Memantine Inhibits α3β2-nAChRs-Mediated Nitrergic Neurogenic Vasodilation in Porcine Basilar Arteries

PubMed Central

Wu, Celeste Yin-Chieh; Chen, Po-Yi; Chen, Mei-Fang; Kuo, Jon-Son; Lee, Tony Jer-Fu

2012-01-01

Memantine, an NMDA receptor antagonist used for treatment of Alzheimer’s disease (AD), is known to block the nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS). In the present study, we examined by wire myography if memantine inhibited α3β2-nAChRs located on cerebral perivascular sympathetic nerve terminals originating in the superior cervical ganglion (SCG), thus, leading to inhibition of nicotine-induced nitrergic neurogenic dilation of isolated porcine basilar arteries. Memantine concentration-dependently blocked nicotine-induced neurogenic dilation of endothelium-denuded basilar arteries without affecting that induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, memantine significantly inhibited nicotine-elicited inward currents in Xenopous oocytes expressing α3β2-, α7- or α4β2-nAChR, and nicotine-induced calcium influx in cultured rat SCG neurons. These results suggest that memantine is a non-specific antagonist for nAChR. By directly inhibiting α3β2-nAChRs located on the sympathetic nerve terminals, memantine blocks nicotine-induced neurogenic vasodilation of the porcine basilar arteries. This effect of memantine is expected to reduce the blood supply to the brain stem and possibly other brain regions, thus, decreasing its clinical efficacy in the treatment of Alzheimer’s disease. PMID:22792283

Inhibitory effect of ebselen on cerebral acetylcholinesterase activity in vitro: kinetics and reversibility of inhibition.

PubMed

Martini, Franciele; Bruning, César Augusto; Soares, Suelen Mendonca; Nogueira, Cristina Wayne; Zeni, Gilson

2015-01-01

Ebselen is a synthetic organoselenium compound that has been considered a potential pharmacological agent with low toxicity, showing antioxidant, anti-inflammatory and neuroprotective effects. It is bioavailable, blood-brain barrier permeant and safe based on cellular toxicity and Phase I-III clinical trials. There is evidence that ebselen inhibits acetylcholinesterase (AChE) activity, an enzyme that plays a key role in the cholinergic system by hydrolyzing acetylcholine (ACh), in vitro and ex vivo. This system has a well-known relationship with cognitive process, and AChE inhibitors, such as donepezil and galantamine, have been used to treat cognitive deficits, mainly in the Alzheimer's Disease (AD). However, these drugs have poor bioavailability and a number of side effects, including gastrointestinal upsets and hepatotoxicity. In this way, this study aimed to evaluate the effect of ebselen on cerebral AChE activity in vitro and to determine the kinetic profile and the reversibility of inhibition by dialysis. Ebselen inhibited the cerebral AChE activity with an IC50 of 29 µM, similar to IC50 found with pure AChE from electric eel, demonstrating a mixed and reversible inhibition of AChE, since it increased Km and decreased Vmax. The AChE activity was recovered within 60 min of dialysis. Therefore, the use of ebselen as a therapeutic agent for treatment of AD should be considered, although memory behavior tasks are needed to support such hypothesis.

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity

NASA Astrophysics Data System (ADS)

Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Gurung, Arun Bahadur; Bhattacharjee, Atanu; Aguan, Kripamoy; Mitra, Sivaprasad

2018-01-01

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

Tertiary amine derivatives of chlorochalcone as acetylcholinesterase (AChE) and buthylcholinesterase (BuChE) inhibitors: the influence of chlorine, alkyl amine side chain and α,β-unsaturated ketone group.

PubMed

Gao, Xiao-Hui; Zhou, Chao; Liu, Hao-Ran; Liu, Lin-Bo; Tang, Jing-Jing; Xia, Xin-Hua

2017-12-01

A new series of tertiary amine derivatives of chlorochalcone (4a∼4l) were designed, synthesized and evaluated for the effect on acetylcholinesterase (AChE) and buthylcholinesterase (BuChE). The results indicated that all compounds revealed moderate or potent inhibitory activity against AChE, and some possessed high selectivity for AChE over BuChE. The structure-activity investigation showed that the substituted position of chlorine significantly influenced the activity and selectivity. The alteration of tertiary amine group also leads to obvious change in bioactivity. Among them, IC 50 of compound 4l against AChE was 0.17 ± 0.06 µmol/L, and the selectivity was 667.2 fold for AChE over BuChE. Molecular docking and enzyme kinetic study on compound 4l suggested that it simultaneously binds to the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. Further study showed that the pyrazoline derivatives synthesized from chlorochalcones had weaker activity and lower selectivity in inhibiting AChE compared to that of chlorochalcone derivatives.

Reciprocal activation of α5-nAChR and STAT3 in nicotine-induced human lung cancer cell proliferation.

PubMed

Zhang, Yao; Jia, Yanfei; Li, Ping; Li, Huanjie; Xiao, Dongjie; Wang, Yunshan; Ma, Xiaoli

2017-07-20

Cigarette smoking is the top environmental risk factor for lung cancer. Nicotine, the addictive component of cigarettes, induces lung cancer cell proliferation, invasion and migration via the activation of nicotinic acetylcholine receptors (nAChRs). Genome-wide association studies (GWAS) show that CHRNA5 gene encoding α5-nAChR is especially relevant to lung cancer. However, the mechanism of this subunit in lung cancer is not clear. In the present study, we demonstrate that the expression of α5-nAChR is correlated with phosphorylated STAT3 (pSTAT3) expression, smoking history and lower survival of non-small cell lung cancer (NSCLC) samples. Nicotine increased the levels of α5-nAChR mRNA and protein in NSCLC cell lines and activated the JAK2/STAT3 signaling cascade. Nicotine-induced activation of JAK2/STAT3 signaling was inhibited by the silencing of α5-nAChR. Characterization of the CHRNA5 promoter revealed four STAT3-response elements. ChIP assays confirmed that the CHRNA5 promoter contains STAT3 binding sites. By silencing STAT3 expression, nicotine-induced upregulation of α5-nAChR was suppressed. Downregulation of α5-nAChR and/or STAT3 expression inhibited nicotine-induced lung cancer cell proliferation. These results suggest that there is a feedback loop between α5-nAChR and STAT3 that contributes to the nicotine-induced tumor cell proliferation, which indicates that α5-nAChR is an important therapeutic target involved in tobacco-associated lung carcinogenesis. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Phytochemical and ethnomedicinal study of Huperzia species used in the traditional medicine of Saraguros in Southern Ecuador; AChE and MAO inhibitory activity.

PubMed

Armijos, Chabaco; Gilardoni, Gianluca; Amay, Luis; Lozano, Antonio; Bracco, Francesco; Ramirez, Jorge; Bec, Nicole; Larroque, Christian; Finzi, Paola Vita; Vidari, Giovanni

2016-12-04

This study concerns seven Huperzia species (Lycopodiaceae), namely H. brevifolia, H. columnaris, H. compacta, H. crassa, H. espinosana, H. tetragona, H. weberbaueri, which are considered sacred plants by the Saraguro community, living in the Southern Andes of Ecuador; these plants are widely used in traditional medicine and ritual ceremonies. The plants were selected on the basis of written interviews with 10 visionary healers (yachak) (2 women, 8 men), indicated as the most credible by the Saraguro Healers Council. The Informant Consensus Factor (F ic ) was determined. The first phytochemical study of the plants was performed by standard procedures, while the AChE and MAO-A inhibition by fractions enriched in high MW alkaloids, was measured in vitro. i) to investigate the uses of some Huperzia plants in healing and magical-religious practices of Saraguros; ii) to identify the main components of plant hydromethanolic extracts; iiì) to test the effects of alkaloidal fractions on the activity of two enzymes linked to mental health. All the interviewed Saraguro yachak showed a high consensus about the uses of the seven Huperzia plants as purgatives and against supernatural diseases, such as the "espanto" (startle). In admixtures with other plants, some species also induce a state of trance or hallucinations in participants in magical-religious rituals. GC-MS of the volatile alkaloid fractions allowed the identification of some lycodine-type and lycopodine-type alkaloids (1-5) in H. compacta, H. columnaris, and H. tetragona. The flavones selgin) (6) and tricin (7) were isolated from H. brevifolia and H. espinosana. Tricin (7) was also detected in the other five species. The rare serratene triterpenes serratenediol (8) serratenediol-3-O-acetate (9), 21-episerratenediol (10), and 21-episerratenediol-3-O-acetate (11) were isolated from H. crassa. In addition, the presence of an unprecedented group of high molecular weight alkaloids has been determined. Alkaloid fractions

Differential effects of lysophosphatidylcholine and ACh on muscarinic K(+),non-selective cation and Ca(2+) currents in guinea-pig atrial cells.

PubMed

Li, Libing; Matsuoka, Isao; Sakamoto, Kazuho; Kimura, Junko

2016-06-08

We compared the effects of lysophosphatidylcholine (LPC) and acetylcholine (ACh) on IK(ACh), ICa and a non-selective cation current (INSC) in guinea-pig atrial myocytes to clarify whether LPC and ACh activate similar Gi/o-coupled effector systems.　IK(ACh), ICa and INSC were analyzed in single atrial myocytes by the whole cell patch-clamp.　LPC induced INSC in a concentration-dependent manner in atrial cells.　ACh activated IK(ACh), but failed to evoke INSC.　LPC also activated IK(ACh) but with significantly less potency than ACh.　The effects of both ligands on IK(ACh) were inhibited by intracellular loading of pre-activated PTX.　This treatment also inhibited LPC-induced INSC, indicating that IK(ACh) and INSC induced by LPC are both mediated by Gi/o.　LPC and ACh had similar potencies in inhibiting ICa, which was pre-augmented by forskolin, indicating that LPC and ACh activate similar amounts of α-subunits of Gi/o.　The different effects of LPC and ACh on IK(ACh) and INSC may suggest that LPC and ACh activate Gi/o having different types of βγ subunits, and that LPC-induced INSC may be mediated by βγ subunits of Gi/o, which are less effective in inducing IK(ACh).

Acetylcholinesterase Regulates Skeletal In Ovo Development of Chicken Limbs by ACh-Dependent and -Independent Mechanisms

PubMed Central

Spieker, Janine; Ackermann, Anica; Salfelder, Anika; Vogel-Höpker, Astrid; Layer, Paul G.

2016-01-01

Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS). Here, we first analyzed the expression of acetylcholinesterase (AChE) by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37). AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA), respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Both acetylcholine (ACh)- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein. PMID:27574787

Acetylcholinesterase Regulates Skeletal In Ovo Development of Chicken Limbs by ACh-Dependent and -Independent Mechanisms.

PubMed

Spieker, Janine; Ackermann, Anica; Salfelder, Anika; Vogel-Höpker, Astrid; Layer, Paul G

2016-01-01

Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS). Here, we first analyzed the expression of acetylcholinesterase (AChE) by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37). AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA), respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Both acetylcholine (ACh)- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein.

Postsynaptic activity reverses the sign of the acetylcholine-induced long-term plasticity of GABAA inhibition

PubMed Central

Domínguez, Soledad; Fernández de Sevilla, David; Buño, Washington

2014-01-01

Acetylcholine (ACh) regulates forms of plasticity that control cognitive functions but the underlying mechanisms remain largely unknown. ACh controls the intrinsic excitability, as well as the synaptic excitation and inhibition of CA1 hippocampal pyramidal cells (PCs), cells known to participate in circuits involved in cognition and spatial navigation. However, how ACh regulates inhibition in function of postsynaptic activity has not been well studied. Here we show that in rat PCs, a brief pulse of ACh or a brief stimulation of cholinergic septal fibers combined with repeated depolarization induces strong long-term enhancement of GABAA inhibition (GABAA-LTP). Indeed, this enhanced inhibition is due to the increased activation of α5βγ2 subunit-containing GABAA receptors by the GABA released. GABAA-LTP requires the activation of M1-muscarinic receptors and an increase in cytosolic Ca2+. In the absence of PC depolarization ACh triggered a presynaptic depolarization-induced suppression of inhibition (DSI), revealing that postsynaptic activity gates the effects of ACh from presynaptic DSI to postsynaptic LTP. These results provide key insights into mechanisms potentially linked with cognitive functions, spatial navigation, and the homeostatic control of abnormal hyperexcitable states. PMID:24938789

Photolabeling a Nicotinic Acetylcholine Receptor (nAChR) with an (α4)3(β2)2 nAChR-Selective Positive Allosteric Modulator

PubMed Central

Deba, Farah; Wang, Ze-Jun; Cohen, Jonathan B.

2016-01-01

Positive allosteric modulators (PAMs) of nicotinic acetylcholine (ACh) receptors (nAChRs) have potential clinical applications in the treatment of nicotine dependence and many neuropsychiatric conditions associated with decreased brain cholinergic activity, and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI) has been identified as a PAM selective for neuronal nAChRs containing the α4 subunit. In this report, we compare CMPI interactions with low-sensitivity (α4)3(β2)2 and high-sensitivity (α4)2(β2)3 nAChRs, and with muscle-type nAChRs. In addition, we use the intrinsic reactivity of [3H]CMPI upon photolysis at 312 nm to identify its binding sites in Torpedo nAChRs. Recording from Xenopus oocytes, we found that CMPI potentiated maximally the responses of (α4)3(β2)2 nAChR to 10 μM ACh (EC10) by 400% and with an EC50 of ∼1 µM. CMPI produced a left shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (∼35% at 10 µM) ACh responses of (α4)2(β2)3 nAChRs and fully inhibited human muscle and Torpedo nAChRs with IC50 values of ∼0.5 µM. Upon irradiation at 312 nm, [3H]CMPI photoincorporated into each Torpedo [(α1)2β1γδ] nAChR subunit. Sequencing of peptide fragments isolated from [3H]CMPI-photolabeled nAChR subunits established photolabeling of amino acids contributing to the ACh binding sites (αTyr190, αTyr198, γTrp55, γTyr111, γTyr117, δTrp57) that was fully inhibitable by agonist and lower-efficiency, state-dependent [3H]CMPI photolabeling within the ion channel. Our results establish that CMPI is a potent potentiator of nAChRs containing an α4:α4 subunit interface, and that its intrinsic photoreactivy makes it of potential use to identify its binding sites in the (α4)3(β2)2 nAChR. PMID:26976945

Synthesis, biological evaluation, and computational studies of Tri- and tetracyclic nitrogen-bridgehead compounds as potent dual-acting AChE inhibitors and hH3 receptor antagonists.

PubMed

Darras, Fouad H; Pockes, Steffen; Huang, Guozheng; Wehle, Sarah; Strasser, Andrea; Wittmann, Hans-Joachim; Nimczick, Martin; Sotriffer, Christoph A; Decker, Michael

2014-03-19

Combination of AChE inhibiting and histamine H3 receptor antagonizing properties in a single molecule might show synergistic effects to improve cognitive deficits in Alzheimer's disease, since both pharmacological actions are able to enhance cholinergic neurotransmission in the cortex. However, whereas AChE inhibitors prevent hydrolysis of acetylcholine also peripherally, histamine H3 antagonists will raise acetylcholine levels mostly in the brain due to predominant occurrence of the receptor in the central nervous system. In this work, we designed and synthesized two novel classes of tri- and tetracyclic nitrogen-bridgehead compounds acting as dual AChE inhibitors and histamine H3 antagonists by combining the nitrogen-bridgehead moiety of novel AChE inhibitors with a second N-basic fragment based on the piperidinylpropoxy pharmacophore with different spacer lengths. Intensive structure-activity relationships (SARs) with regard to both biological targets led to compound 41 which showed balanced affinities as hAChE inhibitor with IC50 = 33.9 nM, and hH3R antagonism with Ki = 76.2 nM with greater than 200-fold selectivity over the other histamine receptor subtypes. Molecular docking studies were performed to explain the potent AChE inhibition of the target compounds and molecular dynamics studies to explain high affinity at the hH3R.

