol and Nicotine Addictions

    Allison Anne Feduccia

    2012-08-01

    Full Text Available Addictive drugs can activate systems involved in normal reward-related learning, creating long-lasting memories of the drug’s reinforcing effects and the environmental cues surrounding the experience. These memories significantly contribute to the maintenance of compulsive drug use as well as cue-induced relapse which can occur even after long periods of abstinence. Synaptic plasticity is thought to be a prominent molecular mechanism underlying drug-induced learning and memories. Ethanol and nicotine are both widely abused drugs that share a common molecular target in the brain, the neuronal nicotinic acetylcholine receptors (nAChRs. The nAChRs are ligand-gated ion channels that are vastly distributed throughout the brain and play a key role in synaptic neurotransmission. In this review, we will delineate the role of nAChRs in the development of ethanol and nicotine addiction. We will characterize both ethanol and nicotine’s effects on nAChR-mediated synaptic transmission and plasticity in several key brain areas that are important for addiction. Finally, we will discuss some of the behavioral outcomes of drug-induced synaptic plasticity in animal models. An understanding of the molecular and cellular changes that occur following administration of ethanol and nicotine will lead to better therapeutic strategies.

  18. Metabolic stabilization of acetylcholine receptors in vertebrate neuromuscular junction by muscle activity

    The effects of muscle activity on the growth of synaptic acetylcholine receptor (AChR) accumulations and on the metabolic AChR stability were investigated in rat skeletal muscle. Ectopic end plates induced surgically in adult soleus muscle were denervated early during development when junctional AChR number and stability were still low and, subsequently, muscles were either left inactive or they were kept active by chronic exogenous stimulation. AChR numbers per ectopic AChR cluster and AChR stabilities were estimated from the radioactivity and its decay with time, respectively, of end plate sites whose AChRs had been labeled with 125I-alpha-bungarotoxin (alpha-butx). The results show that the metabolic stability of the AChRs in ectopic clusters is reversibly increased by muscle activity even when innervation is eliminated very early in development. 1 d of stimulation is sufficient to stabilize the AChRs in ectopic AChR clusters. Muscle stimulation also produced an increase in the number of AChRs at early denervated end plates. Activity-induced cluster growth occurs mainly by an increase in area rather than in AChR density, and for at least 10 d after denervation is comparable to that in normally developing ectopic end plates. The possible involvement of AChR stabilization in end plate growth is discussed

  19. Meandering and unstable reentrant wave fronts induced by acetylcholine in isolated canine right atrium.

    Ikeda, T; Wu, T J; Uchida, T; Hough, D; Fishbein, M C; Mandel, W J; Chen, P S; Karagueuzian, H S

    1997-07-01

    The mechanism(s) by which acetylcholine (ACh) increases atrial vulnerability to reentry and maintains its activity for longer durations remains poorly defined. In the present study we used high-resolution activation maps to test the hypothesis that ACh promotes meandering of atrial reentrant wave fronts, resulting in breakup and the generation of new wave fronts that sustain the activity. Reentry was induced in 11 isolated canine right atrial tissues (3.8 x 3.2 cm) by a premature point stimulus (S2) before and after superfusion with ACh (15 x 10(-6) M). Endocardial isochronal activation maps were constructed with the use of 509 bipolar electrodes (1.6-mm spatial resolution), and the dynamics of the activation wave fronts were visualized with animation. A vulnerable period was found during which an S2 current strength > 4.4 +/- 2.5 mA [lower limit of vulnerability (LLV)] and vulnerability (ULV)] induced a single stationary reentrant wave front that lasted 3 +/- 2.5 s with a period of 159 +/- 17 ms (16 episodes). AC shortened the refractory period from 100 +/- 12 to 59 +/- 9 ms (P vulnerability to reentry induction by simultaneous decrease in the LLV (0.7 +/- 0.2 mA, P single reentrant wave front, leading to breakup and the generation of new wave fronts. Single meandering and complex wave front dynamics cause fibrillation-like activity and sustain the activity for longer duration. PMID:9249511

  20. Alpha9 alpha10 nicotinic acetylcholine receptors as target for the treatment of chronic pain.

    Del Bufalo, Alessandra; Cesario, Alfredo; Salinaro, Gianluca; Fini, Massimo; Russo, Patrizia

    2014-01-01

    Chronic pain is a widespread healthcare problem affecting not only the patient but in many ways all the society. Chronic pain is a disease itself that endures for a long period of time and it is resistant to the majority of medical treatments that provide modest improvements in pain and minimum improvements in physical and emotional functioning. More co-existing chronic pain conditions may be present in the same individual (patient). The α9α10 nicotinic acetylcholine receptor (nAChR) may be a potential target in the pathophysiology of chronic pain, as well in the development of breast and lung cancers. α-conotoxins (α-CNT) are small peptides used offensively by carnivorous marine snails known as Conus that target nAChR. Among α-CNT there are potent and selective antagonists of α9α10 nAChR such as RgIA and Vc1.1 that produces both acute and long lasting analgesia. Moreover, these peptides accelerate the recovery of nerve function after injury, likely through immune/inflammatory-mediated mechanisms. We review the background, findings, implications and problems in using compounds that act on α9α10 nAChR. PMID:24641230

  1. Effective loci and roles of acetylcholine in temperature regulation of goldfish.

    Crawshaw, L I; Wollmuth, L P

    1992-09-01

    Microinjections of acetylcholine (ACh) and carbachol were made into discrete forebrain loci in goldfish (Carassius auratus) to evaluate the importance of cholinergic mechanisms for behavioral thermoregulation. Injections of 5, 10, 25, and 50 micrograms ACh into the far anterior nucleus preopticus periventricularis (NPP) (R. Peter and V. Gill. J. Comp. Neurol. 159: 69-102, 1975) and immediately adjacent ventral telencephalon led to consistent dose-dependent decrease in selected temperature. No effect was observed following injections of 2 micrograms ACh or 0.7% NaCl. Injections of ACh into a different portion of the ventral telencephalon led to increases in the selected temperature. Lower doses of carbachol (0.5 and 1.0 micrograms) injected into the NPP produced decreases in selected temperature similar to the highest doses of ACh. Injections of ACh into loci other than those mentioned above either had no thermoregulatory effect or had lesser thermoregulatory effects which, in comparison with injections into the most effective sites, were inconsistent and required larger doses to obtain. The site where cholinergic stimulation led to decreases in the selected temperature exactly overlapped the effective site of ethanol hypothermia in the goldfish. PMID:1415647

  2. Impaired acetylcholine release from the myenteric plexus of Trichinella-infected rats

    We examined the release of acetylcholine (ACh) from jejunal longitudinal muscle-myenteric plexus preparations in noninfected control rats and in rats infected 6, 23, or 40 days previously with Trichinella spiralis. ACh release was assessed by preincubating the tissue with [3H]choline and measuring the evoked release of tritium. The uptake of 3H was significantly less in tissue from T. spiralis-infected rats compared with control. In tissues from either infected or control animals, electrical field stimulation (30 V, 0.5 ms, 10 Hz for 1 min), or veratridine (6-30 microM) induced 3H release that was tetrodotoxin sensitive. Depolarization by KCl (25-75 mM) also caused 3H release, but this was only partially reduced by tetrodotoxin. Radiochromatographic analysis indicated evoked release of 3H to be almost entirely [3H]ACh. In rats infected 6 days previously with T. spiralis, [3H]ACh release induced by KCl, veratridine, and field stimulation were decreased at least 80%. The suppression of [3H]ACh release induced by veratridine or KCl was fully reversible after 40 days postinfection, but field-stimulated responses remained approximately 50% of control values. These results indicate that T. spiralis infection in the rat is accompanied by a reversible suppression of ACh release from the longitudinal muscle-myenteric plexus of the jejunum

  3. Elemental maps in human allantochorial placental vessels cells: 1. High K + and acetylcholine effects

    Michelet-Habchi, C.; Barberet, Ph.; Dutta, R. K.; Guiet-Bara, A.; Bara, M.; Moretto, Ph.

    2003-09-01

    Regulation of vascular tone in the fetal extracorporeal circulation most likely depends on circulating hormones, local paracrine mechanisms and changes in membrane potential of vascular smooth muscle cells (VSMCs) and of vascular endothelial cells (VECs). The membrane potential is a function of the physiological activities of ionic channels (particularly, K + and Ca 2+ channels in these cells). These channels regulate the ionic distribution into these cells. Micro-particle induced X-ray emission (PIXE) analysis was applied to determine the ionic composition of VSMC and of VEC in the placental human allantochorial vessels in a physiological survival medium (Hanks' solution) modified by the addition of acetylcholine (ACh: which opens the calcium-sensitive K + channels, K Ca) and of high concentration of K + (which blocks the voltage-sensitive K + channels, K df). In VSMC (media layer), the addition of ACh induced no modification of the Na, K, Cl, P, S, Mg and Ca concentrations and high K + medium increased significantly the Cl and K concentrations, the other ion concentrations remaining constant. In endothelium (VEC), ACh addition implicated a significant increase of Na and K concentration, and high K + medium, a significant increase in Cl and K concentration. These results indicated the importance of K df, K Ca and K ATP channels in the regulation of K + intracellular distribution in VSMC and VEC and the possible intervention of a Na-K-2Cl cotransport and corroborated the previous electrophysiological data.