Design of multi-target compounds as AChE, BACE1, and amyloid-β(1-42) oligomerization inhibitors: in silico and in vitro studies.

PubMed

Hernández-Rodríguez, Maricarmen; Correa-Basurto, José; Martínez-Ramos, Federico; Padilla-Martínez, Itzia Irene; Benítez-Cardoza, Claudia G; Mera-Jiménez, Elvia; Rosales-Hernández, Martha Cecilia

2014-01-01

Despite great efforts to develop new therapeutic strategies against Alzheimer's disease (AD), the acetylcholinesterase inhibitors (AChEIs): donepezil, rivastigmine, and galantamine, have been used only as a palliative therapeutic approach. However, the pathogenesis of AD includes several factors such as cholinergic hypothesis, amyloid-β (Aβ) aggregation, and oxidative stress. For this reason, the design of compounds that target the genesis and progression of AD could offer a therapeutic benefit. We have designed a set of compounds (M-1 to M-5) with pharmacophore moieties to inhibit the release, aggregation, or toxicity of Aβ, act as AChEIs and have antioxidant properties. Once the compounds were designed, we analyzed their physicochemical parameters and performed docking studies to determine their affinity values for AChE, β-site amyloid-protein precursor cleaving enzyme 1 (BACE1), and the Aβ monomer. The best ligands, M-1 and M-4, were then synthesized, chemically characterized, and evaluated in vitro. The in vitro studies showed that these compounds inhibit AChE (M-1 Ki = 0.12 and M-4 Ki = 0.17 μM) and BACE1 (M-1 IC50 = 15.1 and M-4 IC50 = 15.4 nM). They also inhibit Aβ oligomerization and exhibit antioxidant activity. In addition, these compounds showed low cytotoxicity in microglial cells. For these reasons, they are promising for future use as drugs in AD mice transgenic models.

Characterization of acetylcholinesterase-inhibition by itopride.

PubMed

Iwanaga, Y; Kimura, T; Miyashita, N; Morikawa, K; Nagata, O; Itoh, Z; Kondo, Y

1994-11-01

Itopride is a gastroprokinetic benzamide derivative. This agent inhibited both electric eel acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BuChE). The IC50 of itopride with AChE (2.04 +/- 0.27 microM) was, however, 100-fold less than that with BuChE, whereas in the case of neostigmine with AChE (11.3 +/- 3.4 nM), it was 10-fold less. The recovery of AChE activity inhibited by 10(-7) M neostigmine was partial, but that inhibited by up to 3 x 10(-5) M itopride was complete when the reaction mixture was subjected to ultrafiltration. Double reciprocal plots of the experimental data showed that both Km and Vmax were affected by itopride, suggesting that the inhibition is a "mixed" type, although primarily being an uncompetitive one. The inhibitory effect of itopride on cholinesterase (ChE) activity in guinea pig gastrointestine was much weaker than that on pure AChE. However, in the presence of a low dose of diisopropyl fluorophosphate, just enough to inhibit BuChE but not AChE, the IC50s of itopride against ChE activities were found to be about 0.5 microM. In conclusion, itopride exerts reversible and a "mixed" type of inhibition preferably against AChE. The IC50 of itopride for electric eel and guinea pig gastrointestinal AChE inhibition was 200 times and 50 times as large as that of neostigmine, respectively.

Chlorpyrifos and chlorpyrifos-oxon inhibit axonal growth by interfering with the morphogenic activity of acetylcholinesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang Dongren; Howard, Angela; Bruun, Donald

2008-04-01

A primary role of acetylcholinesterase (AChE) is regulation of cholinergic neurotransmission by hydrolysis of synaptic acetylcholine. In the developing nervous system, however, AChE also functions as a morphogenic factor to promote axonal growth. This raises the question of whether organophosphorus pesticides (OPs) that are known to selectively bind to and inactivate the enzymatic function of AChE also interfere with its morphogenic function to perturb axonogenesis. To test this hypothesis, we exposed primary cultures of sensory neurons derived from embryonic rat dorsal root ganglia (DRG) to chlorpyrifos (CPF) or its oxon metabolite (CPFO). Both OPs significantly decreased axonal length at concentrationsmore » that had no effect on cell viability, protein synthesis or the enzymatic activity of AChE. Comparative analyses of the effects of CPF and CPFO on axonal growth in DRG neurons cultured from AChE nullizygous (AChE{sup -/-}) versus wild type (AChE{sup +/+}) mice indicated that while these OPs inhibited axonal growth in AChE{sup +/+} DRG neurons, they had no effect on axonal growth in AChE{sup -/-} DRG neurons. However, transfection of AChE{sup -/-} DRG neurons with cDNA encoding full-length AChE restored the wild type response to the axon inhibitory effects of OPs. These data indicate that inhibition of axonal growth by OPs requires AChE, but the mechanism involves inhibition of the morphogenic rather than enzymatic activity of AChE. These findings suggest a novel mechanism for explaining not only the functional deficits observed in children and animals following developmental exposure to OPs, but also the increased vulnerability of the developing nervous system to OPs.« less

Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in Vitro and in Silico Assays.

PubMed

Liu, Yanyan; Yan, Bing; Winkler, David A; Fu, Jianjie; Zhang, Aiqian

2017-06-07

Acetylcholinesterase (AChE) activity regulation by chemical agents or, potentially, nanomaterials is important for both toxicology and pharmacology. Competitive inhibition via direct catalytic active sites (CAS) binding or noncompetitive inhibition through interference with substrate and product entering and exiting has been recognized previously as an AChE-inhibition mechanism for bespoke nanomaterials. The competitive inhibition by peripheral anionic site (PAS) interaction without CAS binding remains unexplored. Here, we proposed and verified the occurrence of a presumed competitive inhibition of AChE without CAS binding for hydrophobically functionalized C 60 nanoparticles (NPs) by employing both experimental and computational methods. The kinetic inhibition analysis distinguished six competitive inhibitors, probably targeting the PAS, from the pristine and hydrophilically modified C 60 NPs. A simple quantitative nanostructure-activity relationship (QNAR) model relating the pocket accessible length of substituent to inhibition capacity was then established to reveal how the geometry of the surface group decides the NP difference in AChE inhibition. Molecular docking identified the PAS as the potential binding site interacting with the NPs via a T-shaped plug-in mode. Specifically, the fullerene core covered the enzyme gorge as a lid through π-π stacking with Tyr72 and Trp286 in the PAS, while the hydrophobic ligands on the fullerene surface inserted into the AChE active site to provide further stability for the complexes. The modeling predicted that inhibition would be severely compromised by Tyr72 and Trp286 deletions, and the subsequent site-directed mutagenesis experiments proved this prediction. Our results demonstrate AChE competitive inhibition of NPs without CAS participation to gain further understanding of both the neurotoxicity and the curative effect of NPs.

Chlorpyrifos and malathion have opposite effects on behaviors and brain size that are not correlated to changes in AChE activity.

PubMed

Richendrfer, Holly; Creton, Robbert

2015-07-01

Organophosphates, a type of neurotoxicant pesticide, are used globally for the treatment of pests on croplands and are therefore found in a large number of conventional foods. These pesticides are harmful and potentially deadly if ingested or inhaled in large quantities by causing a significant reduction in acetylcholinesterase (AChE) activity in the central and peripheral nervous system. However, much less is known about the effects of exposure to small quantities of the pesticides on neural systems and behavior during development. In the current study we used zebrafish larvae in order to determine the effects of two of the most widely used organophosphates, chlorpyrifos and malathion, on zebrafish behavior and AChE activity. Embryos and larvae were exposed to the organophosphates during different time points in development and then tested at 5 days post-fertilization for behavioral, neurodevelopmental and AChE abnormalities. The results of the study indicate that chlorpyrifos and malathion cause opposing behaviors in the larvae such as swim speed (hypoactivity vs. hyperactivity) and rest. Additionally, the pesticides affect only certain behaviors, such as thigmotaxis, during specific time points in development that are unrelated to changes in AChE activity. Larvae treated with malathion but not chlorpyrifos also had significantly smaller forebrain and hindbrain regions compared to controls by 5 days post-fertilization. We conclude that exposure to very low concentrations of organophosphate pesticides during development cause abnormalities in behavior and brain size. Copyright © 2015 Elsevier Inc. All rights reserved.

Chlorpyrifos and Malathion have opposite effects on behaviors and brain size that are not correlated to changes in AChE activity

PubMed Central

Richendrfer, Holly; Creton, Robbert

2015-01-01

Organophosphates, a type of neurotoxicant pesticide, are used globally for the treatment of pests on croplands and are therefore found in a large number of conventional foods. These pesticides are harmful and potentially deadly if ingested or inhaled in large quantities by causing a significant reduction in acetylcholinesterase (AChE) activity in the central and peripheral nervous system. However, much less is known about the effects of exposure to small quantities of the pesticides on neural systems and behavior during development. In the current study we used zebrafish larvae in order to determine the effects of two of the most widely used organophosphates, chlorpyrifos and malathion, on zebrafish behavior and AChE activity. Embryos and larvae were exposed to the organophosphates during different time points in development and then tested at 5 days post-fertilization for behavioral, neurodevelopmental and AChE abnormalities. The results of the study indicate that chlorpyrifos and malathion cause opposing behaviors in the larvae such as swim speed (hypoactivity vs. hyperactivity) and rest. Additionally, the pesticides affect only certain behaviors, such as thigmotaxis, during specific time points in development that are unrelated to changes in AChE activity. Larvae treated with malathion but not chlorpyrifos also had significantly smaller forebrain and hindbrain regions compared to controls by 5 days post-fertilization. We conclude that exposure to very low concentrations of organophosphate pesticides during development cause abnormalities in behavior and brain size. PMID:25983063

Sesquiterpenes and a monoterpenoid with acetylcholinesterase (AchE) inhibitory activity from Valeriana officinalis var. latiofolia in vitro and in vivo.

PubMed

Chen, Heng-Wen; He, Xuan-Hui; Yuan, Rong; Wei, Ben-Jun; Chen, Zhong; Dong, Jun-Xing; Wang, Jie

2016-04-01

Acetylcholinesterase Inhibitor (AchEI) is the most extensive in all anti-dementia drugs. The extracts and isolated compounds from the Valeriana genus have shown anti-dementia bioactivity. Four new sesquiterpenoids (1-4) and a new monoterpenoid (5) were isolated from the root of Valeriana officinalis var. latiofolia. The acetylcholinesterase (AchE) inhibitory activity of isolates was evaluated by modified Ellman method in vitro. Learning and memory ability of compound 4 on mice was evaluated by the Morris water maze. The contents of acetylcholine (Ach), acetylcholine transferase (ChAT) and AchE in mice brains were determined by colorimetry. The results showed IC50 of compound 4 was 0.161 μM in vitro. Compared with the normal group, the learning and memory ability of mice and the contents of Ach and ChAT decreased in model group mice (P<0.01), while the AchE increased (P<0.01). Compared with the model group, Ach and ChAT in the positive control group, the high-dose group and the medium-dose group increased (P<0.01), while the AchE decreased (P<0.01). Compound 4 can improve the learning and memory abilities of APPswe/PSΔE9 double-transgenic mice, and the mechanism may be related to the regulation of the relative enzyme in the cholinergic system. Copyright © 2016 Elsevier B.V. All rights reserved.

Ebselen inhibits the activity of acetylcholinesterase globular isoform G4 in vitro and attenuates scopolamine-induced amnesia in mice.

PubMed

Martini, Franciele; Pesarico, Ana P; Brüning, César A; Zeni, Gilson; Nogueira, Cristina W

2018-02-05

There is a well-known relationship between the cholinergic system and learning, memory, and other common cognitive processes. The process for researching and developing new drugs has lead researchers to repurpose older ones. This study investigated the effects of ebselen on the activity of acethylcholinesterase (AChE) isoforms in vitro and in an amnesia model induced by scopolamine in Swiss mice. In vitro, ebselen at concentrations equal or higher than 10 μM inhibited the activity of cortical and hippocampal G4/AChE, but not G1/AChE isoform. Treatment of mice with ebselen (50 mg/kg, i.p.) was effective against impairment of spatial recognition memory in both Y-maze and novel object recognition tests induced by scopolamine (1 mg/kg, i.p.). Ebselen (50 mg/kg) inhibited hippocampal AChE activity in mice. The present study demonstrates that ebselen inhibited the G4/AChE isoform in vitro and elicited an anti-amnesic effect in a mouse model induced by scopolamine. These findings reveal ebselen as a potential compound in terms of opening up valid therapeutic avenues for the treatment of memory impairment diseases. © 2018 Wiley Periodicals, Inc.

Acotiamide hydrochloride (Z-338) enhances gastric motility and emptying by inhibiting acetylcholinesterase activity in rats.

PubMed

Kawachi, Masanao; Matsunaga, Yugo; Tanaka, Takao; Hori, Yuko; Ito, Katsunori; Nagahama, Kenji; Ozaki, Tomoko; Inoue, Naonori; Toda, Ryoko; Yoshii, Kazuyoshi; Hirayama, Masamichi; Kawabata, Yoshihiro; Takei, Mineo

2011-09-01

In clinical trials, acotiamide hydrochloride (acotiamide: Z-338) has been reported to be useful in the treatment of functional dyspepsia. Here, we investigated the effects of acotiamide on gastric contraction and emptying activities in rats in comparison with itopride hydrochloride (itopride) and mosapride citrate (mosapride). We also examined in vitro the compound's inhibitory effect on acetylcholinesterase (AChE) activity derived from rat stomach. In in vivo studies, acotiamide (30 and 100mg/kg s.c.) and itopride (100mg/kg s.c.) markedly enhanced normal gastric antral motility in rats. In gastric motility dysfunction models, acotiamide (100mg/kg s.c.) and itopride (100mg/kg s.c.) improved both gastric antral hypomotility and the delayed gastric emptying induced by clonidine, an α(2)-adrenoceptor agonist. In contrast, mosapride (10mg/kg s.c.) had no effect on these models. Like the AChE inhibitors itopride (30 mg/kg s.c.) and neostigmine (10 μg/kg s.c.), acotiamide (10mg/kg s.c.) also clearly enhanced gastric body contractions induced by electrical stimulation of the vagus, which were abolished by atropine and hexamethonium, whereas mosapride (3 and 10mg/kg s.c.) did not. In in vitro studies, acotiamide concentration-dependently inhibited rat stomach-derived AChE activity (IC(50)=2.3 μmol/l). In addition, stomach tissue concentrations of acotiamide after administration at 10mg/kg s.c. were sufficient to produce inhibition of AChE activity in rat stomach. These results suggest that acotiamide stimulates gastric motility and improves gastric motility dysfunction in rats by inhibiting AChE activity, and may suggest a role for acotiamide in improving gastric motility dysfunction in patients with functional dyspepsia. Copyright © 2011 Elsevier B.V. All rights reserved.

Acotiamide Hydrochloride, a Therapeutic Agent for Functional Dyspepsia, Enhances Acetylcholine-induced Contraction via Inhibition of Acetylcholinesterase Activity in Circular Muscle Strips of Guinea Pig Stomach.

PubMed

Ito, K; Kawachi, M; Matsunaga, Y; Hori, Y; Ozaki, T; Nagahama, K; Hirayama, M; Kawabata, Y; Shiraishi, Y; Takei, M; Tanaka, T

2016-04-01

Acotiamide is a first-in-class prokinetic drug approved in Japan for the treatment of functional dyspepsia. Given that acotiamide enhances gastric motility in conscious dogs and rats, we assessed the in vitro effects of this drug on the contraction of guinea pig stomach strips and on acetylcholinesterase (AChE) activity in stomach homogenate following fundus removal. We also investigated the serotonin 5-HT4 receptor agonist mosapride, dopamine D2 receptor and AChE inhibitor itopride, and representative AChE inhibitor neostigmine. Acotiamide (0.3 and 1 μM) and itopride (1 and 3 μM) significantly enhanced the contraction of gastric body strips induced by electrical field stimulation (EFS), but mosapride (1 and 10 μM) did not. Acotiamide and itopride significantly enhanced the contraction of gastric body and antrum strips induced by acetylcholine (ACh), but not that induced by carbachol (CCh). Neostigmine also significantly enhanced the contraction of gastric body strips induced by ACh, but not that by CCh. In contrast, mosapride failed to enhance contractions induced by either ACh or CCh in gastric antrum strips. Acotiamide exerted mixed inhibition of AChE, and the percentage inhibition of acotiamide (100 μM) against AChE activity was markedly reduced after the reaction mixture was dialyzed. In contrast, itopride exerted noncompetitive inhibition on AChE activity. These results indicate that acotiamide enhances ACh-dependent contraction in gastric strips of guinea pigs via the inhibition of AChE activity, and that it exerts mixed and reversible inhibition of AChE derived from guinea pig stomach. © Georg Thieme Verlag KG Stuttgart · New York.

Inhibition effect of graphene oxide on the catalytic activity of acetylcholinesterase enzyme.

PubMed

Wang, Yong; Gu, Yao; Ni, Yongnian; Kokot, Serge

2015-11-01

Variations in the enzyme activity of acetylcholinesterase (AChE) in the presence of the nano-material, graphene oxide (GO), were investigated with the use of molecular spectroscopy UV-visible and fluorescence methods. From these studies, important kinetic parameters of the enzyme were extracted; these were the maximum reaction rate, Vm , and the Michaelis constant, Km . A comparison of these parameters indicated that GO inhibited the catalytic activity of the AChE because of the presence of the AChE-GO complex. The formation of this complex was confirmed with the use of fluorescence data, which was resolved with the use of the MCR-ALS chemometrics method. Furthermore, it was found that the resonance light-scattering (RLS) intensity of AChE changed in the presence of GO. On this basis, it was demonstrated that the relationship between AChE and GO was linear and such models were used for quantitative analyses of GO. Copyright © 2015 John Wiley & Sons, Ltd.

Extracts and constituents of Leontopodium alpinum enhance cholinergic transmission: Brain ACh increasing and memory improving properties

PubMed Central

Hornick, Ariane; Schwaiger, Stefan; Rollinger, Judith M.; Vo, Nguyen Phung; Prast, Helmut; Stuppner, Hermann

2012-01-01

Leontopodium alpinum (‘Edelweiss’) was phytochemically investigated for constituents that might enhance cholinergic neurotransmission. The potency to increase synaptic availability of acetylcholine (ACh) in rat brain served as key property for the bioguided isolation of cholinergically active compounds using different chromatographic techniques. The dichlormethane (DCM) extract of the root, fractions and isolated constituents were injected i.c.v. and the effect on brain ACh was detected via the push–pull technique. The DCM extract enhanced extracellular ACh concentration in rat brain and inhibited acetylcholinesterase (AChE) in vitro. The extracellular level of brain ACh was significantly increased by the isolated sesquiterpenes, isocomene and 14-acetoxyisocomene, while silphiperfolene acetate and silphinene caused a small increasing tendency. Only silphiperfolene acetate showed in vitro AChE inhibitory activity, thus suggesting the other sesquiterpenes to stimulate cholinergic transmission by an alternative mechanism of action. Isocomene was further investigated with behavioural tasks in mice. It restored object recognition in scopolamine-impaired mice and showed nootropic effects in the T-maze alternation task in normal and scopolamine-treated mice. Additionally, this sesquiterpene reduced locomotor activity of untreated mice in the open field task, while the activity induced by scopolamine was abolished. The enhancement of synaptic availability of ACh, the promotion of alternation, and the amelioration of scopolamine-induced deficit are in accordance with a substance that amplifies cholinergic transmission. Whether the mechanism of action is inhibition of AChE or another pro-cholinergic property remains to be elucidated. Taken together, isocomene and related constituents of L. alpinum deserve further interest as potential antidementia agents in brain diseases associated with cholinergic deficits. PMID:18541221

α7 Nicotinic Acetylcholine Receptor Signaling Inhibits Inflammasome Activation by Preventing Mitochondrial DNA Release

PubMed Central

Lu, Ben; Kwan, Kevin; Levine, Yaakov A; Olofsson, Peder S; Yang, Huan; Li, Jianhua; Joshi, Sonia; Wang, Haichao; Andersson, Ulf; Chavan, Sangeeta S; Tracey, Kevin J

2014-01-01

The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide–induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release. PMID:24849809

Exploration of the susceptibility of AChE from the poultry red mite Dermanyssus gallinae (Acari: Mesostigmata) to organophosphates in field isolates from France.

PubMed

Roy, Lise; Chauve, Claude; Delaporte, Jean; Inizan, Gilbert; Buronfosse, Thierry

2009-06-01

The red fowl mite Dermanyssus gallinae (De Geer, 1778) is a hematophagous mite species, which is very commonly found in layer facilities in Europe. The economic and animal health impact of this parasite is quite important. In laying hen houses, organophosphates are almost the only legally usable chemicals. Detecting a target resistance can be useful in order to limit the emergence of resistant populations. The acetylcholinesterase (AChE) activity and the enzyme sensitivity to paraoxon was investigated in 39 field samples and compared to a susceptible reference strain (SSK). Insensitivity factor values (expressed as IC50 ratio) obtained from field isolates compared to SSK revealed some polymorphism but not exceeding a 6-fold difference. The kinetic characteristics of AChE from some field samples showed some difference in KM values for acetylthiocholine and inhibition kinetics performed with diethyl paraoxon exhibited a 5.5-fold difference in the bimolecular rate constant in one field isolate. Taken together, these data suggested that differences in AChE susceptibility to organophosphates may exist in D. gallinae but no resistant population was found.

Inhibition of cholinesterase activity by extracts, fractions and compounds from Calceolaria talcana and C. integrifolia (Calceolariaceae: Scrophulariaceae).

PubMed

Cespedes, Carlos L; Muñoz, Evelyn; Salazar, Juan R; Yamaguchi, Lydia; Werner, Enrique; Alarcon, Julio; Kubo, Isao

2013-12-01

Extracts, fractions and compounds from Calceolaria talcana and C. integrifolia exhibited strong inhibitory effects of the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the in vitro Ellman's method. The most active samples were from the ethyl acetate extract, which caused a mixed-type inhibition against AChE (69.8% and 79.5% at 100 and 200 μg/ml, respectively) and against BChE (98.5% and 99.8% at 100 and 200 μg/ml, respectively) and its major components verbascoside 8 (50.9% and 70.0% at 200 μg/ml, against AChE and BChE, respectively), martynoside 9, and fraction F-7 (which corresponds to a mixture of 8, 9, and other phenylethanoids and phenolics that remain unidentified) (80.2% and 85.3% at 100 and 200 μg/ml, against AChE, respectively and 99.1% and 99.7% at 100 and 200 μg/ml, against BChE, respectively) inhibited the acetylcholinesterase enzyme competitively. The most polar fraction F-5 from n-hexane extract (a mixture of naphthoquinones: 2-hydroxy-3-(1,1-dimethylallyl-1,4-naphthoquinone) 6, α-dunnione 7 and other polar compounds that remain unidentified) showed a mixed-type inhibition (71.5% and 72.1% against AChE and BChE at 200 μg/ml, respectively). Finally, the methanol-soluble residue presented a complex, mixed-type inhibition (39.9% and 67.9% against AChE and BChE at 200 μg/ml, respectively). The mixture F-3 with diterpenes was obtained from the n-hexane extract: (1,10-cyclopropyl-9-epi-ent-isopimarol) 1, 19-α-hydroxy-abietatriene 2, and F-4 a mixture of triterpenes α-lupeol 3, β-sitosterol 4, ursolic acid 5 together with a complex mixture of terpenes that did not show activity. In summary, extracts and natural compounds from C. talcana and C. integrifolia were isolated, identified and characterized as cholinesterase inhibitors.

MMB-4 Inhibition of Aceylcholinesterase Is Similar across Species

DTIC Science & Technology

2014-11-01

version 5.4). An IC50 value was determined for AChE from each animal species by fitting the percent of AChE activity with respect to MMB 4 concentration...in GraphPad Prism (version 5) using a nonlinear regression dose response model for inhibition (normalized response with variable slope). Assessing the...Therefore, AChE activity and inhibition studies were carried out at 435 nm to reduce interference from MMB 4. Comparison of IC50 Values for MMB 4 with AChE

Inhibition of neutral endopeptidase increases airway responsiveness to ACh in nonsensitized normal rats.

PubMed

Chiba, Y; Misawa, M

1995-02-01

The effects of sensory neuropeptides on the airway responsiveness to acetylcholine (ACh) were investigated in normal nonsensitized rats. The airway responsiveness to inhaled ACh was significantly increased after treatment with neurokinin A (NKA; 0.001%) or substance P (SP; 0.01%) aerosol in the presence of the neutral endopeptidase (NEP) inhibitor. NKA had a more potent effect than SP. Interestingly, the intravenous treatment with NEP inhibitor alone also induced airway hyperresponsiveness (AHR) to inhaled ACh. This AHR was significantly attenuated by pretreatment with a nonselective NK-receptor antagonist, [D-Pro2,D-Trp7,9]SP, systemic capsaicin, or bilateral cervical vagotomy, indicating that decreased NEP activity results in accumulation of endogenous sensory neuropeptide(s) and enhancement of vagal reflex to cause AHR. The airway responsiveness to ACh of isolated left main bronchus was also increased after treatment with 10(-6) M NKA, but not SP, together with 10(-6) M phosphoramidon. This in vitro AHR to ACh induced by phosphoramidon plus NKA was significantly attenuated by pretreatment with 10(-6) M tetrodotoxin. These findings suggest that overaccumulated sensory neuropeptides, especially NKA, may enhance the probability of transmitter release, probably via NK2 receptors, and that the enhanced transmitter release might be involved in AHR in rats.

Cyperus rotundus extract inhibits acetylcholinesterase activity from animal and plants as well as inhibits germination and seedling growth in wheat and tomato.

PubMed

Sharma, Rashmi; Gupta, Rajendra

2007-05-30

Cyperus rotundus (nutgrass) is the world's worst invasive weed through tubers. Its success in dominating natural habitats depends on its ability to prevent herbivory, and to kill or suppress other plants growing in its vicinity. The present study was done to investigate whether chemicals in nutgrass target neuronal and non-neuronal acetylcholinesterases to affect surrounding animals and plants respectively. Methanolic extract of tubers of nutgrass strongly inhibited activity of AChE from electric eel, wheat and tomato. It also inhibited seed germination and seedling growth in wheat and tomato. Our results suggest that inhibitor of AChE in nutgrass possibly acts as agent of plant's war against (a) herbivore animals, and (b) other plants trying to grow in the same habitat. An antiAChE from nutgrass has been purified by employing chromatography and crystallization. The structural determination of the purified inhibitor is in progress.

Crystal structure, phytochemical study and enzyme inhibition activity of Ajaconine and Delectinine

NASA Astrophysics Data System (ADS)

Ahmad, Shujaat; Ahmad, Hanif; Khan, Hidayat Ullah; Shahzad, Adnan; Khan, Ezzat; Ali Shah, Syed Adnan; Ali, Mumtaz; Wadud, Abdul; Ghufran, Mehreen; Naz, Humera; Ahmad, Manzoor

2016-11-01

The Crystal structure, comparative DFT study and phytochemical investigation of atisine type C-20 diterpenoid alkaloid ajaconine (1) and lycoctonine type C-19 diterpenoid alkaloid delectinine (2) is reported here. These compounds were isolated from Delphinium chitralense. Both the natural products 1 and 2 crystallize in orthorhombic crystal system with identical space group of P212121. The geometric parameters of both compounds were calculated with the help of DFT using B3LYP/6-31+G (p) basis set and HOMO-LUMO energies, optimized band gaps, global hardness, ionization potential, electron affinity and global electrophilicity are calculated. The compounds 1 and 2 were screened for acetyl cholinesterase and butyryl cholinesterase inhibition activities in a dose dependent manner followed by molecular docking to explore the possible inhibitory mechanism of ajaconine (1) and delectinine (2). The IC50 values of tested compounds against AChE were observed as 12.61 μM (compound 1) and 5.04 μM (compound 2). The same experiments were performed for inhibition of BChE and IC50 was observed to be 10.18 μM (1) and 9.21 μM (2). Promising inhibition activity was shown by both the compounds against AChE and BChE in comparison with standard drugs available in the market such as allanzanthane and galanthamine. The inhibition efficiency of both the natural products was determined in a dose dependent manner.

Understanding the conformational flexibility and electrostatic properties of curcumin in the active site of rhAChE via molecular docking, molecular dynamics, and charge density analysis.

PubMed

Saravanan, Kandasamy; Kalaiarasi, Chinnasamy; Kumaradhas, Poomani

2017-12-01

Acetylcholinesterase (AChE) is an important enzyme responsible for Alzheimer's disease, as per report, keto-enol form of curcumin inhibits this enzyme. The present study aims to understand the binding mechanism of keto-enol curcumin with the recombinant human Acetylcholinesterase (rhAChE) from its conformational flexibility, intermolecular interactions, charge density distribution, and the electrostatic properties at the active site of rhAChE. To accomplish this, a molecular docking analysis of curcumin with the rhAChE was performed, which gives the structure and conformation of curcumin in the active site of rhAChE. Further, the charge density distribution and the electrostatic properties of curcumin molecule (lifted from the active site of rhAChE) were determined from the high level density functional theory (DFT) calculations coupled with the charge density analysis. On the other hand, the curcumin molecule was optimized (gas phase) using DFT method and further, the structure and charge density analysis were also carried out. On comparing the conformation, charge density distribution and the electrostatic potential of the active site form of curcumin with the corresponding gas phase form reveals that the above said properties are significantly altered when curcumin is present in the active site of rhAChE. The conformational stability and the interaction of curcumin in the active site are also studied using molecular dynamics simulation, which shows a large variation in the conformational geometry of curcumin as well as the intermolecular interactions.

Synthesis, DNA Cleavage Activity, Cytotoxicity, Acetylcholinesterase Inhibition, and Acute Murine Toxicity of Redox-Active Ruthenium(II) Polypyridyl Complexes.