  4. Elemental maps in human allantochorial placental vessels cells: 1. High K+ and acetylcholine effects

    Regulation of vascular tone in the fetal extracorporeal circulation most likely depends on circulating hormones, local paracrine mechanisms and changes in membrane potential of vascular smooth muscle cells (VSMCs) and of vascular endothelial cells (VECs). The membrane potential is a function of the physiological activities of ionic channels (particularly, K+ and Ca2+ channels in these cells). These channels regulate the ionic distribution into these cells. Micro-particle induced X-ray emission (PIXE) analysis was applied to determine the ionic composition of VSMC and of VEC in the placental human allantochorial vessels in a physiological survival medium (Hanks' solution) modified by the addition of acetylcholine (ACh: which opens the calcium-sensitive K+ channels, KCa) and of high concentration of K+ (which blocks the voltage-sensitive K+ channels, Kdf). In VSMC (media layer), the addition of ACh induced no modification of the Na, K, Cl, P, S, Mg and Ca concentrations and high K+ medium increased significantly the Cl and K concentrations, the other ion concentrations remaining constant. In endothelium (VEC), ACh addition implicated a significant increase of Na and K concentration, and high K+ medium, a significant increase in Cl and K concentration. These results indicated the importance of Kdf, KCa and KATP channels in the regulation of K+ intracellular distribution in VSMC and VEC and the possible intervention of a Na-K-2Cl cotransport and corroborated the previous electrophysiological data

  5. Segregation of Acetylcholine and GABA in the Rat Superior Cervical Ganglia: Functional Correlation

    Elinos, Diana; Rodríguez, Raúl; Martínez, Luis Andres; Zetina, María Elena; Cifuentes, Fredy; Morales, Miguel Angel

    2016-01-01

    Sympathetic neurons have the capability to segregate their neurotransmitters (NTs) and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh) and other classical NTs such as gamma aminobutyric acid (GABA). Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX). We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level of segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region showed larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons. PMID:27092054

  6. Dithiocarbamate propineb induces acetylcholine release through cytoskeletal actin depolymerization in PC12 cells.

    Viviani, Barbara; Bartesaghi, Stefano; Binaglia, Marco; Corsini, Emanuela; Boraso, Mariaserena; Grazi, Enrico; Galli, Corrado L; Marinovich, Marina

    2008-11-10

    Neurological complications as well as movement disorders are relevant symptoms in animals and humans chronically exposed to dithiocarbamates. Using rat pheochromocytoma cells differentiated by NGF (PC12), we investigated whether propineb affects acetylcholine (Ach) release and the molecular mechanisms involved. Propineb (0.001-100 nM) dose-dependently increased Ach release from PC12. Thus, 0.001-1 nM propineb-induced Ach release, reaching a maximal effect ( approximately 50%) at 0.1-1 nM. Higher concentrations of propineb (10-100 nM) caused a progressive disappearance of the effect. Chelation of extra- and intracellular Ca(2+) did not affect Ach release by propineb, which was prevented by the actin stabilizer jasplakinolide (500 nM). Accordingly, actin depolymerization was observed after exposure of differentiated PC12 to 0.1-1 nM propineb, a loss of effect was evident at higher concentrations (100 nM), and the effect was Ca(2+)-independent. Disulfiram, a related dithiocarbamate not coordinated with Zn(2+), also depolymerized actin, suggesting the involvement of the organic structure of dithiocarbamates rather than the leakage of Zn(2+). Nevertheless, propineb did not depolymerize actin in a cell-free system. These data suggest that dithiocarbamates, through the activation of intracellular cascade(s), impair cytoskeletal actin. This effect may contribute to affect synaptic vesicles processing resulting in an impaired cholinergic transmission. PMID:18822360

  7. Interaction of bupropion with muscle-type nicotinic acetylcholine receptors in different conformational states.

    Arias, Hugo R; Gumilar, Fernanda; Rosenberg, Avraham; Targowska-Duda, Katarzyna M; Feuerbach, Dominik; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W; Bouzat, Cecilia

    2009-06-01

    To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion (a) inhibits epibatidine-induced Ca(2+) influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with approximately 100-fold higher potency compared to that in the open state, (c) increases the desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits binding of [(3)H]TCP and [(3)H]imipramine to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/alpha-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy-driven process, and (f) interacts with a binding domain located between the serine (position 6') and valine (position 13') rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively, our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process. PMID:19334677

  8. Interaction of Bupropion with Muscle-Type Nicotinic Acetylcholine Receptors in Different Conformational States†

    Arias, Hugo R.; Gumilar, Fernanda; Rosenberg, Avraham; Targowska-Duda, Katarzyna M.; Feuerbach, Dominik; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W.; Bouzat, Cecilia

    2009-01-01

    To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion: (a) inhibits epibatidine-induced Ca2+ influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with ~100-fold higher potency compared to that in the open state, (c) increases desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits [3H]TCP and [3H]imipramine binding to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/α-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy–driven process, and (f) interacts with a binding domain located between the serine (position 6’) and valine (position 13’) rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process. PMID:19334677

  9. Interaction of selective serotonin reuptake inhibitors with neuronal nicotinic acetylcholine receptors.

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Russell, Megan; Jozwiak, Krzysztof

    2010-07-13

    We compared the interaction of fluoxetine and paroxetine, two selective serotonin reuptake inhibitors (SSRIs), with the human (h) alpha4beta2, alpha3beta4, and alpha7 nicotinic acetylcholine receptors (AChRs) in different conformational states, using Ca(2+) influx, radioligand binding, and molecular docking approaches. The results established that (1) fluoxetine was more potent than paroxetine in inhibiting agonist-activated Ca(2+) influx on halpha4beta2 and halpha7 AChRs, whereas the potency of both SSRIs was practically the same in the halpha3beta4 AChR. [corrected] (2) SSRIs bind to the [(3)H]imipramine locus with a [corrected] higher affinity when the AChRs are in the desensitized states compared to the resting states. (3) The different receptor specificity for fluoxetine determined by their inhibitory potencies or binding affinities suggests different modes of interaction when the AChR is in the closed or activated state. (4) Neutral and protonated fluoxetine interacts with a binding domain located in the middle of the AChR ion channel. In conclusion, SSRIs inhibit the most important neuronal AChRs with potencies and affinities that are clinically relevant by binding to a luminal site that is shared with tricyclic antidepressants. PMID:20527991

  10. A New Role for Attentional Corticopetal Acetylcholine in Cortical Memory Dynamics

    Fujii, Hiroshi; Kanamaru, Takashi; Aihara, Kazuyuki; Tsuda, Ichiro

    2011-09-01

    Although the role of corticopetal acetylcholine (ACh) in higher cognitive functions is increasingly recognized, the questions as (1) how ACh works in attention(s), memory dynamics and cortical state transitions, and also (2) why and how loss of ACh is involved in dysfunctions such as visual hallucinations in dementia with Lewy bodies and deficit of attention(s), are not well understood. From the perspective of a dynamical systems viewpoint, we hypothesize that transient ACh released under top-down attention serves to temporarily invoke attractor-like memories, while a background level of ACh reverses this process returning the dynamical nature of the memory structure back to attractor ruins (quasi-attractors). In fact, transient ACh loosens inhibitions of py ramidal neurons (PYRs) by P V+ fas t spiking (FS) i nterneurons, while a baseline ACh recovers inhibitory actions of P V+ FS. Attentional A Ch thus dynamically modifies brain's connectivity. Th e core of this process is in the depression of GABAergic inhibitory currents in PYRs due to muscarinic (probably M2 subtype) presyn aptic effects on GABAergic synapses of PV+ FS neurons

  11. Segregation of Acetylcholine and GABA in the Rat Superior Cervical Ganglia: Functional Correlation.

    Elinos, Diana; Rodríguez, Raúl; Martínez, Luis Andres; Zetina, María Elena; Cifuentes, Fredy; Morales, Miguel Angel

    2016-01-01

    Sympathetic neurons have the capability to segregate their neurotransmitters (NTs) and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh) and other classical NTs such as gamma aminobutyric acid (GABA). Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX). We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level of segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region showed larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons. PMID:27092054

  12. The subpopulation of microglia expressing functional muscarinic acetylcholine receptors expands in stroke and Alzheimer's disease.

    Pannell, Maria; Meier, Maria Almut; Szulzewsky, Frank; Matyash, Vitali; Endres, Matthias; Kronenberg, Golo; Prinz, Vincent; Waiczies, Sonia; Wolf, Susanne A; Kettenmann, Helmut

    2016-03-01

    Microglia undergo a process of activation in pathology which is controlled by many factors including neurotransmitters. We found that a subpopulation (11 %) of freshly isolated adult microglia respond to the muscarinic acetylcholine receptor agonist carbachol with a Ca(2+) increase and a subpopulation of similar size (16 %) was observed by FACS analysis using an antibody against the M3 receptor subtype. The carbachol-sensitive population increased in microglia/brain macrophages isolated from tissue of mouse models for stroke (60 %) and Alzheimer's disease (25 %), but not for glioma and multiple sclerosis. Microglia cultured from adult and neonatal brain contained a carbachol-sensitive subpopulation (8 and 9 %), which was increased by treatment with interferon-γ to around 60 %. This increase was sensitive to blockers of protein synthesis and correlated with an upregulation of the M3 receptor subtype and with an increased expression of MHC-I and MHC-II. Carbachol was a chemoattractant for microglia and decreased their phagocytic activity. PMID:25523105

  13. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Marlet Martinez-Archundia

    2012-01-01

    Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

  14. Gating of long-term potentiation by nicotinic acetylcholine receptors at the cerebellum input stage.

    Francesca Prestori

    Full Text Available The brain needs mechanisms able to correlate plastic changes with local circuit activity and internal functional states. At the cerebellum input stage, uncontrolled induction of long-term potentiation or depression (LTP or LTD between mossy fibres and granule cells can saturate synaptic capacity and impair cerebellar functioning, which suggests that neuromodulators are required to gate plasticity processes. Cholinergic systems innervating the cerebellum are thought to enhance procedural learning and memory. Here we show that a specific subtype of acetylcholine receptors, the α7-nAChRs, are distributed both in cerebellar mossy fibre terminals and granule cell dendrites and contribute substantially to synaptic regulation. Selective α7-nAChR activation enhances the postsynaptic calcium increase, allowing weak mossy fibre bursts, which would otherwise cause LTD, to generate robust LTP. The local microperfusion of α7-nAChR agonists could also lead to in vivo switching of LTD to LTP following sensory stimulation of the whisker pad. In the cerebellar flocculus, α7-nAChR pharmacological activation impaired vestibulo-ocular-reflex adaptation, probably because LTP was saturated, preventing the fine adjustment of synaptic weights. These results show that gating mechanisms mediated by specific subtypes of nicotinic receptors are required to control the LTD/LTP balance at the mossy fibre-granule cell relay in order to regulate cerebellar plasticity and behavioural adaptation.

  15. Expression of somatostatin receptor genes and acetylcholine receptor development in rat skeletal muscle during postnatal development.

    Peng, M; Conforti, L; Millhorn, D E

    1998-05-01

    Our laboratory reported previously that somatostatin (SST) is transiently expressed in rat motoneurons during the first 14 days after birth. We investigated the possibility that the SST receptor (SSTR) is expressed in skeletal muscle. We found that two of the five subtypes of SSTR (SSTR3 and SSTR4) are expressed in skeletal muscle with a time course that correlates with the transient expression of SST in motoneurons. In addition, SSTR2A is expressed from birth to adulthood in skeletal muscle. Both SSTR2A and SSTR4 are also expressed in L6 cells, a skeletal muscle cell line. Somatostatin acting through its receptors has been shown to stimulate tyrosine phosphatase activity in a number of different tissues. We found that several proteins (50, 65, 90, 140, 180 and 200 kDa) exhibited a reduced degree of tyrosine phosphorylation following SST treatment. Inhibition of tyrosine phosphatase activity with sodium orthovanadate increased expression of the nicotinic acetyl-choline receptor (nAChR) epsilon subunit mRNA by three fold. Somatostatin reversed the elevated epsilon mRNA following orthovanadate treatment. These findings show that SSTR is expressed in skeletal muscle and that SST acting via the SSTR regulates tyrosine phosphorylation and expression of the epsilon subunit of the AChR in the rat skeletal muscle. PMID:9852305

  16. Synergistic effect between 5-HT4 receptor agonist and phosphodiesterase 4-inhibitor in releasing acetylcholine in pig gastric circular muscle in vitro.

    Lefebvre, Romain A; Van Colen, Inge; Pauwelyn, Vicky; De Maeyer, Joris H

    2016-06-15

    5-HT4 receptor agonists have a gastroprokinetic effect by facilitating acetylcholine release from cholinergic nerves innervating gastrointestinal smooth muscle. The role of phosphodiesterase (PDE) 4 in the signal transduction pathway of the 5-HT4 receptors located on the cholinergic neurons towards the circular muscle layer in pig stomach was investigated by analysis of acetylcholine release. Circular muscle strips were prepared from pig proximal stomach and tritium outflow, induced by electrical field stimulation, was studied as a marker for acetylcholine release after incubation with [(3)H]-choline. The PDE4-inhibitor roflumilast concentration-dependently (0.1-1µM) enhanced the facilitating effect of a submaximally effective concentration of the 5-HT4 receptor agonist prucalopride (0.01µM) on electrically induced acetylcholine release. Roflumilast (0.3µM) enhanced acetylcholine release per se but in the combined presence of roflumilast and prucalopride, acetylcholine release was enhanced more than the sum of the effect of the 2 compounds alone. The 5-HT4 receptor agonist velusetrag concentration-dependently (0.01-0.1µM) enhanced acetylcholine release; the effect of the minimally effective concentration (0.01µM) was significantly enhanced by 1µM of the PDE4-inhibitor rolipram, again to a level higher than the sum of the effect of the 2 compounds alone. The synergistic effect between 5-HT4 receptor agonists and PDE4-inhibitors demonstrates that the intracellular pathway of the 5-HT4 receptors located on cholinergic neurons towards pig gastric circular muscle is controlled by PDE4. Combining a 5-HT4 receptor agonist with a PDE4-inhibitor might thus enhance its gastroprokinetic effect. PMID:27060014

  17. Effect of tissue-specific acetylcholinesterase inhibitor C-547 on α3β4 and αβεδ acetylcholine receptors in COS cells.

    Lindovský, Jiří; Petrov, Konstantin; Krůšek, Jan; Reznik, Vladimir S; Nikolsky, Eugeny E; Vyskočil, František

    2012-08-01

    The C-547 is the most effective muscle and tissue-specific anticholinesterase among alkylammonium derivatives of 6-methyluracil (ADEMS) acting in nanomolar concentrations on locomotor muscles but not on respiratory muscles, smooth muscles and heart and brain acetylcholine esterases (AChE). When applied systematically it could influence peripheral acetylcholine receptors. The aim of the present study was to investigate the effect of C-547 on rat α3β4 (ganglionic type) and αβεδ (muscle type) nicotinic receptors expressed in COS cells. Currents evoked by rapid application of acetylcholine or nicotine were recorded in whole-cell mode by electrophysiological patch-clamp technique 2-4 days after cell transfection by plasmids coding the α3β4 or αβεδ combination of receptor subunits. In cells sensitive to acetylcholine, the application of C-547 evoked no responses. When acetylcholine was applied during an already running application of C-547, acetylcholine responses were only inhibited at concentrations higher than 10(-7)M. This inhibition is not voltage-dependent, but is accompanied by an increased rate of desensitization. Thus in both types of receptors, effective doses are approximately 100 times higher than those inhibiting AChE in leg muscles and similar to those inhibiting respiratory diaphragm muscles and external intercostal muscles. These observations show that C-547 can be considered for symptomatic treatment of myasthenia gravis and other congenital myasthenic syndromes as an inhibitor of AChE in leg muscles at concentrations much lower than those inhibiting muscle and ganglion types of acetylcholine receptors. PMID:22634638

  18. Comparative studies of huperzine A,donepezil,and rivastigmine on brain acetylcholine,dopamine,norepinephrine,and 5-hydroxytryptamine levels in freely-moving rats

    Yan-qi LIANG; Xi-can TANG

    2006-01-01

    Aim: To assess the effects of cholinesterase inhibitors huperzine A, donepezil and rivastigmine on cerebral neurotransmitters in the cortex and hippocampus in freely-moving rats. Methods: Double-probe cerebral microdialysis and HPLC with electrochemical detection were used to detect neurotransmitters. Results: Our results showed that huperzine A (0.25, 0.5, and 0.75 μmol/kg, po) dose-depen-dently elevated extracellular acetylcholine (Ach) levels in the medial prefrontal cortex (Mpfc) and hippocampus. Oral administration of donepezil (5.4 μmol/kg) or rivastigmine (1 μmol/kg) also elicited significant increases in Ach in the Mpfc and hippocampus. The time course of cortical acetylcholinesterase (AChE) inhibition with the 3 inhibitors mirrored the increases of Ach at the same dose. The marked elevation of Ach after oral administration of huperzine A (0.5 μmol/kg) and donepezil (5.4 μmol/kg) was associated with a significantly increased release of dopamine (DA) in the Mpfc or hippocampus. None of the 3 inhibitors affected norepinephrine (NE) and 5-hydroxytryptamine (5-HT) levels in the Mpfc and hippocampus. The effects of huperzine A and rivastigmine did not depend on the route of administration, but donepezil was less efficacious by the oral route than by ip injection. The ability of huperzine A to increase Ach levels was unchanged when tests were performed after multiple oral administration of the drug at 0.5 μmol/kg, once per day for 30 d. Conclusion: The present findings showed that, in molar terms, huperzine A had similar potency on increasing Mpfc Ach and DA levels as compared to the 11- and 2-fold dosages of donepezil and rivastigmine, respectively, and had longer lasting effects after oral dosing.