PubMed

Alatrash, Nagham; Narh, Eugenia S; Yadav, Abhishek; Kim, Mahn-Jong; Janaratne, Thamara; Gabriel, James; MacDonnell, Frederick M

2017-07-06

Four mononuclear [(L-L) 2 Ru(tatpp)] 2+ and two dinuclear [(L-L) 2 Ru(tatpp)Ru(L-L) 2 ] 4+ ruthenium(II) polypyridyl complexes (RPCs) containing the 9,11,20,22-tetraazatetrapyrido[3,2-a:2',3'-c:3'',2''-l:2''',3'''-n]pentacene (tatpp) ligand were synthesized, in which L-L is a chelating diamine ligand such as 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me 4 phen) or 4,7-diphenyl-1,10-phenanthroline (Ph 2 phen). These Ru-tatpp analogues all undergo reduction reactions with modest reducing agents, such as glutathione (GSH), at pH 7. These, plus several structurally related but non-redox-active RPCs, were screened for DNA cleavage activity, cytotoxicity, acetylcholinesterase (AChE) inhibition, and acute mouse toxicity, and their activities were examined with respect to redox activity and lipophilicity. All of the redox-active RPCs show single-strand DNA cleavage in the presence of GSH, whereas none of the non-redox-active RPCs do. Low-micromolar cytotoxicity (IC 50 ) against malignant H358, CCL228, and MCF7 cultured cell lines was mainly restricted to the redox-active RPCs; however, they were substantially less toxic toward nonmalignant MCF10 cells. The IC 50 values for AChE inhibition in cell-free assays and the acute toxicity of RPCs in mice revealed that whereas most RPCs show potent inhibitory action against AChE (IC 50 values <15 μm), Ru-tatpp complexes as a class are surprisingly well tolerated in animals relative to other RPCs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The rs662 polymorphism of paraoxonase 1 affects the difference in the inhibition of butyrylcholinesterase activity by organophosphorus pesticides in human blood.

PubMed

Nam, Dae Cheol; Ha, Yu Mi; Park, Min Kyu; Cho, Sung Kweon

2016-08-01

Organophosphorus pesticides (OPs) are a human health hazard. OPs inhibit acetylcholinesterase (AChE) at nerve endings and accumulate acetylcholine (ACh) at these sites. High levels of ACh and long exposure promote cholinergic crisis. The hydrolysis of OPs by serum paraoxonase 1 (PON1) plays a role in cholinergic crisis in humans. Human serum PON1 can break down organophosphate before binding to ChE. We investigated the effect of PON1 polymorphisms on AChE activity after OP treatment. 50 healthy volunteers were randomly recruited with informed consent. We investigated butyrylcholinesterase (BuChE) activity changes in plasma as a biomarker of AChE after OP treatment in human blood samples immediately following blood sampling. After the standardization of BuChE activity in human blood, we correlated changes in BuChE activity with changes in blood pH. We analyzed the PON1 polymorphisms (rs854560 and rs662) of 50 participants to retrospectively investigate the interindividual variability of changes in BuChE activity. Changes in BuChE activity are strongly correlated with pH changes after OP treatment (R2 = 0.913). We used changes in pH as a surrogate marker for BuChE inhibition after OP treatment. OP treatment significantly decreased BuChE activity by 56.4 ± 5.1% (p < 0.001). The degree of BuChE inhibition was significantly different in the PON1 rs662 genotype (56.10 ± 4.74% vs. 57.96 ± 5.67% vs. 52.34 ± 1.51%; GG vs. GA vs. AA, respectively). Changes in pH can be used as a surrogate marker for the detection of BuChE inhibition after OP exposure. The rs662 polymorphism of PON1 may explain the inter-individual variability in BuChE inhibition.

Evaluation of enzymes inhibition activities of medicinal plant from Burkina Faso.

PubMed

Bangou, Mindiédiba Jean; Kiendrebeogo, Martin; Meda, Nâg-Tiero Roland; Coulibaly, Ahmed Yacouba; Compaoré, Moussa; Zeba, Boukaré; Millogo-Rasolodimby, Jeanne; Nacoulma, Odile Germaine

2011-01-15

The aim of the present study was to evaluate some enzymes inhibitory effects of 11 plant species belonging to 9 families from Burkina Faso. Methanolic extracts were used for their Glutathione-s-transferase (GST), Acetylcholinesterase (AChE), Carboxylesterase (CES) and Xanthine Oxidase (XO) inhibitory activities at final concentration of 100 microg mL(-1). The total phenolics, flavonoids and tannins were also determined spectrophotometrically using Folin-Ciocalteu, AlCl3 and ammonium citrate iron reagents, respectively. Among the 11 species tested, the best inhibitory percentages were found with Euphorbia hirta, Sclerocarya birrea and Scoparia dulcis (inhibition > 40%) followed by Annona senegalensis, Annona squamosa, Polygala arenaria and Ceratotheca sesamoides (inhibition > 25%). The best total phenolic and tannin contents were found with S. birrea with 56.10 mg GAE/100 mg extract and 47.75 mg TAE/100 mg extract, respectively. E hirta presented the higher total flavonoids (9.96 mg QE/100 mg extract). It's was found that Sclerocarya birrea has inhibited all enzymes at more than 30% and this activity is correlated to total tannins contents. Contrary to S. birrea, the enzymatic activities of E. hirta and S. dulcis are correlated to total flavonoids contents. Present findings suggest that the methanolic extracts of those plant species are potential inhibitors of GST, AChE, CES and XO and confirm their traditional uses in the treatment of mental disorders, gout, painful inflammations and cardiovascular diseases.

The inhibition, reactivation and mechanism of VX-, sarin-, fluoro-VX and fluoro-sarin surrogates following their interaction with HuAChE and HuBuChE.

PubMed

Chao, Chih-Kai; Balasubramanian, Narayanaganesh; Gerdes, John M; Thompson, Charles M

2018-06-16

In this study, the mechanisms of HuAChE and HuBChE inhibition by Me-P(O) (OPNP) (OR) [PNP = p-nitrophenyl; R = CH 2 CH 3 , CH 2 CH 2 F, OCH(CH 3 ) 2 , OCH(CH 3 ) (CH 2 F)] representing surrogates and fluoro-surrogates of VX and sarin were studied by in vitro kinetics and mass spectrometry. The in vitro measures showed that the VX- and fluoro-VX surrogates were relatively strong inhibitors of HuAChE and HuBChE (k i  ∼ 10 5 -10 6  M -1 min -1 ) and underwent spontaneous and 2-PAM-mediated reactivation within 30 min. The sarin surrogates were weaker inhibitors of HuAChE and HuBChE (k i  ∼ 10 4 -10 5  M -1 min -1 ), and in general did not undergo spontaneous reactivation, although HuAChE adducts were partially reactivatable at 18 h using 2-PAM. The mechanism of HuAChE and HuBChE inhibition by the surrogates was determined by Q-TOF and MALDI-TOF mass spectral analyses. The surrogate-adducted proteins were trypsin digested and the active site-containing peptide bearing the OP-modified serine identified by Q-TOF as triply- and quadruply-charged ions representing the respective increase in mass of the attached OP moiety. Correspondingly, monoisotopic ions of the tryptic peptides representing the mass increase of the OP-adducted peptide was identified by MALDI-TOF. The mass spectrometry analyses validated the identity of the OP moiety attached to HuAChE or HuBChE as MeP(O) (OR)-O-serine peptides (loss of the PNP leaving group) via mechanisms consistent with those found with chemical warfare agents. MALDI-TOF MS analyses of the VX-modified peptides versus time showed a steady reduction in adduct versus parent peptide (reactivation), whereas the sarin-surrogate-modified peptides remained largely intact over the course of the experiment (24 h). Overall, the presence of a fluorine atom on the surrogate modestly altered the rate constants of inhibition and reactivation, however, the mechanism of inhibition (ejection of PNP group) did not change

Effect of atherosclerosis on endothelium-dependent inhibition of platelet activation in humans.

PubMed

Diodati, J G; Dakak, N; Gilligan, D M; Quyyumi, A A

1998-07-07

We investigated whether luminal release of nitric oxide (NO) contributes to inhibition of platelet activation and whether these effects are reduced in patients with atherosclerosis. Femoral blood flow velocity and ex vivo whole blood platelet aggregation by impedance aggregometry were measured in femoral venous blood during femoral arterial infusion of acetylcholine (ACh; 30 microg/min) in 30 patients, 19 of whom had angiographic atherosclerosis. Measurements were repeated with sodium nitroprusside (40 microg/min), L-arginine (160 micromol/min), and N(G)-monomethyl-L-arginine (L-NMMA; 16 micromol/min). There was significant inhibition of collagen-induced platelet aggregation with ACh (45+/-9.5% lower, P<0.001), and this inhibition was greater in patients without atherosclerosis (68.7+/-10.4% reduction) than in those with atherosclerosis (32.5+/-8.1%, P=0.04). The magnitude of inhibition correlated with vasodilation with ACh, indicating an association between the smooth muscle and antiplatelet effects of endothelium-dependent stimulation. Neither L-NMMA nor sodium nitroprusside altered platelet aggregation. L-Arginine inhibited platelet aggregation equally in vitro (34+/-8% reduction, P<0.01) and in vivo (37+/-13% reduction, P<0.01). Stimulation of NO release into the vascular lumen with ACh inhibits platelet aggregation, an effect that is attenuated in patients with atherosclerosis and endothelial dysfunction. Basal NO release does not appear to contribute to platelet passivation in vivo. L-Arginine inhibited platelet aggregation by its direct action on platelets. These findings provide a pathophysiological basis for the observed increase in thrombotic events in atherosclerosis. Use of L-arginine and other strategies to improve endothelial NO activity may impact favorably on thrombotic events in atherosclerosis.

Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells

PubMed Central

Hasebe, Masaharu

2016-01-01

The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na+-activated K+ (KNa) channel activity in Kenyon cells of crickets (Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells. PMID:26984419

Biphasic photoelectrochemical sensing strategy based on in situ formation of CdS quantum dots for highly sensitive detection of acetylcholinesterase activity and inhibition.

PubMed

Hou, Ting; Zhang, Lianfang; Sun, Xinzhi; Li, Feng

2016-01-15

Herein, we reported a facile and highly sensitive biphasic photoelectrochemical (PEC) sensing strategy based on enzymatic product-mediated in situ formation of CdS quantum dots (QDs), and assayed the activity and inhibition of acetylcholinesterase (AChE) in its optimal state. Upon the hydrolysis of acetylthiocholine catalyzed by AChE, the product thiocholine stabilizes the in situ formation of CdS QDs in homogenous solution. Due to the electrostatic attraction, the resulting tertiary amino group-functionalized CdS QDs are attached to the surface of the negatively charged indium tin oxide (ITO) electrode, generating significant PEC response upon illumination in the presence of electron donors. By taking full advantage of the in situ formation of CdS QDs in homogenous solution, this strategy is capable of detecting AChE activity and inhibition in its optimal state. A directly measured detection limit of 0.01mU/mL for AChE activity is obtained, which is superior to those obtained by some fluorescence methods. The inhibition of AChE activity by aldicarb is successfully detected, and the corresponding IC50 is determined to be 13μg/L. In addition to high sensitivity and good selectivity, this strategy also exhibits additional advantages of simplicity, low cost and easy operation. To the best of our knowledge, the as-proposed strategy is the first example demonstrating the application of CdS QDs formed in situ for biphasic PEC detection of enzyme activity and inhibition. More significantly, it opens up a new horizon for the development of homogenous PEC sensing platforms, and has great potential in probing many other analytes. Copyright © 2015 Elsevier B.V. All rights reserved.

Acetylcholine ameliorates endoplasmic reticulum stress in endothelial cells after hypoxia/reoxygenation via M3 AChR-AMPK signaling.

PubMed

Bi, Xueyuan; He, Xi; Xu, Man; Zhao, Ming; Yu, Xiaojiang; Lu, Xingzhu; Zang, Weijin

2015-08-03

Endoplasmic reticulum (ER) stress is associated with various cardiovascular diseases. However, its pathophysiological relevance and the underlying mechanisms in the context of hypoxia/reoxygenation (H/R) in endothelial cells are not fully understood. Previous findings have suggested that acetylcholine (ACh), the major vagal nerve neurotransmitter, protected against cardiomyocyte injury by activating AMP-activated protein kinase (AMPK). This study investigated the role of ER stress in endothelial cells during H/R and explored the beneficial effects of ACh. Our results showed that H/R triggered ER stress and apoptosis in endothelial cells, evidenced by the elevation of glucose-regulated protein 78, cleaved caspase-12 and C/EBP homologous protein expression. ACh significantly decreased ER stress and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling positive cells and restored ER ultrastructural changes induced by H/R, possibly via protein kinase-like ER kinase and inositol-requiring kinase 1 pathways. Additionally, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3 AChR) inhibitor, abolished ACh-mediated increase in AMPK phosphorylation during H/R. Furthermore, M3 AChR or AMPK siRNA abrogated the ACh-elicited the attenuation of ER stress in endothelial cells, indicating that the salutary effects of ACh were likely mediated by M3 AChR-AMPK signaling. Overall, ACh activated AMPK through M3 AChR, thereby inhibited H/R-induced ER stress and apoptosis in endothelial cells. We have suggested for the first time that AMPK may function as an essential intermediate step between M3 AChR stimulation and inhibition of ER stress-associated apoptotic pathway during H/R, which may help to develop novel therapeutic approaches targeting ER stress to prevent or alleviate ischemia/reperfusion injury.

Association of health symptoms with low-level exposure to organophosphates, DNA damage, AChE activity, and occupational knowledge and practice among rice, corn, and double-crop farmers

PubMed Central

Hongsibsong, Surat; Sittitoon, Nalin; Sapbamrer, Ratana

2017-01-01

Objectives: This study aims to determine (1) total dialkylphosphate (ΣDAP) levels, occupational knowledge and practice, DNA damage, AChE activity, and health symptoms in rice, corn, and double-crop farmers; (2) the association of health symptoms with ΣDAP levels, occupational knowledge and practice, DNA damage, and AChE activity in farmers; and (3) the prevalence of health symptoms between farmers and non-farmers. Methods: A cross-sectional study was conducted by interviewing as well as analyzing urine and blood samples during July to August 2014. Results: There were no differences in ΣDAP levels, AChE activity, and occupational knowledge and practice scores among all farmer groups. In terms of health symptoms related to ΣDAP, AChE activity, DNA damage, and occupational knowledge and practice, pesticide-related symptoms were determined, including breathlessness, chest pain, dry throat, numbness, muscle weakness, cramp, headache, dizziness, eye irritation, white/red rash, and white/red pimple, which were classified as respiratory, muscle, nervous, and epithelial symptoms. A remarkable finding was that farmers had a significantly higher prevalence of muscle weakness (odds ratio (OR)=3.79) and numbness (OR=3.45) as compared with non-farmers. Conclusion: Our findings, therefore, suggest that a long-term low-level exposure to organophosphates (OPs) may be associated with an increasing prevalence of muscle symptoms. However, a further cohort study incorporating sensitive health outcomes and measurement of multiple pesticides monitoring on a larger scale is warranted. PMID:28077823

Association of health symptoms with low-level exposure to organophosphates, DNA damage, AChE activity, and occupational knowledge and practice among rice, corn, and double-crop farmers.