  19. Different modulation by Ca2+-activated K+ channel blockers and herbimycin of acetylcholine- and flow-evoked vasodilatation in rat mesenteric small arteries

    Thorsgaard, Michael; Lopez, Vanesa; Buus, Niels H; Simonsen, Ulf

    2003-01-01

    The present study addressed whether endothelium-dependent vasodilatation evoked by acetylcholine and flow are mediated by the same mechanisms in isolated rat mesenteric small arteries, suspended in a pressure myograph for the measurement of internal diameter. In pressurized arterial segments contracted with U46619 in the presence of indomethacin, shear stress generated by the flow evoked relaxation. Thus, in endothelium-intact segments low (5.1±0.6 dyn cm−2) and high (19±2 dyn cm−2) shear stress evoked vasodilatations that were reduced by, respectively, 68±11 and 68±8% (P<0.05, n=7) by endothelial cell removal. Acetylcholine (0.01–1 μM) evoked concentration-dependent vasodilatation that was abolished by endothelial cell removal. Incubation with indomethacin alone did not change acetylcholine and shear stress-evoked vasodilatation, while the combination of indomethacin with the nitric oxide (NO) synthase inhibitor, NG,NG-asymmetric dimethyl-L-arginine (ADMA 1 mM), reduced low and high shear stress-evoked vasodilatation with, respectively, 52±15 and 58±10% (P<0.05, n=9), but it did not change acetylcholine-evoked vasodilatation. Inhibition of Ca2+-activated K+ channels with a combination of apamin (0.5 μM) and charybdotoxin (ChTX) (0.1 μM) did not change shear stress- and acetylcholine-evoked vasodilatation. In the presence of indomethacin and ADMA, the combination of apamin (0.5 μM) and ChTx (0.1 μM) increased contraction induced by U46619, but these blockers did not change the vasodilatation evoked by shear stress. In contrast, acetylcholine-evoked vasodilatation was abolished by the combination of apamin and charybdotoxin. In the presence of indomethacin, the tyrosine kinase inhibitor, herbimycin A (1 μM), inhibited low and high shear stress-evoked vasodilatation with, respectively, 32±12 and 68±14% (P<0.05, n=8), but it did not change vasodilatation induced by acetylcholine. In the presence of indomethacin and ADMA, herbimycin A neither

  20. Highly selective and sensitive detection of neurotransmitters using receptor-modified single-walled carbon nanotube sensors

    We present receptor-modified carbon nanotube sensors for the highly selective and sensitive detection of acetylcholine (ACh), one kind of neurotransmitter. Here, we successfully expressed the M1 muscarinic acetylcholine receptor (M1 mAChR), a family of G protein-coupled receptors (GPCRs), in E. coli and coated single-walled carbon nanotube (swCNT)-field effect transistors (FETs) with lipid membrane including the receptor, enabling highly selective and sensitive ACh detection. Using this sensor, we could detect ACh at 100 pM concentration. Moreover, we showed that this sensor could selectively detect ACh among other neurotransmitters. This is the first demonstration of the real-time detection of ACh using specific binding between ACh and M1 mAChR, and it may lead to breakthroughs for various applications such as disease diagnosis and drug screening. (paper)

  1. Highly selective and sensitive detection of neurotransmitters using receptor-modified single-walled carbon nanotube sensors

    Kim, Byeongju; Song, Hyun Seok; Jin, Hye Jun; Park, Eun Jin; Lee, Sang Hun; Lee, Byung Yang; Park, Tai Hyun; Hong, Seunghun

    2013-07-01

    We present receptor-modified carbon nanotube sensors for the highly selective and sensitive detection of acetylcholine (ACh), one kind of neurotransmitter. Here, we successfully expressed the M1 muscarinic acetylcholine receptor (M1 mAChR), a family of G protein-coupled receptors (GPCRs), in E. coli and coated single-walled carbon nanotube (swCNT)-field effect transistors (FETs) with lipid membrane including the receptor, enabling highly selective and sensitive ACh detection. Using this sensor, we could detect ACh at 100 pM concentration. Moreover, we showed that this sensor could selectively detect ACh among other neurotransmitters. This is the first demonstration of the real-time detection of ACh using specific binding between ACh and M1 mAChR, and it may lead to breakthroughs for various applications such as disease diagnosis and drug screening.

  2. “Warming yang and invigorating qi” acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis

    Huang, Hai-peng; Pan, Hong; Wang, Hong-feng

    2016-01-01

    Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction. “Warming yang and invigorating qi” acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis. However, few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction. Here, we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following “warming yang and invigorating qi” acupuncture therapy. Needles were inserted at acupressure points Shousanli (LI10), Zusanli (ST36), Pishu (BL20), and Shenshu (BL23) once daily for 7 consecutive days. The treatment was repeated after 1 day of rest. We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment. This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide. These findings suggest that “warming yang and invigorating qi” acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis. PMID:27127487

  3. "Warming yang and invigorating qi" acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis.

    Huang, Hai-Peng; Pan, Hong; Wang, Hong-Feng

    2016-03-01

    Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction. "Warming yang and invigorating qi" acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis. However, few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction. Here, we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following "warming yang and invigorating qi" acupuncture therapy. Needles were inserted at acupressure points Shousanli (LI10), Zusanli (ST36), Pishu (BL20), and Shenshu (BL23) once daily for 7 consecutive days. The treatment was repeated after 1 day of rest. We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment. This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide. These findings suggest that "warming yang and invigorating qi" acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis. PMID:27127487

  4. "Warming yang and invigorating qi" acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis

    Hai-peng Huang

    2016-01-01

    Full Text Available Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction. "Warming yang and invigorating qi" acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis. However, few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction. Here, we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following "warming yang and invigorating qi" acupuncture therapy. Needles were inserted at acupressure points Shousanli (LI10, Zusanli (ST36, Pishu (BL20, and Shenshu (BL23 once daily for 7 consecutive days. The treatment was repeated after 1 day of rest. We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment. This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide. These findings suggest that "warming yang and invigorating qi" acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis.

  5. Aspects of dopamine and acetylcholine release induced by glutamate receptors; Aspectos das liberacoes de dopamina e acetilcolina mediadas por receptores de glutamato

    Paes, Paulo Cesar de Arruda

    2002-07-01

    The basal ganglia play an important role in the motor control of rats and humans. This control involves different neurotransmitters and the mutual control of these key elements has been subject to several studies. In this work we determined the role of glutamate on the release of radioactively labelled dopamine and acetylcholine from chopped striatal tissue in vitro. The values of Effective Concentration 50% for glutamate, NMDA, kainic, quisqualic acids and AMPA on the release of dopamine and acetylcholine were obtained. The inhibitory effects of magnesium, tetrodotoxin, MK-801, AP5 and MCPG, as well as the effects of glycin were evaluated. The results suggested that dopamine is influenced by the NMDA type glutamate receptor while acetylcholine seems to be influenced by NMDA, kainate and AMPA receptors. Tetrodotoxin experiments suggested that kainate receptors are both present in cholinergic terminals and cell bodies while AMPA and NMDA receptors are preferentially distributed in cell bodies. Magnesium effectively blocked the NMDA stimulation and unexpectedly also AMPA- and quisqualate-induced acetylcholine release. The latter could not be blocked by MCPG ruling out the participation of methabotropic receptors. MK-801 also blocked NMDA-receptors. Results point out the importance of the glutamic acid control of dopamine and acetylcholine release in striatal tissue. (author)

  6. Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus.

    Vázquez-Gómez, Elizabeth; Arias, Hugo R; Feuerbach, Dominik; Miranda-Morales, Marcela; Mihailescu, Stefan; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof; García-Colunga, Jesús

    2014-10-01

    The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation. PMID:25016090

  7. The generation and breaking of internal waves on a lutocline in the Ems estuary (Germany), their hydro-acoustic detectability and their contribution to sediment transport dynamics

    Held, Philipp

    2014-01-01

    This thesis deals with internal waves propagating along a lutocline and their influence on the local sediment transport dynamics in the German Ems estuary. Additionally, the operability of oceanographic instruments in highly turbid waters, which is the case in the Ems estuary, is verified.

  8. Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: Pharmacological and biochemical properties

    The pharmacological and biochemical properties of rat m1 and m3 muscarinic acetylcholine receptors (mAChR) stably transfected into Chinese hamster ovary-K1 (CHO) cells were characterized with ligand binding, affinity labeling and biochemical assays. Both mAChR subtypes display saturable, high affinity binding of [3H]-quinuclidinyl benzilate (QNB) and a rank order of antagonist potency of QNB greater than atropine greater than pirenzepine greater than AF-DX 116. Carbachol displacement of [3H]-QNB binding to the m3 mAChR revealed an approximate 17-fold higher affinity than observed with the m1 mAChR. [3H]-propylbenzilylcholine mustard (PrBCM) labeling of mAChR revealed that m1 and m3 mAChR migrated on SDS-polyacrylamide gels with apparent molecular masses of 80,000 and 94,000 daltons, respectively, consistent with the known differences in their molecular sizes. Both m1 and m3 mAChR elicited dose-dependent increases in the hydrolysis of phosphoinositides; however, the maximal increase in total inositol phosphates elicited with the m1 mAChR was approximately 2-fold greater than that observed in cells expressing similar densities of m3 mAChR. Agonist activation of the m1 mAChR also elicited increases in basal and forskolin-stimulated cAMP, whereas the m3 mAChR had no effect on intracellular cAMP levels. These data suggest that although m1 and m3 mAChR display a considerable degree of structural homology, they exhibit distinct pharmacological and biochemical properties

  9. Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: Pharmacological and biochemical properties

    Buck, M.A.; Fraser, C.M. (National Institute on Alcohol Abuse and Alcoholism, Rockville, MD (USA))

    1990-12-14

    The pharmacological and biochemical properties of rat m1 and m3 muscarinic acetylcholine receptors (mAChR) stably transfected into Chinese hamster ovary-K1 (CHO) cells were characterized with ligand binding, affinity labeling and biochemical assays. Both mAChR subtypes display saturable, high affinity binding of (3H)-quinuclidinyl benzilate (QNB) and a rank order of antagonist potency of QNB greater than atropine greater than pirenzepine greater than AF-DX 116. Carbachol displacement of (3H)-QNB binding to the m3 mAChR revealed an approximate 17-fold higher affinity than observed with the m1 mAChR. (3H)-propylbenzilylcholine mustard (PrBCM) labeling of mAChR revealed that m1 and m3 mAChR migrated on SDS-polyacrylamide gels with apparent molecular masses of 80,000 and 94,000 daltons, respectively, consistent with the known differences in their molecular sizes. Both m1 and m3 mAChR elicited dose-dependent increases in the hydrolysis of phosphoinositides; however, the maximal increase in total inositol phosphates elicited with the m1 mAChR was approximately 2-fold greater than that observed in cells expressing similar densities of m3 mAChR. Agonist activation of the m1 mAChR also elicited increases in basal and forskolin-stimulated cAMP, whereas the m3 mAChR had no effect on intracellular cAMP levels. These data suggest that although m1 and m3 mAChR display a considerable degree of structural homology, they exhibit distinct pharmacological and biochemical properties.

  10. alpha4beta2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief

    McGranahan, Tresa M.; Patzlaff, Natalie E.; Grady, Sharon R.; Heinemann, Stephen F.; Booker, T.K.

    2012-01-01

    Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addition, mental illness and movement control in humans. We developed a unique model system to examine the role of alpha4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the alpha4 subunit from dopaminergic neurons in mice. The loss alpha4 mRNA and alpha4beta2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of alpha4beta2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. Alpha4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. Alpha4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze and elimination of alpha4-beta2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of alpha4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression, however nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine behaviors. PMID:21795541

  11. α4β2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief.

    McGranahan, Tresa M; Patzlaff, Natalie E; Grady, Sharon R; Heinemann, Stephen F; Booker, T K

    2011-07-27

    Nicotine is the primary psychoactive substance in tobacco, and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the α4β2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal as well as nicotine-induced behaviors. Although α4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addiction, mental illness, and movement control in humans. We developed a unique model system to examine the role of α4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the α4 subunit from dopaminergic neurons in mice. The loss α4 mRNA and α4β2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of α4β2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. α4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. α4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze, and elimination of α4β2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of α4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression; however, nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine-related behaviors. PMID:21795541

  12. Centrally injected histamine increases posterior hypothalamic acetylcholine release in hemorrhage-hypotensive rats.

    Altinbas, Burcin; Yilmaz, Mustafa S; Savci, Vahide; Jochem, Jerzy; Yalcin, Murat

    2015-01-01

    Histamine, acting centrally as a neurotransmitter, evokes a reversal of hemorrhagic hypotension in rats due to the activation of the sympathetic and the renin-angiotensin systems as well as the release of arginine vasopressin and proopiomelanocortin-derived peptides. We demonstrated previously that central nicotinic cholinergic receptors are involved in the pressor effect of histamine. The aim of the present study was to examine influences of centrally administrated histamine on acetylcholine (ACh) release at the posterior hypothalamus-a region characterized by location of histaminergic and cholinergic neurons involved in the regulation of the sympathetic activity in the cardiovascular system-in hemorrhage-hypotensive anesthetized rats. Hemodynamic and microdialysis studies were carried out in Sprague-Dawley rats. Hemorrhagic hypotension was induced by withdrawal of a volume of 1.5 ml blood/100 g body weight over a period of 10 min. Acute hemorrhage led to a severe and long-lasting decrease in mean arterial pressure (MAP), heart rate (HR), and an increase in extracellular posterior hypothalamic ACh and choline (Ch) levels by 56% and 59%, respectively. Intracerebroventricularly (i.c.v.) administered histamine (50, 100, and 200 nmol) dose- and time-dependently increased MAP and HR and caused an additional rise in extracellular posterior hypothalamic ACh and Ch levels at the most by 102%, as compared to the control saline-treated group. Histamine H1 receptor antagonist chlorpheniramine (50 nmol; i.c.v.) completely blocked histamine-evoked hemodynamic and extracellular posterior hypothalamic ACh and Ch changes, whereas H2 and H3/H4 receptor blockers ranitidine (50 nmol; i.c.v.) and thioperamide (50 nmol; i.c.v.) had no effect. In conclusion, centrally administered histamine, acting via H1 receptors, increases ACh release at the posterior hypothalamus and causes a pressor and tachycardic response in hemorrhage-hypotensive anesthetized rats. PMID:25468497

  13. Developmental adaptation of central nervous system to extremely high acetylcholine levels.

    Vladimir Farar

    Full Text Available Acetylcholinesterase (AChE is a key enzyme in termination of fast cholinergic transmission. In brain, acetylcholine (ACh is produced by cholinergic neurons and released in extracellular space where it is cleaved by AChE anchored by protein PRiMA. Recently, we showed that the lack of AChE in brain of PRiMA knock-out (KO mouse increased ACh levels 200-300 times. The PRiMA KO mice adapt nearly completely by the reduction of muscarinic receptor (MR density. Here we investigated changes in MR density, AChE, butyrylcholinesterase (BChE activity in brain in order to determine developmental period responsible for such adaptation. Brains were studied at embryonal day 18.5 and postnatal days (pd 0, 9, 30, 120, and 425. We found that the AChE activity in PRiMA KO mice remained very low at all studied ages while in wild type (WT mice it gradually increased till pd120. BChE activity in WT mice gradually decreased until pd9 and then increased by pd120, it continually decreased in KO mice till pd30 and remained unchanged thereafter. MR number increased in WT mice till pd120 and then became stable. Similarly, MR increased in PRiMA KO mice till pd30 and then remained stable, but the maximal level reached is approximately 50% of WT mice. Therefore, we provide the evidence that adaptive changes in MR happen up to pd30. This is new phenomenon that could contribute to the explanation of survival and nearly unchanged phenotype of PRiMA KO mice.

  14. Structure-activity relationship of ibogaine analogs interacting with nicotinic acetylcholine receptors in different conformational states.

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof

    2011-09-01

    The interaction of ibogaine analogs with nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that ibogaine analogs: (a) inhibit (±)-epibatidine-induced Ca²⁺ influx in human embryonic muscle AChRs with the following potency sequence (IC(50) in μM): (±)-18-methylaminocoronaridine (5.9±0.3)∼(±)-18-methoxycoronaridine (18-MC) (6.8±0.8)>(-)-ibogaine (17±3)∼(+)-catharanthine (20±1)>(±)-albifloranine (46±13), (b) bind to the [³H]TCP binding site with higher affinity when the Torpedo AChR is in the desensitized state compared to that in the resting state. Similar results were obtained using [³H]18-MC. These and docking results suggest a steric interaction between TCP and ibogaine analogs for the same site, (c) enhance [³H]cytisine binding to resting but not to desensitized AChRs, with desensitizing potencies (apparent EC₅₀) that correlate very well with the pK(i) values in the desensitized state, and (d) there are good bilinear correlations between the ligand molecular volumes and their affinities in the desensitized and resting states, with an optimal volume of ∼345 ų for the ibogaine site. These results indicate that the size of the binding sites for ibogaine analogs, located between the serine and nonpolar rings and shared with TCP, is an important structural feature for binding and for inducing desensitization. PMID:21642011

  15. Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.

    Tillman, Tommy S; Alvarez, Frances J D; Reinert, Nathan J; Liu, Chuang; Wang, Dawei; Xu, Yan; Xiao, Kunhong; Zhang, Peijun; Tang, Pei

    2016-08-26

    Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human α7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design. PMID:27385587

  16. Brain α7 nicotinic acetylcholine receptors in MPTP-lesioned monkeys and parkinsonian patients.

    Morissette, Marc; Morin, Nicolas; Grégoire, Laurent; Rajput, Alex; Rajput, Ali H; Di Paolo, Thérèse

    2016-06-01

    L-DOPA-induced dyskinesias (LID) appear in the majority of Parkinson's disease (PD) patients. Nicotinic acetylcholine (nACh) receptor-mediated signaling has been implicated in PD and LID and modulation of brain α7 nACh receptors might be a potential therapeutic target for PD. This study used [(125)I]α-Bungarotoxin autoradiography to investigate α7 nACh receptors in LID in post-mortem brains from PD patients (n=14) and control subjects (n=11), and from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys treated with saline (n=5), L-DOPA (n=4) or L-DOPA+2-methyl-6-(phenylethynyl)pyridine (MPEP) (n=5), and control monkeys (n=4). MPEP is the prototypal metabotropic glutamate 5 (mGlu5) receptor antagonist; it reduced the development of LID in these monkeys. [(125)I]α-Bungarotoxin specific binding to striatal and pallidal α7 nACh receptors were only increased in L-DOPA-treated dyskinetic MPTP monkeys as compared to controls, saline and L-DOPA+MPEP MPTP monkeys; dyskinesia scores correlated positively with this binding. The total group of Parkinsonian patients had higher [(125)I]α-Bungarotoxin specific binding compared to controls in the caudate nucleus but not in the putamen. PD patients without motor complications had higher [(125)I]α-Bungarotoxin specific binding compared to controls only in the caudate nucleus. PD patients with LID only had higher [(125)I]α-Bungarotoxin specific binding compared to controls in the caudate nucleus and compared to those without motor complications and controls in the putamen. PD patients with wearing-off only, had [(125)I]α-Bungarotoxin specific binding at control values in the caudate nucleus and lower in the putamen. Reduced motor complications were associated with normal striatal α7 nACh receptors, suggesting the potential of this receptor to manage motor complications in PD. PMID:27038656

  17. Alpha7 nicotinic acetylcholine receptor agonists and PAMs as adjunctive treatment in schizophrenia. An experimental study.