PubMed

Hongsibsong, Surat; Sittitoon, Nalin; Sapbamrer, Ratana

2017-03-28

This study aims to determine (1) total dialkylphosphate (ΣDAP) levels, occupational knowledge and practice, DNA damage, AChE activity, and health symptoms in rice, corn, and double-crop farmers; (2) the association of health symptoms with ΣDAP levels, occupational knowledge and practice, DNA damage, and AChE activity in farmers; and (3) the prevalence of health symptoms between farmers and non-farmers. A cross-sectional study was conducted by interviewing as well as analyzing urine and blood samples during July to August 2014. There were no differences in ΣDAP levels, AChE activity, and occupational knowledge and practice scores among all farmer groups. In terms of health symptoms related to ΣDAP, AChE activity, DNA damage, and occupational knowledge and practice, pesticide-related symptoms were determined, including breathlessness, chest pain, dry throat, numbness, muscle weakness, cramp, headache, dizziness, eye irritation, white/red rash, and white/red pimple, which were classified as respiratory, muscle, nervous, and epithelial symptoms. A remarkable finding was that farmers had a significantly higher prevalence of muscle weakness (odds ratio (OR)=3.79) and numbness (OR=3.45) as compared with non-farmers. Our findings, therefore, suggest that a long-term low-level exposure to organophosphates (OPs) may be associated with an increasing prevalence of muscle symptoms. However, a further cohort study incorporating sensitive health outcomes and measurement of multiple pesticides monitoring on a larger scale is warranted.

The structure-AChE inhibitory activity relationships study in a series of pyridazine analogues.

PubMed

Saracoglu, M; Kandemirli, F

2009-07-01

The structure-activity relationships (SAR) are investigated by means of the Electronic-Topological Method (ETM) followed by the Neural Networks application (ETM-NN) for a class of anti-cholinesterase inhibitors (AChE, 53 molecules) being pyridazine derivatives. AChE activities of the series were measured in IC(50) units, and relative to the activity levels, the series was partitioned into classes of active and inactive compounds. Based on pharmacophores and antipharmacophores calculated by the ETM-software as sub-matrices containing important spatial and electronic characteristics, a system for the activity prognostication is developed. Input data for the ETM were taken as the results of conformational and quantum-mechanics calculations. To predict the activity, we used one of the most well known neural networks, namely, the feed-forward neural networks (FFNNs) trained with the back propagation algorithm. The supervised learning was performed using a variant of FFNN known as the Associative Neural Networks (ASNN). The result of the testing revealed that the high ETM's ability of predicting both activity and inactivity of potential AChE inhibitors. Analysis of HOMOs for the compounds containing Ph1 and APh1 has shown that atoms with the highest values of the atomic orbital coefficients are mainly those atoms that enter into the pharmacophores. Thus, the set of pharmacophores and antipharmacophores found as the result of this study forms a basis for a system of the anti-cholinesterase activity prediction.

Ibogaine and the inhibition of acetylcholinesterase.

PubMed

Alper, Kenneth; Reith, Maarten E A; Sershen, Henry

2012-02-15

Ibogaine is a psychoactive monoterpine indole alkaloid extracted from the root bark of Tabernanthe iboga Baill. that is used globally in medical and nonmedical settings to treat drug and alcohol addiction, and is of interest as an ethnopharmacological prototype for experimental investigation and pharmaceutical development. The question of whether ibogaine inhibits acetylcholinesterase (AChE) is of pharmacological and toxicological significance. AChE activity was evaluated utilizing reaction with Ellman's reagent with physostigmine as a control. Ibogaine inhibited AChE with an IC(50) of 520±40 μM. Ibogaine's inhibition of AChE is physiologically negligible, and does not appear to account for observations of functional effects in animals and humans that might otherwise suggest the possible involvement of pathways linked to muscarinic acetylcholine transmission. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Synthesis, spectroscopic, computational (DFT/B3LYP), AChE inhibition and antioxidant studies of imidazole derivative

NASA Astrophysics Data System (ADS)

Ahmad, Faheem; Alam, Mohammad Jane; Alam, Mahboob; Azaz, Shaista; Parveen, Mehtab; Park, Soonheum; Ahmad, Shabbir

2018-01-01

The present study reports the synthesis and evaluation of biological properties of 3a,8a-dihydroxy-8-oxo-1,3,3a,8a-tetrahydroindeno[1,2-d]imidazol-2(1H)-iminium chloride (3). The structure was confirmed by the FTIR, NMR, MS, CHN microanalysis and X-ray crystallographic analysis. Quantum chemical calculations have been performed at B3LYP-D3/6-311++G(d,p) level of theory to study the molecular geometry, IR, (1H and 13C) NMR, UV/Vis spectra and other molecular parameters of the asymmetric unit of crystal of imidazole compound (3). An empirical dispersion correction to hybrid functional (B3LYP-D3) has been incorporated in the present calculations due to presence of non-covalent interaction, Cl⋯H-O, in the present compound. The remarkable agreement has been observed between theoretical data and those measured experimentally. Moreover, the Hirshfeld analysis was carried out to ascertain the secondary interactions and associated 2D fingerprint plots. The synthesized imidazole derivative showed promising antioxidant property and inhibitory activity against acetylcholinesterase (AChE). Molecular docking was also performed in order to explain in silico antioxidant studies and to ascertain the probable binding mode of compound with the amino acid residues of protein.

PhKv a toxin isolated from the spider venom induces antinociception by inhibition of cholinesterase activating cholinergic system.

PubMed

Rigo, Flavia Karine; Rossato, Mateus Fortes; Trevisan, Gabriela; De Prá, Samira Dal-Toé; Ineu, Rafael Porto; Duarte, Mariane Bernardo; de Castro Junior, Célio José; Ferreira, Juliano; Gomez, Marcus Vinicius

2017-10-01

capsaicin (1nmol/paw) caused a maximal antinociceptive effect of 69.5±4.9% and 85±2.5%, respectively. A pretreatment in mice with atropine; 5mg/kg, i.p. or mecamylamine 0.001mg/kg, i.p. inhibited a neostigimine and PhKv-induced antinociception, suggesting a cholinergic mechanism. Spinal acetylcholinesterase was inhibited by PhKv with ED 50 of 7.6 (4.6-12.6pmol/site, i.t.). PhKv also inhibited the in vitro AChE activity of spinal cord homogenates with an EC 50 of 20.8 (11.6-37.3nM), shifting the Km value from 0.06mM to 18.5mM, characterizing a competitive inhibition of AChE activity by PhKv. Our findings provide, to our knowledge, the first evidence that PhKv caused inhibition of AChE, it increased the ACh content at the neuronal synapses, leading to an activation of the cholinergic system and an antinociceptive response. Studies regarding the nociceptive mechanisms and the identification of potential targets for the treatment of pain have become top priorities. PhKv, by its action of stimulating the cholinergic receptors muscarinic and nicotinic system, reduces pain it may be an alternative for controlling the pain processes. Copyright © 2017 Scandinavian Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine.

PubMed

Ohta, Kazumasa; Takahashi, Chifumi; Tosuji, Hiroaki

2009-08-01

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.

The lignicolous fungus Trametes versicolor (L.) Lloyd (1920): a promising natural source of antiradical and AChE inhibitory agents.

PubMed

Janjušević, Ljiljana; Karaman, Maja; Šibul, Filip; Tommonaro, Giuseppina; Iodice, Carmine; Jakovljević, Dragica; Pejin, Boris

2017-12-01

This study aimed to determine antiradical (DPPH • and • OH) and acetylcholinesterase (AChE) inhibitory activities along with chemical composition of autochtonous fungal species Trametes versicolor (Serbia). A total of 38 phenolic compounds with notable presence of phenolic acids were identified using HPLC/MS-MS. Its water extract exhibited the highest antiradical activity against • OH (3.21 μg/mL), among the rest due to the presence of gallic, p-coumaric and caffeic acids. At the concentration of 100 μg/mL, the same extract displayed a profound AChE inhibitory activity (60.53%) in liquid, compared to donepezil (89.05%), a drug in clinical practice used as positive control. The flavonoids baicalein and quercetin may be responsible compounds for the AChE inhibitory activity observed. These findings have demonstrated considerable potential of T. versicolor water extract as a natural source of antioxidant(s) and/or AChE inhibitor(s) to be eventually used as drug-like compounds or food supplements in the treatment of Alzheimer's disease.

Synthesis and cholinesterase inhibition of cativic acid derivatives.

PubMed

Alza, Natalia P; Richmond, Victoria; Baier, Carlos J; Freire, Eleonora; Baggio, Ricardo; Murray, Ana Paula

2014-08-01

Alzheimer's disease (AD) is a neurodegenerative disorder associated with memory impairment and cognitive deficit. Most of the drugs currently available for the treatment of AD are acetylcholinesterase (AChE) inhibitors. In a preliminary study, significant AChE inhibition was observed for the ethanolic extract of Grindelia ventanensis (IC₅₀=0.79 mg/mL). This result prompted us to isolate the active constituent, a normal labdane diterpenoid identified as 17-hydroxycativic acid (1), through a bioassay guided fractionation. Taking into account that 1 showed moderate inhibition of AChE (IC₅₀=21.1 μM), selectivity over butyrylcholinesterase (BChE) (IC₅₀=171.1 μM) and that it was easily obtained from the plant extract in a very good yield (0.15% w/w), we decided to prepare semisynthetic derivatives of this natural diterpenoid through simple structural modifications. A set of twenty new cativic acid derivatives (3-6) was prepared from 1 through transformations on the carboxylic group at C-15, introducing a C2-C6 linker and a tertiary amine group. They were tested for their inhibitory activity against AChE and BChE and some structure-activity relationships were outlined. The most active derivative was compound 3c, with an IC₅₀ value of 3.2 μM for AChE. Enzyme kinetic studies and docking modeling revealed that this inhibitor targeted both the catalytic active site and the peripheral anionic site of this enzyme. Furthermore, 3c showed significant inhibition of AChE activity in SH-SY5Y human neuroblastoma cells, and was non-cytotoxic. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Novel Antifungal Is Active against Candida albicans Biofilms and Inhibits Mutagenic Acetaldehyde Production In Vitro

PubMed Central

Nieminen, Mikko T.; Novak-Frazer, Lily; Rautemaa, Vilma; Rajendran, Ranjith; Sorsa, Timo; Ramage, Gordon; Bowyer, Paul; Rautemaa, Riina

2014-01-01

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm

Continuous flow immobilized enzyme reactor-tandem mass spectrometry for screening of AChE inhibitors in complex mixtures.

PubMed

Forsberg, Erica M; Green, James R A; Brennan, John D

2011-07-01

A method is described for identifying bioactive compounds in complex mixtures based on the use of capillary-scale monolithic enzyme-reactor columns for rapid screening of enzyme activity. A two-channel nanoLC system was used to continuously infuse substrate coupled with automated injections of substrate/small molecule mixtures, optionally containing the chromogenic Ellman reagent, through sol-gel derived acetylcholinesterase (AChE) doped monolithic columns. This is the first report of AChE encapsulated in monolithic silica for use as an immobilized enzyme reactor (IMER), and the first use of such IMERs for mixture screening. AChE IMER columns were optimized to allow rapid functional screening of compound mixtures based on changes in the product absorbance or the ratio of mass spectrometric peaks for product and substrate ions in the eluent. The assay had robust performance and produced a Z' factor of 0.77 in the presence of 2% (v/v) DMSO. A series of 52 mixtures consisting of 1040 compounds from the Canadian Compound Collection of bioactives was screened and two known inhibitors, physostigmine and 9-aminoacridine, were identified from active mixtures by manual deconvolution. The activity of the compounds was confirmed using the enzyme reactor format, which allowed determination of both IC(50) and K(I) values. Screening results were found to correlate well with a recently published fluorescence-based microarray screening assay for AChE inhibitors.

Synthesis and in vitro reactivation study of isonicotinamide derivatives of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide as reactivators of Sarin and VX inhibited human acetylcholinesterase (hAChE).

PubMed

Karade, Hitendra N; Raviraju, G; Acharya, B N; Valiveti, Aditya Kapil; Bhalerao, Uma; Acharya, Jyotiranjan

2016-09-15

Previously (Karade et al., 2014), we have reported the synthesis and in vitro evaluation of bis-pyridinium derivatives of pyridine-3-yl-(2-hydroxyimino acetamide), as reactivators of sarin and VX inhibited hAChE. Few of the molecules showed superior in vivo protection efficacy (mice model) (Kumar et al., 2014; Swami et al., 2016) in comparison to 2-PAM against DFP and sarin poisoning. Encouraged by these results, herein we report the synthesis and in vitro evaluation of isonicotinamide derivatives of pyridine-3-yl-(2-hydroxyimino acetamide) (4a-4d) against sarin and VX inhibited erythrocyte ghost hAChE. Reactivation kinetics of these compounds was studied and the determined kinetic parameters were compared with that of commercial reactivators viz. 2-PAM and obidoxime. In comparison to 2-PAM and obidoxime, oxime 4a and 4b exhibited enhanced reactivation efficacy toward sarin inhibited hAChE while oxime 4c showed far greater reactivation efficacy toward VX inhibited hAChE. The acid dissociation constant and IC50 values of these oximes were determined and correlated with the observed reactivation potential. Copyright © 2016 Elsevier Ltd. All rights reserved.

Isolation of cholinesterase and β-secretase 1 inhibiting compounds from Lycopodiella cernua.

PubMed

Nguyen, Van Thu; To, Dao Cuong; Tran, Manh Hung; Oh, Sang Ho; Kim, Jeong Ah; Ali, Md Yousof; Woo, Mi-Hee; Choi, Jae Sue; Min, Byung Sun

2015-07-01

Three new serratene-type triterpenoids (1-3) and a new hydroxy unsaturated fatty acid (13) together with nine known compounds (4-12) were isolated from Lycopodiella cernua. The chemical structures were established using NMR, MS, and Mosher's method. Compound 13 showed the most potent inhibitory activity against acetylcholinesterase (AChE) with an IC50 value of 0.22μM. For butyrylcholinesterase (BChE) inhibitory activity, 5 showed the most potent activity with an IC50 value of 0.42μM. Compound 2 showed the most potent activity with an IC50 of 0.23μM for BACE-1 inhibitory activity. The kinetic activities were investigated to determine the type of enzyme inhibition involved. The types of AChE inhibition shown by compounds 4, 5, and 13 were mixed; BChE inhibition by 5 was competitive, while 2 and 6 showed mixed-types. In addition, molecular docking studies were performed to investigate the interaction of these compounds with the pocket sites of AChE. The docking results revealed that the tested inhibitors 3, 4, and 13 were stably present in several pocket domains of the AChE residue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fucoxanthin, a Marine Carotenoid, Reverses Scopolamine-Induced Cognitive Impairments in Mice and Inhibits Acetylcholinesterase in Vitro

PubMed Central

Lin, Jiajia; Huang, Ling; Yu, Jie; Xiang, Siying; Wang, Jialing; Zhang, Jinrong; Yan, Xiaojun; Cui, Wei; He, Shan; Wang, Qinwen

2016-01-01

Fucoxanthin, a natural carotenoid abundant in edible brown seaweeds, has been shown to possess anti-cancer, anti-oxidant, anti-obesity and anti-diabetic effects. In this study, we report for the first time that fucoxanthin effectively protects against scopolamine-induced cognitive impairments in mice. In addition, fucoxanthin significantly reversed the scopolamine-induced increase of acetylcholinesterase (AChE) activity and decreased both choline acetyltransferase activity and brain-derived neurotrophic factor (BDNF) expression. Using an in vitro AChE activity assay, we discovered that fucoxanthin directly inhibits AChE with an IC50 value of 81.2 μM. Molecular docking analysis suggests that fucoxanthin likely interacts with the peripheral anionic site within AChE, which is in accordance with enzymatic activity results showing that fucoxanthin inhibits AChE in a non-competitive manner. Based on our current findings, we anticipate that fucoxanthin might exhibit great therapeutic efficacy for the treatment of Alzheimer’s disease by acting on multiple targets, including inhibiting AChE and increasing BDNF expression. PMID:27023569

Calcium-activated butyrylcholinesterase in human skin protects acetylcholinesterase against suicide inhibition by neurotoxic organophosphates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schallreuter, Karin U.; Institute for Pigmentary Disorders in Association with EM Arndt University of Greifswald; University of Bradford

The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5 x 10{sup -3}M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. {sup 45}Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H{sub 2}O{sub 2}-mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8 m{sup 2} surfacemore » area with its calcium gradient in the 10{sup -3}M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue.« less

Inhibition of allergen-induced basophil activation by ASM-024, a nicotinic receptor ligand.