    Marcus, Monica M; Björkholm, Carl; Malmerfelt, Anna; Möller, Annie; Påhlsson, Ninni; Konradsson-Geuken, Åsa; Feltmann, Kristin; Jardemark, Kent; Schilström, Björn; Svensson, Torgny H

    2016-09-01

    Nicotine has been found to improve cognition and reduce negative symptoms in schizophrenia and a genetic and pathophysiological link between the α7 nicotinic acetylcholine receptors (nAChRs) and schizophrenia has been demonstrated. Therefore, there has been a large interest in developing drugs affecting the α7 nAChRs for schizophrenia. In the present study we investigated, in rats, the effects of a selective α7 agonist (PNU282987) and a α7 positive allosteric modulator (PAM; NS1738) alone and in combination with the atypical antipsychotic drug risperidone for their utility as adjunct treatment in schizophrenia. Moreover we also investigated their utility as adjunct treatment in depression in combination with the SSRI citalopram. We found that NS1738 and to some extent also PNU282987, potentiated a subeffective dose of risperidone in the conditioned avoidance response test. Both drugs also potentiated the effect of a sub-effective concentration of risperidone on NMDA-induced currents in pyramidal cells of the medial prefrontal cortex. Moreover, NS1738 and PNU282987 enhanced recognition memory in the novel object recognition test, when given separately. Both drugs also potentiated accumbal but not prefrontal risperidone-induced dopamine release. Finally, PNU282987 reduced immobility in the forced swim test, indicating an antidepressant-like effect. Taken together, our data support the utility of drugs targeting the α7 nAChRs, perhaps especially α7 PAMs, to potentiate the effect of atypical antipsychotic drugs. Moreover, our data suggest that α7 agonists and PAMs can be used to ameliorate cognitive symptoms in schizophrenia and depression. PMID:27474687

  18. [Treatment approach to congenital myasthenic syndrome in a patient with acetylcholine receptor deficiency].

    Ishigaki, Keiko; Murakami, Terumi; Ito, Yasushi; Yanagisawa, Akiko; Kodaira, Kayano; Shishikura, Keiko; Suzuki, Haruko; Hirayama, Yoshito; Osawa, Makiko

    2009-01-01

    Congenital myasthenic syndromes (CMS) are rare heterogeneous disorders of neurotransmission caused by genetic defects of neuromuscular junction molecules. While CMS patients have been reported worldwide, in Japan there have been only a few descriptions of adult CMS patients with acetylcholinesterase (AChE) deficiency and slow channel syndrome. Herein, we report a Japanese CMS patient with acetylcholine receptor (AChR) deficiency, diagnosed during childhood, and our treatment approach to the patient. This 13-year-old Japanese boy had had severe myasthenic symptoms since infancy. Ptosis, his first symptom, appeared at 5 months and nasal voice was recognized at 2 years of age. AchR and anti-muscle-specific tyrosine kinase (Musk) antibody remained negative. A positive tensilon test and decremental response on electromyogram supported the diagnosis of sero-negative myasthenia gravis. Despite thymectomy and strong immunosuppressive therapy including steroid pulse and FK 506, he gradually deteriorated and became wheelchair bound. Genetic analyses for AchR, Rapsyn, Musk and AChE were negative. At age 11 years, a muscle biopsy was performed in the deltoid muscle for neuromuscular junction sampling. Electron microscopic and confocal microscopic analysis of endplates showed almost complete loss of AChR and the diagnosis of CMS with AChR deficiency was confirmed. All immunosuppressive therapies were discontinued. Instead, we started Ubretide and 3,4-diaminopyridine (DAP) after obtaining informed consent. Although not approved in Japan for this use, 3,4-DAP is reportedly effective in refractory cases of CMS. The patient experienced no side effects. Despite all of the objective data were improving, his subjective symptoms and ADL remained poor. There are still many challenges in the treatment of the patient. PMID:19172815

  19. Association of nicotinic acetylcholine receptor subunit alpha-4 polymorphisms with smoking behaviors in Chinese male smokers

    CHU Cheng-jing; YANG Yan-chun; WEI Jin-xue; ZHANG Lan

    2011-01-01

    Background It has been reported that the nicotinic acetylcholine receptor subunit a4 gene (CHRNA4) might be associated with smoking behaviors in the previous studies. Up to now, there are few reports on the relationship between CHRNA4 and smoking initiation. In this study, we tried to explore the role of two polymorphisms in CHRNA4 (rs 1044396 and rs 1044397) in smoking initiation and nicotine dependence in Chinese male smokers.Methods Nine hundred and sixty-six Chinese male lifetime nonsmokers and smokers were assessed by the Fagerstr(o)m test for nicotine dependence (FTND), smoking quantity (SQ) and the heaviness of smoking index (HSI). All subjects were divided into four groups based on their tobacco use history and the FTND scores. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find two polymorphisms of CHRNA4 in these subjects.Results The x2 test showed that rs1044396 was significantly associated with smoking initiation (x2=4.65, P=0.031),while both rs1044396 and rs1044397 were significantly associated with nicotine dependence (x2=5.42, P=0.020; x2=758,P=0.005). Furthermore, the T-G (3.9%) haplotype of rs1044396-rs1044397 showed significant association with smoking initiation (x2=6.30, P=0.012) and the C-G haplotype (58.9%) remained positive association with nicotine dependence (x2=8.64, P=0.003) after Bonferroni correction. The C-G haplotype also significantly increased the HSI (P=0.002) and FTND scores (P=0.001) after Bonferroni correction.Conclusion These findings suggest that CHRNA4 may be associated with smoking initiation and the C-G haplotype of rs1044396-rs1044397 might increase the vulnerability to nicotine dependence in Chinese male smokers.

  20. Physiological and biochemical studies of newly synthesized muscarinic acetylcholine receptors in embryonic chicken heart

    Exposure of either chicken embryos in ovo or cultured embryonic chicken cardiac cells in vitro to the muscarinic agonist carbachol results in a 70-90% decrease in the number of muscarinic acetylcholine receptors (mAChR) expressed in cardiac cells. Block of agonist-receptor interactions in ovo with the antagonist atropine or removal of the agonist in vitro results in a gradual increase in mAChR number, reaching the control level in 14 hr. Measurements of physiological sensitivity of atria or cultured cells show that, even after the complete recovery of receptor number, the sensitivity to agonist is reduced. The sensitivity of the mAChR-mediated inhibition of adenylate cyclase is also decreased at this time. Newly synthesized mAChR which appear following affinity alkylation in cultured cells are also poorly coupled to the stimulation of 86Rb+ efflux, indicating that decreased physiological sensitivity is not due to an unknown effect of long-term agonist exposure on general cellular function, but rather reflects an intrinsic property of newly synthesized mAChR. This increase in sensitivity is also not blocked by cycloheximide. The increase in sensitivity of the mAChR-mediated responses is due neither to a lack of expression of newly synthesized mAChR on the surface nor to reduced agonist affinity of the mAChR. The diminished sensitivity and subsequent maturation observed in cells containing newly synthesized receptors is due either to a small change in mAChR, or to a change in an as-yet-undefined component of the mAChR transduction system; this alteration represents a novel locus for modulation of cholinergic signals in the heart

  1. Inducibility of human atrial fibrillation in an in silico model reflecting local acetylcholine distribution and concentration.

    Hwang, Minki; Lee, Hyun-Seung; Pak, Hui-Nam; Shim, Eun Bo

    2016-01-01

    Vagal nerve activity has been known to play a crucial role in the induction and maintenance of atrial fibrillation (AF). However, it is unclear how the distribution and concentration of local acetylcholine (ACh) promotes AF. In this study, we investigated the effect of the spatial distribution and concentration of ACh on fibrillation patterns in an in silico human atrial model. A human atrial action potential model with an ACh-dependent K(+) current (IKAch) was used to examine the effect of vagal activation. A simulation of cardiac wave dynamics was performed in a realistic 3D model of the atrium. A model of the ganglionated plexus (GP) and nerve was developed based on the "octopus hypothesis". The pattern of cardiac wave dynamics was examined by applying vagal activation to the GP areas or randomly. AF inducibility in the octopus hypothesis-based GP and nerve model was tested. The effect of the ACh concentration level was also examined. In the single cell simulation, an increase in the ACh concentration shortened APD90 and increased the maximal slope of the restitution curve. In the 3D simulation, a random distribution of vagal activation promoted wavebreaks while ACh secretion limited to the GP areas did not induce a noticeable change in wave dynamics. The octopus hypothesis-based model of the GP and nerve exhibited AF inducibility at higher ACh concentrations. In conclusion, a 3D in silico model of the GP and parasympathetic nerve based on the octopus model exhibited higher AF inducibility with higher ACh concentrations. PMID:26807030

  2. Spontaneous release of acetylcholine and acetylhomocholine from mouse forebrain minces: cytoplasmic or vesicular origin

    The objective of this study was to determine the subcellular origin of cholinergic transmitter released spontaneously from mouse forebrain minces. To accomplish this objective, minces were pretreated in ionic media and then loaded with [14C]homocholine, an analog of choline, to form the false transmitter [14Cy]acetylhomocholine [( 14C]AHCh). The ratio of the false transmitter [14C]AHCh to the true transmitter ACh was then used as an index of cholinergic transmitter contents for both the cytoplasmic (S3) and vesicle-bound (P3) fractions. Three different pretreatment procedures were used to cause the following changes in S3 and P3 false to true transmitter ratios prior to spontaneous release: 1) a small increase in the S3 ratio of [14C]AHCh to acetylcholine (ACh) and a large increase in the P3 ratio of [14C] AHCh to ACh; 2) a decrease in the S3 ratio of [14C]AHCh to ACh and an increase in the P3 ratio of [14C]AHCh to ACh; 3) an increase in the P3 ratio of [14C]AHCh to ACh without affecting the S3 ratio of [14C]AHCh to ACh. The influence of each pretreatment on these subcellular ratios was then compared with its influence on the spontaneous release ratio of [14C]AHCh to ACh. In all 3 instances, the influence of pretreatment on the ratio of spontaneously released false and true cholinergic transmitters from minces coincided with the effect of pretreatment on the pre-release ratio of false to true transmitter in the S3 fraction. These results suggest that much of the cholinergic transmitter which is spontaneously released from mouse forebrain occurs from the cytoplasmic fraction

  3. Propofol and AZD3043 Inhibit Adult Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

    Malin Jonsson Fagerlund

    2016-02-01

    Full Text Available Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1β1δε, α3β2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1β1δε, α3β2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses.