PubMed

Watson, Brittany M; Oliveria, John Paul; Nusca, Graeme M; Smith, Steven G; Beaudin, Sue; Dua, Benny; Watson, Rick M; Assayag, Evelynne Israël; Cormier, Yvon F; Sehmi, Roma; Gauvreau, Gail M

2014-01-01

Nicotinic acetylcholine receptors (nAChRs) were identified on eosinophils and shown to regulate inflammatory responses, but nAChR expression on basophils has not been explored yet. We investigated surface receptor expression of nAChR α4, α7 and α1/α3/α5 subunits on basophils. Furthermore, we examined the effects of ASM-024, a synthetic nicotinic ligand, on in vitro anti-IgE and in vivo allergen-induced basophil activation. Basophils were enriched from the peripheral blood of allergic donors and the expression of nAChR subunits and muscarinic receptors was determined. Purified basophils were stimulated with anti-IgE in the presence of ASM-024 with or without muscarinic or nicotinic antagonists for the measurement of CD203c expression and histamine release. The effect of 9 days of treatment with 50 and 200 mg ASM-024 on basophil CD203c expression was examined in the blood of mild allergic asthmatics before and after allergen inhalation challenge. nAChR α4, α7 and α1/α3/α5 receptor subunit expression was detected on basophils. Stimulation of basophils with anti-IgE increased CD203c expression and histamine release, which was inhibited by ASM-024 (10(-5) to 10(-)(3) M, p < 0.05). The effect of ASM-024 was reversed in the presence of muscarinic and nicotinic antagonists. In subjects with mild asthma, ASM-024 inhalation significantly inhibited basophil CD203c expression measured 24 h after allergen challenge (p = 0.03). This study shows that ASM-024 inhibits IgE- and allergen-induced basophil activation through both nicotinic and muscarinic receptors, and suggests that ASM-024 may be an efficacious agent for modulating allergic asthma responses. © 2015 S. Karger AG, Basel.

Voltage sensitivity of M2 muscarinic receptors underlies the delayed rectifier-like activation of ACh-gated K(+) current by choline in feline atrial myocytes.

PubMed

Navarro-Polanco, Ricardo A; Aréchiga-Figueroa, Iván A; Salazar-Fajardo, Pedro D; Benavides-Haro, Dora E; Rodríguez-Elías, Julio C; Sachse, Frank B; Tristani-Firouzi, Martin; Sánchez-Chapula, José A; Moreno-Galindo, Eloy G

2013-09-01

Choline (Ch) is a precursor and metabolite of the neurotransmitter acetylcholine (ACh). In canine and guinea pig atrial myocytes, Ch was shown to activate an outward K(+) current in a delayed rectifier fashion. This current has been suggested to modulate cardiac electrical activity and to play a role in atrial fibrillation pathophysiology. However, the exact nature and identity of this current has not been convincingly established. We recently described the unique ligand- and voltage-dependent properties of muscarinic activation of ACh-activated K(+) current (IKACh) and showed that, in contrast to ACh, pilocarpine induces a current with delayed rectifier-like properties with membrane depolarization. Here, we tested the hypothesis that Ch activates IKACh in feline atrial myocytes in a voltage-dependent manner similar to pilocarpine. Single-channel recordings, biophysical profiles, specific pharmacological inhibition and computational data indicate that the current activated by Ch is IKACh. Moreover, we show that membrane depolarization increases the potency and efficacy of IKACh activation by Ch and thus gives the appearance of a delayed rectifier activating K(+) current at depolarized potentials. Our findings support the emerging concept that IKACh modulation is both voltage- and ligand-specific and reinforce the importance of these properties in understanding cardiac physiology.

Synthesis and in-vitro reactivation screening of imidazolium aldoximes as reactivators of sarin and VX-inhibited human acetylcholinesterase (hAChE).

PubMed

Sharma, Rahul; Gupta, Bhanushree; Sahu, Arvind Kumar; Acharya, Jyotiranjan; Satnami, Manmohan L; Ghosh, Kallol K

2016-11-25

Post-treatment of organophosphate (OP) poisoning involves the application of oxime reactivator as an antidote. Structurally different oximes are widely studied to examine their kinetic and mechanistic behavior against OP-inhibited cholinesterase enzyme. A series of structurally related 1,3-disubstituted-2-[(hydroxyiminomethyl)alkyl]imidazolium halides (5a-5e, 9a-9c) were synthesized and further evaluated for their in-vitro reactivation ability to reactivate sarin- and VX-inhibited human acetylcholinesterase (hAChE). The observed results were compared with the reactivation efficacy of standard reactivators; 2-PAM, obidoxime and HI-6. Amongst the synthesized oximes, 5a, 9a and 9b were found to be most potent reactivators against sarin-inhibited hAChE while in case of VX only 9a exhibited comparable reactivity with 2-PAM. Incorporation of pyridinium ring to the imidazole ring resulted in substantial increase in the reactivation strength of prepared reactivator. Physicochemical properties of synthesized reactivators have also been evaluated. Copyright © 2016. Published by Elsevier Ireland Ltd.

Buprofezin inhibits acetylcholinesterase activity in B-biotype Bemisia tabaci.

PubMed

Cottage, Emma L A; Gunning, Robin V

2006-01-01

B-biotype Bemisia tabaci is a severe insect pest worldwide in many ornamental, agricultural, and horticultural industries. Control of this insect is hampered by resistance to many acetylcholinesterase (AChE)-inhibiting insecticides, such as organophosphates and carbamates. Consequently, insect growth regulators such as buprofezin, which act by inhibiting chitin synthesis, are being investigated for use against B-biotype B. tabaci in Australia. This study discusses the effects of buprofezin on B. tabaciAChE.

In vitro investigation of efficacy of new reactivators on OPC inhibited rat brain acetylcholinesterase.

PubMed

Atanasov, Vasil N; Petrova, Iskra; Dishovsky, Christophor

2013-03-25

Organophosphorus compounds (OPC) were developed as warfare nerve agents. They are also widely used as pesticides. The drug therapy of intoxication with OPC includes mainly combination of cholinesterase (ChE) reactivators and cholinolytics. There is no single ChE reactivator having an ability to reactivate sufficiently the inhibited enzyme due to the high variability of chemical structure of the inhibitors. The difficulties in reactivation of ChE activity and slight antidote effect regarding intoxication with some OPC are some of the reasons for continuous efforts to obtain new reactivators of ChE. The aim of the present study was to evaluate the efficacy of some ChE reactivators against OPC intoxication (tabun, paraoxon and dichlorvos) in in vitro experiments and to compare their activity to that known for some currently used oximes (obidoxime, HI-6, 2-PAM). Experiments were carried out using rat brain acetylcholinesterase (AChE). Reactivators showed different activity in the reactivation of rat brain AChE after dichlorvos, paraoxon and tabun inhibition. AChE was easier reactivated after paraoxon treatment. The best effect showed BT-07-4M, obidoxime, TMB-4 and BT-08 from the group of symmetric oximes, and Toxidin, BT-05 and BT-03 from asymmetric compounds. The reactivation of brain AChE inhibited with tabun demonstrated better activity of new compound BT-07-4M, TMB-4 and obidoxime from symmetric oximes, and BT-05 and BT-03 possessing asymmetric structure. All compounds showed low activity toward inhibition of AChE caused by dichlorvos. Comparison of two main structure types (symmetric/asymmetric) showed that the symmetric compounds reactivated better AChE, inhibited with this OPC, than asymmetric ones. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Antioxidant, antityrosinase, anticholinesterase, and nitric oxide inhibition activities of three malaysian macaranga species.

PubMed

Mazlan, Nor Aishah; Mediani, Ahmed; Abas, Faridah; Ahmad, Syahida; Shaari, Khozirah; Khamis, Shamsul; Lajis, N H

2013-01-01

The methanol extracts of three Macaranga species (M. denticulata, M. pruinosa, and M. gigantea) were screened to evaluate their total phenolic contents and activities as cholinesterase inhibitors, nitric oxide (NO) production inhibitors, tyrosinase inhibitors, and antioxidants. The bark of M. denticulata showed the highest total phenolic content (2682 mg gallic acid equivalent (GAE)/100 g) and free radical scavenging activity (IC50 = 0.063 mg/mL). All of the samples inhibited linoleic acid peroxidation by greater than 80%, with the leaves of M. gigantea exhibiting the highest inhibition of 92.21%. Most of the samples exhibited significant antioxidant potential. The bark of M. denticulata and the leaves of both M. pruinosa and M. gigantea exhibited greater than 50% tyrosinase inhibition, with the bark of M. denticulata having the highest percentage of inhibition (68.7%). The bark and leaves of M. denticulata exhibited greater than 50% inhibition (73.82% and 54.50%, resp.) of the acetylcholinesterase enzyme (AChE), while none of the samples showed any significant inhibition of butyrylcholinesterase (BChE). Only the bark of M. denticulata and M. gigantea displayed greater than 50% inhibition of nitric oxide production in cells (81.79% and 56.51%, resp.). These bioactivities indicate that some Macaranga spp. have therapeutic potential in medicinal research.

Inhibition and Larvicidal Activity of Phenylpropanoids from Piper sarmentosum on Acetylcholinesterase against Mosquito Vectors and Their Binding Mode of Interaction.

PubMed

Hematpoor, Arshia; Liew, Sook Yee; Chong, Wei Lim; Azirun, Mohd Sofian; Lee, Vannajan Sanghiran; Awang, Khalijah

2016-01-01

Aedes aegypti, Aedes albopictus and Culex quinquefasciatus are vectors of dengue fever and West Nile virus diseases. This study was conducted to determine the toxicity, mechanism of action and the binding interaction of three active phenylpropanoids from Piper sarmentosum (Piperaceae) toward late 3rd or early 4th larvae of above vectors. A bioassay guided-fractionation on the hexane extract from the roots of Piper sarmentosum led to the isolation and identification of three active phenylpropanoids; asaricin 1, isoasarone 2 and trans-asarone 3. The current study involved evaluation of the toxicity and acetylcholinesterase (AChE) inhibition of these compounds against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus larvae. Asaricin 1 and isoasarone 2 were highly potent against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus larvae causing up to 100% mortality at ≤ 15 μg/mL concentration. The ovicidal activity of asaricin 1, isoasarone 2 and trans-asarone 3 were evaluated through egg hatching. Asaricin 1 and isoasarone 2 showed potent ovicidal activity. Ovicidal activity for both compounds was up to 95% at 25μg/mL. Asaricin 1 and isoasarone 2 showed strong inhibition on acetylcholinesterase with relative IC50 values of 0.73 to 1.87 μg/mL respectively. These findings coupled with the high AChE inhibition may suggest that asaricin 1 and isoasarone 2 are neuron toxic compounds toward Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. Further computational docking with Autodock Vina elaborates the possible interaction of asaricin 1 and isoasarone 2 with three possible binding sites of AChE which includes catalytic triads (CAS: S238, E367, H480), the peripheral sites (PAS: E72, W271) and anionic binding site (W83). The binding affinity of asaricin 1 and isoasarone 2 were relatively strong with asaricin 1 showed a higher binding affinity in the anionic pocket.

Biochemical effects of glyphosate based herbicide, Excel Mera 71 on enzyme activities of acetylcholinesterase (AChE), lipid peroxidation (LPO), catalase (CAT), glutathione-S-transferase (GST) and protein content on teleostean fishes.

PubMed

Samanta, Palas; Pal, Sandipan; Mukherjee, Aloke Kumar; Ghosh, Apurba Ratan

2014-09-01

Effects of glyphosate based herbicide, Excel Mera 71 at a dose of 17.20mg/l on enzyme activities of acetylcholinesterase (AChE), lipid peroxidation (LPO), catalase (CAT), glutathione-S-transferase (GST) and protein content were measured in different tissues of two Indian air-breathing teleosts, Anabas testudineus (Bloch) and Heteropneustes fossilis (Bloch) during an exposure period of 30 days under laboratory condition. AChE activity was significantly increased in all the investigated tissues of both fish species and maximum elevation was observed in brain of H. fossilis, while spinal cord of A. testudineus showed minimum increment. Fishes showed significant increase LPO levels in all the tissues; highest was observed in gill of A. testudineus but lowest LPO level was observed in muscle of H. fossilis. CAT was also enhanced in both the fishes, while GST activity in liver diminished substantially and minimum was observed in liver of A. testudineus. Total protein content showed decreased value in all the tissues, maximum reduction was observed in liver and minimum in brain of A. testudineus and H. fossilis respectively. The results indicated that Excel Mera 71 caused serious alterations in the enzyme activities resulting into severe deterioration of fish health; so, AChE, LPO, CAT and GST can be used as suitable indicators of herbicidal toxicity. Copyright © 2014 Elsevier Inc. All rights reserved.

Discovery of New Classes of Compounds that Reactivate Acetylcholinesterase Inhibited by Organophosphates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katz, Francine S.; Pecic, Stevan; Tran, Timothy H.

Acetylcholinesterase (AChE) that has been covalently inhibited by organophosphate compounds (OPCs), such as nerve agents and pesticides, has traditionally been reactivated by using nucleophilic oximes. There is, however, a clearly recognized need for new classes of compounds with the ability to reactivate inhibited AChE with improved in vivo efficacy. Here we describe our discovery of new functional groups—Mannich phenols and general bases—that are capable of reactivating OPC-inhibited AChE more efficiently than standard oximes and we describe the cooperative mechanism by which these functionalities are delivered to the active site. These discoveries, supported by preliminary in vivo results and crystallographic data,more » significantly broaden the available approaches for reactivation of AChE.« less

Inhibition of acetylcholinesterase and cytochrome oxidase activity in Fasciola gigantica cercaria by phytoconstituents.

PubMed

Sunita, Kumari; Habib, Maria; Kumar, P; Singh, Vinay Kumar; Husain, Syed Akhtar; Singh, D K

2016-02-01

Fasciolosis is an important cattle and human disease caused by Fasciola hepatica and Fasciola gigantica. One of the possible methods to control this problem is to interrupt the life cycle of Fasciola by killing its larva (redia and cercaria) in host snail. Molecular identification of cercaria larva of F. gigantica was done by comparing the nucleotide sequencing with adult F. gigantica. It was noted that nucleotide sequencing of cercaria larva and adult F. gigantica were 99% same. Every month during the year 2011-2012, in vivo treatment with 60% of 4 h LC50 of phyto cercaricides citral, ferulic acid, umbelliferone, azadirachtin and allicin caused significant inhibition of acetylcholinesterase (AChE) and cytochrome oxidase activity in the treated cercaria larva of F. gigantica. Whereas, activity of both enzymes were not significantly altered in the nervous tissues of vector snail Lymnaea acuminata exposed to same treatments. Maximum reduction in AChE (1.35% of control in month of June) and cytochrome oxidase (3.71% of control in the month of July) activity were noted in the cercaria exposed to 60% of 4 h LC50 of azadirachtin and allicin, respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

Antioxidant, Antityrosinase, Anticholinesterase, and Nitric Oxide Inhibition Activities of Three Malaysian Macaranga Species

PubMed Central

Abas, Faridah; Ahmad, Syahida; Shaari, Khozirah; Khamis, Shamsul; Lajis, N. H.

2013-01-01

The methanol extracts of three Macaranga species (M. denticulata, M. pruinosa, and M. gigantea) were screened to evaluate their total phenolic contents and activities as cholinesterase inhibitors, nitric oxide (NO) production inhibitors, tyrosinase inhibitors, and antioxidants. The bark of M. denticulata showed the highest total phenolic content (2682 mg gallic acid equivalent (GAE)/100 g) and free radical scavenging activity (IC50 = 0.063 mg/mL). All of the samples inhibited linoleic acid peroxidation by greater than 80%, with the leaves of M. gigantea exhibiting the highest inhibition of 92.21%. Most of the samples exhibited significant antioxidant potential. The bark of M. denticulata and the leaves of both M. pruinosa and M. gigantea exhibited greater than 50% tyrosinase inhibition, with the bark of M. denticulata having the highest percentage of inhibition (68.7%). The bark and leaves of M. denticulata exhibited greater than 50% inhibition (73.82% and 54.50%, resp.) of the acetylcholinesterase enzyme (AChE), while none of the samples showed any significant inhibition of butyrylcholinesterase (BChE). Only the bark of M. denticulata and M. gigantea displayed greater than 50% inhibition of nitric oxide production in cells (81.79% and 56.51%, resp.). These bioactivities indicate that some Macaranga spp. have therapeutic potential in medicinal research. PMID:24319356

Inhibitory effects of psychotropic drugs on the acetylcholine receptor-operated potassium current (IK.ACh) in guinea-pig atrial myocytes.

PubMed

Okada, Muneyoshi; Watanabe, Shinya; Matada, Takashi; Asao, Yoko; Hamatani, Ramu; Yamawaki, Hideyuki; Hara, Yukio

2013-01-01

Influences of psychotropic drugs, six antipsychotics and three antidepressants, on acetylcholine receptor-operated potassium current (IK.ACh) were examined by a whole-cell patch clamp method in freshly isolated guinea-pig atrial myocyte. IK.ACh was induced by a superfusion of carbachol (CCh) or by an intracellular application of guanosine 5'-[thio] triphosphate (GTPγS). To elucidate mechanism for anticholinergic action, IC50 ratio, the ratio of IC50 for GTPγS-activated IK.ACh to CCh-induced IK.ACh, was calculated. Antipsychotics and antidepressants inhibited CCh-induced IK.ACh in a concentration-dependent manner. The IC50 values were as follows; chlorpromazine 0.53 μM, clozapine 0.06 μM, fluphenazine 2.69 μM, haloperidol 2.66 μM, sulpiride 42.3 μM, thioridazine 0.07 μM, amitriptyline 0.03 μM, imipramine 0.22 μM and maprotiline 1.81 μM. The drugs, except for sulpiride, inhibited GTPγS-activated IK.ACh with following IC50 values; chlorpromazine 1.71 μM, clozapine 14.9 μM, fluphenazine 3.55 μM, haloperidol 2.73 μM, thioridazine 1.90 μM, amitriptyline 7.55 μM, imipramine 7.09 μM and maprotiline 5.93 μM. The IC50 ratio for fluphenazine and haloperidol was close to unity. The IC50 ratio for chlorpromazine, clozapine, thioridazine, amitriptyline, imipramine and maprotiline was much higher than unity. The present findings suggest that the psychotropics studied suppress IK.ACh. Chlorpromazine, clozapine, thioridazine, amitriptyline, imipramine, maprotiline and sulpiride are preferentially acting on muscarinic receptor. Fluphenazine and haloperidol may act on G protein and/or potassium channel.

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

PubMed

Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M; DeSimone, John A; Lyall, Vijay

2015-01-01

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.

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

PubMed Central

Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M.; DeSimone, John A.; Lyall, Vijay

2015-01-01

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. PMID:26039516

PKCε phosphorylates α4β2 nicotinic ACh receptors and promotes recovery from desensitization

PubMed Central

Lee, A M; Wu, D-F; Dadgar, J; Wang, D; McMahon, T; Messing, R O

2015-01-01

Background and Purpose Nicotinic (ACh) receptor recovery from desensitization is modulated by PKC, but the PKC isozymes and the phosphorylation sites involved have not been identified. We investigated whether PKCε phosphorylation of α4β2 nAChRs regulates receptor recovery from desensitization. Experimental Approach Receptor recovery from desensitization was investigated by electrophysiological characterization of human α4β2 nAChRs. Phosphorylation of the α4 nAChR subunit was assessed by immunoblotting of mouse synaptosomes. Hypothermia induced by sazetidine-A and nicotine was measured in Prkce−/− and wild-type mice. Key Results Inhibiting PKCε impaired the magnitude of α4β2 nAChR recovery from desensitization. We identified five putative PKCε phosphorylation sites in the large intracellular loop of the α4 subunit, and mutating four sites to alanines also impaired recovery from desensitization. α4 nAChR subunit phosphorylation was reduced in synaptosomes from Prkce−/− mice. Sazetidine-A-induced hypothermia, which is mediated by α4β2 nAChR desensitization, was more severe and prolonged in Prkce−/− than in wild-type mice. Conclusions and Implications PKCε phosphorylates the α4 nAChR subunit and regulates recovery from receptor desensitization. This study illustrates the importance of phosphorylation in regulating α4β2 receptor function, and suggests that reducing phosphorylation prolongs receptor desensitization and decreases the number of receptors available for activation. PMID:26103136

Cholinesterase inhibitors modify the activity of intrinsic cardiac neurons.

PubMed

Darvesh, Sultan; Arora, Rakesh C; Martin, Earl; Magee, David; Hopkins, David A; Armour, J Andrew

2004-08-01

Cholinesterase inhibitors used to treat the symptoms of Alzheimer's disease (AD) inhibit both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), albeit to different degrees. Because central and peripheral neurons, including intrinsic cardiac neurons located on the surface of the mammalian heart, express both BuChE and AChE, we studied spontaneously active intrinsic cardiac neurons in the pig as a model to assess the effects of inhibition of AChE compared to BuChE. Neuroanatomical experiments showed that some porcine intrinsic cardiac neurons expressed AChE and/or BuChE. Enzyme kinetic experiments with cholinesterase inhibitors, namely, donepezil, galantamine, (+/-) huperzine A, metrifonate, rivastigmine, and tetrahydroaminoacridine, demonstrated that these compounds differentially inhibited porcine AChE and BuChE. Donepezil and (+/-) huperzine A were better reversible inhibitors of AChE, and galantamine equally inhibited both the enzymes. Tetrahydroaminoacridine was a better reversible inhibitor of BuChE. Rivastigmine caused more rapid inactivation of BuChE as compared to AChE. Neurophysiological studies showed that acetylcholine and butyrylcholine increase or decrease the spontaneous activity of the intrinsic cardiac neurons. Donepezil, galantamine, (+/-) huperzine A, and tetrahydroaminoacridine changed spontaneous neuronal activity by about 30-35 impulses per minute, while rivastigmine changed it by approximately 100 impulses per minute. It is concluded that (i) inhibition of AChE and BuChE directly affects the porcine intrinsic cardiac nervous system, (ii) the intrinsic cardiac nervous system represents a suitable model for examining the effects of cholinesterase inhibitors on mammalian neurons in vivo, and (iii) the activity of intrinsic cardiac neurons may be affected by pharmacological agents that inhibit cholinesterases.

APS8, a Polymeric Alkylpyridinium Salt Blocks α7 nAChR and Induces Apoptosis in Non-Small Cell Lung Carcinoma

PubMed Central

Zovko, Ana; Viktorsson, Kristina; Lewensohn, Rolf; Kološa, Katja; Filipič, Metka; Xing, Hong; Kem, William R.; Paleari, Laura; Turk, Tom

2013-01-01

Naturally occurring 3-alkylpyridinium polymers (poly-APS) from the marine sponge Reniera sarai, consisting of monomers containing polar pyridinium and nonpolar alkyl chain moieties, have been demonstrated to exert a wide range of biological activities, including a selective cytotoxicity against non-small cell lung cancer (NSCLC) cells. APS8, an analog of poly-APS with defined alkyl chain length and molecular size, non-competitively inhibits α7 nicotinic acetylcholine receptors (nAChRs) at nanomolar concentrations that are too low to be acetylcholinesterase (AChE) inhibitory or generally cytotoxic. In the present study we show that APS8 inhibits NSCLC tumor cell growth and activates apoptotic pathways. APS8 was not toxic for normal lung fibroblasts. Furthermore, in NSCLC cells, APS8 reduced the adverse anti-apoptotic, proliferative effects of nicotine. Our results suggest that APS8 or similar compounds might be considered as lead compounds to develop antitumor therapeutic agents for at least certain types of lung cancer. PMID:23880932

Brain stem stimulation and the acetylcholine-evoked inhibition of neurones in the feline nucleus reticularis thalami

PubMed Central

Dingledine, Raymond; Kelly, J. S.

1977-01-01

1. In cats anaesthetized with halothane and nitrous oxide, the responses to iontophoretically applied acetylcholine (ACh) and to high-frequency stimulation of the mid-brain reticular formation (MRF) were tested on spontaneously active neurones in the nucleus reticularis thalami and underlying ventrobasal complex. 2. The initial response to MRF stimulation of 90% of the ACh-inhibited neurones found in the region of the dorsolateral nucleus reticularis was an inhibition. Conversely, the initial response of 82% of the ACh-excited neurones in the ventrobasal complex was an excitation. Neurones in the rostral pole of the nucleus reticularis were inhibited by both ACh and RMF stimulation. 3. The mean latency (and s.e. of mean) for the MRF-evoked inhibition was 13·7 ± 3·2 ms (n = 42) and that for the MRF-evoked excitation, 44.1 ± 4.2 ms (n = 35). 4. The ACh-evoked inhibitions were blocked by iontophoretic atropine, in doses that did not block amino acid-evoked inhibition. In twenty-four ACh-inhibited neurones the effect of iontophoretic atropine was tested on MRF-evoked inhibition. In all twenty-four neurones atropine had no effect on the early phase of MRF-evoked inhibition but weakly antagonized the late phase of inhibition in nine of fourteen neurones. 5. Interspike-interval histograms showed that the firing pattern of neurones in the nucleus reticularis was characterized by periods of prolonged, high-frequency bursting. Both the ACh-evoked inhibitions and the late phase of MRF-evoked inhibitions were accompanied by an increased burst activity. In contrast, iontophoretic atropine tended to suppress burst activity. 6. The possibility is discussed that electrical stimulation of the MRF activates an inhibitory cholinergic projection to the nucleus reticularis. Since neurones of the nucleus reticularis have been shown to inhibit thalamic relay cells, activation of this inhibitory pathway may play a role in MRF-evoked facilitation of thalamo-cortical relay transmission

[Effects of cornel iridoid glycoside on activity of cholinesterases in vitro].

PubMed

Chu, Si-Juan; Zhang, Lan; Liu, Gang; Zhou, Wen-Xia; Li, Lin

2013-05-01

The purpose of the present study was to investigate the effects of cornel iridoid glycoside (CIG) on the activity of cholinesterases in vitro, and to investigate the mechanism of CIG's treating Alzheimer's disease (AD). The sources of cholinesterases were prepared from human blood cells, rat brain homogenate and human blood plasma, respectively. The biochemical methods were used to detect the activity of acetylcholine esterase (AChE) and butyryl cholinesterase (BuChE) to investigate the influence of CIG on cholinesterases. The results showed that CIG inhibited the activity of AChE of human blood cells and rat brain homogenate, with the 50% inhibition rate (IC50) of 1.6 g . L-1 and 3.3 g . L-1, respectively; and the inhibition of AChE of CIG is reversible. CIG also inhibited the activity of BuChE of human blood plasma, with the IC50 of 2.9 g . L-1. In conclusion, CIG can inhibit the activity of AChE and BuChE in vitro, which may be one of the mechanisms of CIG to treat AD.

Inhibition pathways of the potent organophosphate CBDP with cholinesterases revealed by X-ray crystallographic snapshots and mass spectrometry.

PubMed

Carletti, Eugénie; Colletier, Jacques-Philippe; Schopfer, Lawrence M; Santoni, Gianluca; Masson, Patrick; Lockridge, Oksana; Nachon, Florian; Weik, Martin

2013-02-18

Tri-o-cresyl-phosphate (TOCP) is a common additive in jet engine lubricants and hydraulic fluids suspected to have a role in aerotoxic syndrome in humans. TOCP is metabolized to cresyl saligenin phosphate (CBDP), a potent irreversible inhibitor of butyrylcholinesterase (BChE), a natural bioscavenger present in the bloodstream, and acetylcholinesterase (AChE), the off-switch at cholinergic synapses. Mechanistic details of cholinesterase (ChE) inhibition have, however, remained elusive. Also, the inhibition of AChE by CBDP is unexpected, from a structural standpoint, i.e., considering the narrowness of AChE active site and the bulkiness of CBDP. In the following, we report on kinetic X-ray crystallography experiments that provided 2.7-3.3 Å snapshots of the reaction of CBDP with mouse AChE and human BChE. The series of crystallographic snapshots reveals that AChE and BChE react with the opposite enantiomers and that an induced-fit rearrangement of Phe297 enlarges the active site of AChE upon CBDP binding. Mass spectrometry analysis of aging in either H(2)(16)O or H(2)(18)O furthermore allowed us to identify the inhibition steps, in which water molecules are involved, thus providing insights into the mechanistic details of inhibition. X-ray crystallography and mass spectrometry show the formation of an aged end product formed in both AChE and BChE that cannot be reactivated by current oxime-based therapeutics. Our study thus shows that only prophylactic and symptomatic treatments are viable to counter the inhibition of AChE and BChE by CBDP.

Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae

PubMed Central

Alizadeh, Ali; Talebi-Jahromi, Khalil; Hosseininaveh, Vahid; Ghadamyari, Mohammad

2014-01-01

Abstract The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity ( KM ) and hydrolyzing efficiency ( Vmax ) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed

Comparison of the oxime-induced reactivation of rhesus monkey, swine and guinea pig erythrocyte acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity.