  4. Inhibitory mechanisms and binding site location for serotonin selective reuptake inhibitors on nicotinic acetylcholine receptors.

    Arias, Hugo R; Feuerbach, Dominik; Bhumireddy, Pankaj; Ortells, Marcelo O

    2010-05-01

    Functional and structural approaches were used to examine the inhibitory mechanisms and binding site location for fluoxetine and paroxetine, two serotonin selective reuptake inhibitors, on nicotinic acetylcholine receptors (AChRs) in different conformational states. The results establish that: (a) fluoxetine and paroxetine inhibit h alpha1beta1 gammadelta AChR-induced Ca(2+) influx with higher potencies than dizocilpine. The potency of fluoxetine is increased approximately 10-fold after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs elicited by these antidepressants, (b) fluoxetine and paroxetine inhibit the binding of the phencyclidine analog piperidyl-3,4-(3)H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine to the desensitized Torpedo AChR with higher affinities compared to the resting AChR, and (c) fluoxetine inhibits [(3)H]dizocilpine binding to the desensitized AChR, suggesting a mutually exclusive interaction. This is supported by our molecular docking results where neutral dizocilpine and fluoxetine and the conformer of protonated fluoxetine with the highest LUDI score interact with the domain between the valine (position 13') and leucine (position 9') rings. Molecular mechanics calculations also evidence electrostatic interactions of protonated fluoxetine at positions 20', 21', and 24'. Protonated dizocilpine bridges these two binding domains by interacting with the valine and outer (position 20') rings. The high proportion of protonated fluoxetine and dizocilpine calculated at physiological pH suggests that the protonated drugs can be attracted to the channel mouth before binding deeper within the AChR ion channel between the leucine and valine rings, a domain shared with phencyclidine, finally blocking ion flux and inducing AChR desensitization. PMID:20079457

  5. Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor.

    Arias, Hugo R; Gu, Ruo-Xu; Feuerbach, Dominik; Guo, Bao-Bao; Ye, Yong; Wei, Dong-Qing

    2011-06-14

    The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ∼12 Å from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important. PMID:21510634

  6. (-)-Reboxetine inhibits muscle nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites.

    Arias, Hugo R; Ortells, Marcelo O; Feuerbach, Dominik

    2013-11-01

    The interaction of (-)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (-)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca(2+) influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC50=3.86±0.49 and 1.92±0.48 μM, respectively, (b) binds to the [(3)H]TCP site with ~13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [(3)H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6' and 13' in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9' is the minimum structural component for (-)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (-)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants. PMID:23917086

  7. Functional and structural interaction of (-)-reboxetine with the human α4β2 nicotinic acetylcholine receptor.

    Arias, Hugo R; Fedorov, Nikolai B; Benson, Lisa C; Lippiello, Patrick M; Gatto, Greg J; Feuerbach, Dominik; Ortells, Marcelo O

    2013-01-01

    The interaction of the selective norepinephrine reuptake inhibitor (-)-reboxetine with the human α4β2 nicotinic acetylcholine receptor (nAChR) in different conformational states was studied by several functional and structural approaches. Patch-clamp and Ca(2+)-influx results indicate that (-)-reboxetine does not activate hα4β2 nAChRs via interaction with the orthosteric sites, but inhibits agonist-induced hα4β2 activation by a noncompetitive mechanism. Consistently, the results from the electrophysiology-based functional approach suggest that (-)-reboxetine may act via open channel block; therefore, it is capable of producing a use-dependent type of inhibition of the hα4β2 nAChR function. We tested whether (-)-reboxetine binds to the luminal [(3)H]imipramine site. The results indicate that, although (-)-reboxetine binds with low affinity to this site, it discriminates between the resting and desensitized hα4β2 nAChR ion channels. Patch-clamp results also indicate that (-)-reboxetine progressively inhibits the hα4β2 nAChR with two-fold higher potency at the end of one-second application of agonist, compared with the peak current. The molecular docking studies show that (-)-reboxetine blocks the ion channel at the level of the imipramine locus, between M2 rings 6' and 14'. In addition, we found a (-)-reboxetine conformer that docks in the helix bundle of the α4 subunit, near the middle region. According to molecular dynamics simulations, (-)-reboxetine binding is stable for both sites, albeit less stable than imipramine. The interaction of these drugs with the helix bundle might alter allostericaly the functionality of the channel. In conclusion, the clinical action of (-)-reboxetine may be produced (at least partially) by its inhibitory action on hα4β2 nAChRs. PMID:23010362

  8. Nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system

    Jian-xin SHEN; Jerrel L YAKEL

    2009-01-01

    Based on the composition of the five subunits forming functional neuronal nicotinic acetylcholine receptors (nAChRs), they are grouped into either heteromeric (comprising both α and β subunits) or homomeric (comprising only α subunits) recep-tors. The nAChRs are known to be differentially permeable to calcium ions, with the α7 nAChR subtype having one of the highest permeabilities to calcium. Calcium influx through nAChRs, particularly through the α-bungarotoxin-sensitive α7-containing nAChRs, is a very efficient way to raise cytoplasmic calcium levels. The activation of nAChRs can mediate three types of cytoplasmic calcium signals: (1) direct calcium influx through the nAChRs, (2) indirect calcium influx through voltage-dependent calcium channels (VDCCs) which are activated by the nAChR-mediated depolarization, and (3) calcium-induced calcium release (CICR) (triggered by the first two sources) from the endoplasmic reticulum (ER) through the ryanodine receptors and inositol (1,4,5)-triphosphate receptors (IP3Rs). Downstream signaling events mediated by nAChR-mediated calcium responses can be grouped into instantaneous effects (such as neurotransmitter release, which can occur in milliseconds after nAChR activation), short-term effects (such as the recovery of nAChR desensitization through cellular signaling cascades), and long-term effects (such as neuroprotection via gene expression). In addition, nAChR activity can be regulated by cytoplasmic calcium levels, suggesting a complex reciprocal relationship. Further advances in imaging techniques, animal models, and more potent and subtype-selective ligands for neuronal nAChRs would help in understand-ing the neuronal nAChR-mediated calcium signaling, and lead to the development of improved therapeutic treatments.

  9. Complete definite positive spasm on acetylcholine spasm provocation tests: comparison of clinical positive spasm.

    Sueda, Shozo; Miyoshi, Toru; Sasaki, Yasuhiro; Ohshima, Kousei; Sakaue, Tomoki; Habara, Hirokazu; Kohno, Hiroaki

    2016-02-01

    In the clinical grounds, patients with ≥90 % luminal narrowing during acetylcholine (ACh) testing had variable response. We investigated ischemic findings and chest symptoms in patients with ≥90 % luminal narrowing when performing ACh tests, retrospectively. We performed 763 ACh tests over 13 years (2001-2013). We analyzed chest symptoms and positive ischemic ECG changes during ACh tests. More than 90 % luminal narrowing was found in 441 patients (57.8 %) including 355 patients in the right coronary artery (RCA) and 363 patients in the left coronary artery (LCA). Chest symptom was observed in 386 patients (87.5 %) including 293 patients in the RCA and 304 patients in the LCA. ST elevation was found in 161 patients including 110 in the RCA and 85 patients in the LCA, while ST depression was recognized in 146 patients including 119 patients in the RCA and 117 patients in the LCA. Three quarter of patients with ≥90 % luminal narrowing had significant ischemic ECG changes, whereas two-third of patients with ≥90 % luminal narrowing complained usual chest pain accompanied with significant ischemic ECG changes. Unusual chest symptom was complained in 7.3 % patients with ≥90 % luminal narrowing. Neither chest symptom nor ECG changes were found in 30 patients (6.8 %) with ≥90 % luminal narrowing. A third of these patients had ischemic findings on non-invasive tests before catheterization and six patients had subtotal or total occlusion. We should realize some limitation to define positive coronary spasm based on the ischemic ECG change and chest symptom during ACh tests. PMID:25366988

  10. Gender differences in sensitivity of acetylcholine and ergonovine to coronary spasm provocation test.

    Sueda, Shozo; Miyoshi, Toru; Sasaki, Ysuhiro; Sakaue, Tomoki; Habara, Hirokazu; Kohno, Hiroaki

    2016-03-01

    We examined the sex difference concerning the coronary artery response between ACh and ER in this study. We already reported the difference of coronary response between acetylcholine (ACh) and ergonovine (ER). We performed both ACh and ER tests of 461 patients (male 294 patients, female 167 patients, mean age 64.4 ± 11.3 years) during 23 years. Positive coronary spasm was defined as >99 % transient luminal narrowing with usual chest pain and/or ischemic ECG changes. Firstly, ACh was administered in incremental doses of 20/50/(80) μg into the RCA and 20/50/100/(200) μg into the LCA over 20 s. Secondly, ER was administered in a total dose of 40 μg into the RCA and of 64 μg into the LCA over 2-4 min. Intracoronary injection of ACh and ER provoked spasm in 221 patients consisting of 160 male patients and 61 female patients. In female patients, the spasm provoked by ACh was almost perfect except in two patients (59 patients, 96.7 %), while ER provoked spasm in only 20 patients (32.8 %). In male patients, provoked spasm by ACh (129 patients, 80.6 %) was significantly higher than ER (97 patients, 60.6 %). As a spasm provocation test, ACh is more sensitive than ER in both sexes and especially in females. We may select two pharmacological agents by sex differences to provoke coronary artery spasm in the cardiac catheterization laboratory in the future. PMID:25539623

  11. Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis.

    Chernyavsky, Alex; Chen, Yumay; Wang, Ping H; Grando, Sergei A

    2015-11-01

    The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, β2 and β4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity. PMID:25998908

  12. Transport policy

    1980-01-01

    Transport is a fundamental component of all modern economies. Transport Policy presents a wide ranging collection of previously published articles which aim to provide the reader with an understanding of the main elements of transport policy.

  13. Regional pulmonary ventilation and perfusion studies with sup(81m)Kr after acetylcholine inhalation in bronchial asthma

    sup(81m)Kr is a radioactive noble gas with an ultrashort halflife of 13 seconds, and can be produced in gaseous form or in solution from its parent, 81Rb(T1/2=4.7hours). Using sup(81m)Kr both regional pulmonary ventilation and perfusion were studied simultaneously in 32 asthmatic patients during remission just before and after acetylcholine inhalation. Twenty-five patients were studied in the sitting position and 7 patients supine : the former were examined once, the latter repeatedly after acetylcholine inhalation. The results were as follows : 1. Changes in regional ventilation 1) Ventilatory abnormalities after acetylcholine inhalation appeared to be localized. Their appearances were related to the percentage decrease in FEVsub(1.0). 2) The lower zones were involved most frequently and the upper zones least frequently. 3) On serial examination, the time courses of regional ventilatory function showed the same patterns as those of percentage decrease in FEVsub(1.0). in hypoventilated areas. But regional ventilatory recovery times were different in each area. 2. Regional ventilation and perfusion relationships 1) Hypoventilated areas generally showed hypoperfusion (similar distribution), but ventilatory abnormalities were greater than those of perfusion (low regional ventilation /perfusion ratio). These findings indicate that perfusion abnormalities are secondary to the ventilatory abnormalities. 2) Abnormalities of regional ventilation/perfusion ratios were related to the ventilatory abnormalities but almost never to the percentage decrease in FEVsub(1.0). 3) In upper (middle) zones, there were some cases with increased perfusion in spite of decreased ventilation. 4) Hypoperfusion reached a maximum witnin 2-3 minutes after the start of the hypoventilated plateau. (author)

  14. Oseltamivir produces hypothermic and neuromuscular effects by inhibition of nicotinic acetylcholine receptor functions: comparison to procaine and bupropion.

    Fukushima, Akihiro; Chazono, Kaori; Hashimoto, Yuichi; Iwajima, Yui; Yamamoto, Shohei; Maeda, Yasuhiro; Ohsawa, Masahiro; Ono, Hideki

    2015-09-01

    Oseltamivir, an anti-influenza virus drug, induces marked hypothermia in normal mice. We have proposed that the hypothermic effect arises from inhibition of the nicotinic acetylcholine receptor function of sympathetic ganglion neurons which innervate the brown adipose tissue (a heat generator). It has been reported that local anesthetics inhibit nicotinic acetylcholine receptor function by acting on its ionic channels, and that bupropion, a nicotinic antagonist, induces hypothermia. In this study, we compared the effects of oseltamivir, procaine and bupropion on body temperature, cardiovascular function and neuromuscular transmission. Intraperitoneal administration of oseltamivir (100mg/kg), procaine (86.6mg/kg) and bupropion (86.7mg/kg) lowered the core body temperature of normal mice. At lower doses (10-30mg/kg oseltamivir, 8.7-26mg/kg procaine and bupropion), when administered subcutaneously, the three drugs antagonized the hypothermia induced by intraperitoneal injection of nicotine (1mg/kg). In anesthetized rats, intravenous oseltamivir (30-100mg/kg), procaine (10mg/kg) and bupropion (10mg/kg) induced hypotension and bradycardia. Oseltamivir alone (100mg/kg) did not inhibit neuromuscular twitch contraction of rats, but at 3-30mg/kg it augmented the muscle-relaxing effect of d-tubocurarine. Similar effects were observed when lower doses of procaine (10-30mg/kg) and bupropion (3-10mg/kg) were administered, suggesting that systemic administration of oseltamivir inhibits muscular nicotinic acetylcholine receptors. These results support the idea that the hypothermic effect of oseltamivir is due to its effects on sympathetic ganglia which innervate the brown adipose tissue, and suggest that oseltamivir may exert non-selective ion channel blocking effects like those of ester-type local anesthetics. PMID:26049014

  15. Radiation Transport

    Urbatsch, Todd James [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-06-15

    We present an overview of radiation transport, covering terminology, blackbody raditation, opacities, Boltzmann transport theory, approximations to the transport equation. Next we introduce several transport methods. We present a section on Caseology, observing transport boundary layers. We briefly broach topics of software development, including verification and validation, and we close with a section on high energy-density experiments that highlight and support radiation transport.

  16. Reductions in [3H]nicotinic acetylcholine binding in Alzheimer's disease and Parkinson's disease: an autoradiographic study

    In Alzheimer's disease (AD) and Parkinson's disease (PD), dysfunction in the basal forebrain cholinergic system is accompanied by a consistent loss of presynaptic cholinergic markers in cortex, but changes in cholinergic receptor binding sites are poorly understood. In the present study, we used receptor autoradiography to map the distribution of nicotinic [3H]acetylcholine binding sites in cortices of individuals with AD and PD and matched control subjects. In both diseases, a profound loss of nicotinic receptors occurs in all cortical layers, particularly the deepest layers

  17. Constitutively Active Acetylcholine-Dependent Potassium Current Increases Atrial Defibrillation Threshold by Favoring Post-Shock Re-Initiation

    Bingen, Brian O.; Saïd F. A. Askar; Zeinab Neshati; Iolanda Feola; Panfilov, Alexander V.; de Vries, Antoine A F; Pijnappels, Daniël A.

    2015-01-01

    Electrical cardioversion (ECV), a mainstay in atrial fibrillation (AF) treatment, is unsuccessful in up to 10-20% of patients. An important aspect of the remodeling process caused by AF is the constitutive activition of the atrium-specific acetylcholine-dependent potassium current (I-K,I-ACh -> I-K,I-ACh-c), which is associated with ECV failure. This study investigated the role of I-K,I-ACh-c in ECV failure and setting the atrial defibrillation threshold (aDFT) in optically mapped neonatal ra...

  18. Impairment of bidirectional synaptic plasticity in the striatum of a mouse model of DYT1 dystonia: role of endogenous acetylcholine

    Martella, Giuseppina; Tassone, Annalisa; Sciamanna, Giuseppe; Platania, Paola; Cuomo, Dario; Viscomi, Maria Teresa; Bonsi, Paola; Cacci, Emanuele; Biagioni, Stefano; Usiello, Alessandro; Bernardi, Giorgio; Sharma, Nutan

    2009-01-01

    DYT1 dystonia is a severe form of inherited dystonia, characterized by involuntary twisting movements and abnormal postures. It is linked to a deletion in the dyt1 gene, resulting in a mutated form of the protein torsinA. The penetrance for dystonia is incomplete, but both clinically affected and non-manifesting carriers of the DYT1 mutation exhibit impaired motor learning and evidence of altered motor plasticity. Here, we characterized striatal glutamatergic synaptic plasticity in transgenic mice expressing either the normal human torsinA or its mutant form, in comparison to non-transgenic (NT) control mice. Medium spiny neurons recorded from both NT and normal human torsinA mice exhibited normal long-term depression (LTD), whereas in mutant human torsinA littermates LTD could not be elicited. In addition, although long-term potentiation (LTP) could be induced in all the mice, it was greater in magnitude in mutant human torsinA mice. Low-frequency stimulation (LFS) can revert potentiated synapses to resting levels, a phenomenon termed synaptic depotentiation. LFS induced synaptic depotentiation (SD) both in NT and normal human torsinA mice, but not in mutant human torsinA mice. Since anti-cholinergic drugs are an effective medical therapeutic option for the treatment of human dystonia, we reasoned that an excess in endogenous acetylcholine could underlie the synaptic plasticity impairment. Indeed, both LTD and SD were rescued in mutant human torsinA mice either by lowering endogenous acetylcholine levels or by antagonizing muscarinic M1 receptors. The presence of an enhanced acetylcholine tone was confirmed by the observation that acetylcholinesterase activity was significantly increased in the striatum of mutant human torsinA mice, as compared with both normal human torsinA and NT littermates. Moreover, we found similar alterations of synaptic plasticity in muscarinic M2/M4 receptor knockout mice, in which an increased striatal acetylcholine level has been

  19. Acetylcholine sensitivity of biphasic Ca2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate

    Dezaki, Katsuya; Kimura, Ikuko

    1998-01-01

    Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve-diaphragm muscle preparation to measure intracellular free calcium ([Ca2+]i) entry through nicotinic ACh receptors (AChRs) by use of Ca2+-aequorin luminescence.ACh (0.1–3 mM, 20 μl) elicited biphasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization) in muscle cells. The peak amplitude of the slow Ca2+ mobilization (not accompanied by twitch tension) was concentration-dependently increased by ACh, wh...

  20. Expression of the α-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants

    Restriction fragments of DNA derived from a cDNA clone of the α subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2. Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound α-bungarotoxin. Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria. This provides a new approach for gene selection via functional expression-i.e., ligand overlays of colony blots