PubMed

Herkert, Nadja M; Lallement, Guy; Clarençon, Didier; Thiermann, Horst; Worek, Franz

2009-04-28

Recently, a dynamically working in vitro model with real-time determination of membrane-bound human acetylcholinesterase (AChE) activity was shown to be a versatile model to investigate oxime-induced reactivation kinetics of organophosphate- (OP) inhibited enzyme. In this assay, AChE was immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. Subsequently, AChE activity was continuously analyzed in a flow-through detector. Now, it was an intriguing question whether this model could be used with erythrocyte AChE from other species in order to investigate kinetic interactions in the absence of annoying side reactions. Rhesus monkey, swine and guinea pig erythrocytes were a stable and highly reproducible enzyme source. Then, the model was applied to the reactivation of sarin- and paraoxon-inhibited AChE by obidoxime or HI 6 and it could be shown that the derived reactivation rate constants were in good agreement to previous results obtained from experiments with a static model. Hence, this dynamic model offers the possibility to investigate highly reproducible interactions between AChE, OP and oximes with human and animal AChE.

Antioxidant, Acetylcholinesterase, Butyrylcholinesterase, and α-glucosidase Inhibitory Activities of Corchorus depressus

PubMed Central

Afzal, Samina; Chaudhry, Bashir Ahmad; Ahmad, Ashfaq; Uzair, Muhammad; Afzal, Khurram

2017-01-01

Background: Corchorus depressus (Cd) commonly known as Boa-phalee belonging to the family Tiliaceae having 50 genera and 450 species. Cd is not among the studied medicinal agent despite its potential in ethnopharmacology. Objectives: The present study investigated antioxidant, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase inhibitory activities of Cd. The dichloromethane and methanolic extracts of the Cd were evaluated for biological activities such as antioxidant and enzyme inhibitory activities of AChE, BChE, and α-glucosidase. Materials and Methods: Antioxidant activity was evaluated by measuring free radical scavenging potential of Cd using 1,1-diphenyl-2-picrylhydrazyl. Enzyme inhibition activities were done by measuring optical density. Results: The methanol extract of roots of Cd showed potential free radical scavenging activity 99% at concentration 16.1 μg/ml. AChE was inhibited by aerial part of dichloromethane fraction by 46.07% ± 0.45% while dichloromethane extracts of roots of Cd possessed significant activity against BChE with 86% inhibition compared with standard drug Eserine at concentration 0.5 mg/ml. The dichloromethane extract of roots of Cd showed 79% inhibition against α-glucosidase enzyme activity with IC50 62.8 ± 1.5 μg/ml. Conclusion: These findings suggest Cd as useful therapeutic option as antioxidant and inhibition of AChE, BChE, and α-glucosidase activities. SUMMARY The aerial parts and roots of Corchorus depressus (Cd) were extracted in dichloromethane and methanolThe extract of roots of Cd showed free radical scavenging activity 99% at concentration 16.1 mg/ml, Ach inhibition by aerial parts of dichloromethane fraction by 46.07%, and 79% inhibition against a-glucosidase enzyme activity with IC50 62.8 ± 1.5 mg/mlThe dichloromethane and methanolic extracts of Cd exhibited antioxidant inhibition of acetyl cholinesterase, butyrylcholinesterase, and a-glucosidase activities. Abbreviations used: DPPH: 1

Inhibition of Cholinesterases and Some Pro-Oxidant induced Oxidative Stress in Rats Brain by Two Tomato (Lycopersicon Esculentum) Varieties

PubMed Central

Oboh, G.; Bakare, O.O.; Ademosun, A.O.; Akinyemi, A.J.; Olasehinde, T.A.

2015-01-01

This study sought to investigate the effects of two tomato varieties [Lycopersicon esculentum Mill. var. esculentum (ESC) and Lycopersicon esculentum Mill. var. cerasiforme (CER)] on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities in vitro. Phenolics content, carotenoids characterisation, inhibition of Fe2+ and quinolinic acid-induced malondialdehyde (MDA) production in rats brain homogenate and NO* scavenging abilities were also assesed in addition to the AChE and BChE inhibition assays. There was no significant difference in the AChE inhibitory ability of the samples, while CER had significantly higher BChE inhibitory activity. Furthermore, the tomatoes inhibited Fe2+ and quinolinic acid-induced MDA production and further exhibited antioxidant activities through their NO* scavenging abilities. There was no significant difference in the phenolic content of the samples, while significantly high amounts of lycopene were detected in the tomatoes. The cholinesterase-inhibition and antioxidant properties of the “tomatoes” could make them good dietary means for the management of neurodegenerative disorders.

Regulation of ACh release from guinea pig bladder urothelial cells: potential role in bladder filling sensations.

PubMed

McLatchie, L M; Young, J S; Fry, C H

2014-07-01

The aim of this study was to quantify and characterize the mechanism of non-neuronal ACh release from bladder urothelial cells and to determine if urothelial cells could be a site of action of anti-muscarinic drugs. A novel technique was developed whereby ACh could be measured from freshly isolated guinea pig urothelial cells in suspension following mechanical stimulation. Various agents were used to manipulate possible ACh release pathways in turn and to study the effects of muscarinic receptor activation and inhibition on urothelial ATP release. Minimal mechanical stimulus achieved full ACh release, indicating a small dynamic range and possible all-or-none signal. ACh release involved a mechanism dependent on the anion channel CFTR and intracellular calcium concentration, but was independent of extracellular calcium, vesicular trafficking, connexins or pannexins, organic cation transporters and was not affected by botulinum-A toxin. Stimulating ACh receptors increased ATP production and antagonizing them reduced ATP release, suggesting a link between ACh and ATP release. These results suggest that release of non-neuronal ACh from the urothelium is large enough and well located to act as a modulator of ATP release. It is hypothesized that this pathway may contribute to the actions of anti-muscarinic drugs in reducing the symptoms of lower urinary tract syndromes. Additionally the involvement of CFTR in ACh release suggests an exciting new direction for the treatment of these conditions. © 2014 The British Pharmacological Society.

Discovery of potent and selective acetylcholinesterase (AChE) inhibitors: acacetin 7-O-methyl ether Mannich base derivatives synthesised from easy access natural product naringin.

PubMed

Liu, Hao-Ran; Men, Xue; Gao, Xiao-Hui; Liu, Lin-Bo; Fan, Hao-Qun; Xia, Xin-Hua; Wang, Qiu-An

2018-03-01

Naringin, as a component universal existing in the peel of some fruits or medicinal plants, was usually selected as the material to synthesise bioactive derivates since it was easy to gain with low cost. In present investigation, eight new acacetin-7-O-methyl ether Mannich base derivatives (1-8) were synthesised from naringin. The bioactivity evaluation revealed that most of them exhibited moderate or potent acetylcholinesterase (AChE) inhibitory activity. Among them, compound 7 (IC 50 for AChE = 0.82 ± 0.08 μmol•L -1 , IC 50 for BuChE = 46.30 ± 3.26 μmol•L -1 ) showed a potent activity and high selectivity compared with the positive control Rivastigmine (IC 50 for AChE = 10.54 ± 0.86 μmol•L -1 , IC 50 for BuChE = 0.26 ± 0.08 μmol•L -1 ). The kinetic study suggested that compound 7 bind to AChE with mix-type inhibitory profile. Molecular docking study revealed that compound 7 could combine both catalytic active site (CAS) and peripheral active site (PAS) of AChE with four points (Trp84, Trp279, Tyr70 and Phe330), while it could bind with BuChE via only His 20.

Concomitant alpha7 and beta2 nicotinic AChR subunit deficiency leads to impaired energy homeostasis and increased physical activity in mice.

PubMed

Somm, Emmanuel; Guérardel, Audrey; Maouche, Kamel; Toulotte, Audrey; Veyrat-Durebex, Christelle; Rohner-Jeanrenaud, Françoise; Maskos, Uwe; Hüppi, Petra S; Schwitzgebel, Valérie M

2014-05-01

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels well characterized in neuronal signal transmission. Moreover, recent studies have revealed nAChR expression in nonneuronal cell types throughout the body, including tissues involved in metabolism. In the present study, we screen gene expression of nAChR subunits in pancreatic islets and adipose tissues. Mice pancreatic islets present predominant expression of α7 and β2 nAChR subunits but at a lower level than in central structures. Characterization of glucose and energy homeostasis in α7β2nAChR(-/-) mice revealed no major defect in insulin secretion and sensitivity but decreased glycemia apparently unrelated to gluconeogenesis or glycogenolysis. α7β2nAChR(-/-) mice presented an increase in lean and bone body mass and a decrease in fat storage with normal body weight. These observations were associated with elevated spontaneous physical activity in α7β2nAChR(-/-) mice, mainly due to elevation in fine vertical (rearing) activity while their horizontal (ambulatory) activity remained unchanged. In contrast to α7nAChR(-/-) mice presenting glucose intolerance and insulin resistance associated to excessive inflammation of adipose tissue, the present metabolic phenotyping of α7β2nAChR(-/-) mice revealed a metabolic improvement possibly linked to the increase in spontaneous physical activity related to central β2nAChR deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

Design, synthesis and structure-activity relationships of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease.

PubMed

Toda, Narihiro; Tago, Keiko; Marumoto, Shinji; Takami, Kazuko; Ori, Mayuko; Yamada, Naho; Koyama, Kazuo; Naruto, Shunji; Abe, Kazumi; Yamazaki, Reina; Hara, Takao; Aoyagi, Atsushi; Abe, Yasuyuki; Kaneko, Tsugio; Kogen, Hiroshi

2003-05-01

We have designed and synthesized a dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT) as a novel class of treatment drugs for Alzheimer's disease on the basis of a hypothetical model of the AChE active site. Dual inhibitions of AChE and SERT would bring about greater therapeutic effects than AChE inhibition alone and avoid adverse peripheral effects caused by excessive AChE inhibition. Compound (S)-6j exhibited potent inhibitory activities against AChE (IC(50)=101 nM) and SERT (IC(50)=42 nM). Furthermore, (S)-6j showed inhibitory activities of both AChE and SERT in mice brain following oral administration.

Acetylcholinesterase inhibition in cognition-relevant brain areas of mice treated with a nootropic Amazonian herbal (Marapuama).

PubMed

Figueiró, M; Ilha, J; Pochmann, D; Porciúncula, L O; Xavier, L L; Achaval, M; Nunes, D S; Elisabetsky, E

2010-10-01

The goal of acetylcholinesterase inhibitors (AChEIs) used to treat Alzheimer's patients is an improvement in cholinergic transmission. While currently available AChEIs have limited success, a huge impediment to the development of newer ones is access to the relevant brain areas. Promnesic, anti-amnesic and AChEI properties were identified in a standardized ethanol extract from Ptychopetalum olacoides (POEE), a medicinal plant favored by the elderly in Amazon communities. The purpose of this study was to provide conclusive evidence that orally given POEE induces AChE inhibition in brain areas relevant to cognition. Histochemistry experiments confirmed that the anticholinesterase compound(s) present in POEE are orally bioavailable, inducing meaningful AChE inhibition in the hippocampus CA1 (∼33%) and CA3 (∼20%), and striatum (∼17%). Ellman's colorimetric analysis revealed that G1 and G4 AChE isoforms activities were markedly inhibited (66 and 72%, respectively) in hippocampus and frontal cortex (50 and 63%, respectively), while G4 appeared to be selectively inhibited (72%) in the striatum. Western blotting showed that POEE did not induce significant changes in the AChE immunocontent suggesting that its synthesis is not extensively modified. This study provides definitive proof of meaningful anticholinesterase activity compatible with the observed promnesic and anti-amnesic effects of POEE in mice, reaffirming the potential of this extract for treating neurodegenerative conditions where a hypofunctioning cholinergic neurotransmission is prominent. Adequate assessment of the safety and efficacy of this extract and/or its isolated active compound(s) are warranted. 2010 Elsevier GmbH. All rights reserved.

Cigarette toxin 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces experimental pancreatitis through α7 nicotinic acetylcholine receptors (nAChRs) in mice

PubMed Central

Alahmari, A. A.; Sreekumar, B.; Patel, V.; Ashat, M.; Alexandre, M.; Uduman, A. K.; Akinbiyi, E. O.; Ceplenski, A.; Shugrue, C. A.; Kolodecik, T. R.; Messenger, S. W.; Groblewski, G. E.; Gorelick, F. S.

2018-01-01

Clinical studies have shown that cigarette smoking is a dose-dependent and independent risk factor for acute pancreatitis. Cigarette smoke contains nicotine which can be converted to the potent receptor ligand and toxin, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Previously, we have shown that NNK induces premature activation of pancreatic zymogens in rats, an initiating event in pancreatitis, and this activation is prevented by pharmacologic inhibition of nicotinic acetylcholine receptors (nAChR). In this study, we determined whether NNK mediates pancreatitis through the α7 isoform of nAChR using α7nAChR knockout mice. PCR analysis confirmed expression of non-neuronal α7nAChR in C57BL/6 (WT) mouse and human acinar cells. NNK treatment stimulated trypsinogen activation in acini from WT but not α7nAChR-/- mice. NNK also stimulated trypsinogen activation in human acini. To further confirm these findings, WT and α7nAChR-/- mice were treated with NNK in vivo and markers of pancreatitis were measured. As observed in acini NNK treatment induced trypsinogen activation in WT but not α7nAChR-/- mice. NNK also induced other markers of pancreatitis including pancreatic edema, vacuolization and pyknotic nuclei in WT but not α7nAChR-/- animals. NNK treatment led to increased neutrophil infiltration, a marker of inflammation, in WT mice and to a significantly lesser extent in α7nAChR-/- mice. We also examined downstream targets of α7nAChR activation and found that calcium and PKC activation are involved down stream of NNK stimulation of α7nAChR. In this study we used genetic deletion of the α7nAChR to confirm our previous inhibitor studies that demonstrated NNK stimulates pancreatitis by activating this receptor. Lastly, we demonstrate that NNK can also stimulate zymogen activation in human acinar cells and thus may play a role in human disease. PMID:29870540

Inhibition on cholinesterase and tyrosinase by alkaloids and phenolics from Aristotelia chilensis leaves.

PubMed

Cespedes, Carlos L; Balbontin, Cristian; Avila, Jose G; Dominguez, Mariana; Alarcon, Julio; Paz, Cristian; Burgos, Viviana; Ortiz, Leandro; Peñaloza-Castro, Ignacio; Seigler, David S; Kubo, Isao

2017-11-01

It is reported in this study the effect of isolates from leaves of Aristotelia chilensis as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase enzymes. The aim of the paper was to evaluate the activity of A. chilensis towards different enzymes. In addition to pure compounds, extracts rich in alkaloids and phenolics were tested. The most active F5 inhibited AChE (79.5% and 89.8% at 10.0 and 20.0 μg/mL) and against BChE (89.5% and 97.8% at 10.0 and 20.0 μg/mL), showing a strong mixed-type inhibition against AChE and BChE. F3 (a mixture of flavonoids and phenolics acids), showed IC 50 of 90.7 and 59.6 μg/mL of inhibitory activity against AChE and BChE, inhibiting the acetylcholinesterase competitively. Additionally, F3 showed and high potency as tyrosinase inhibitor with IC 50 at 8.4 μg/mL. Sample F4 (anthocyanidins and phenolic composition) presented a complex, mixed-type inhibition of tyrosinase with a IC 50 of 39.8 μg/mL. The findings in this investigation show that this natural resource has a strong potential for future research in the search of new phytotherapeutic treatments for cholinergic deterioration ailments avoiding the side effects of synthetic drugs. This is the first report as cholinesterases and tyrosinase inhibitors of alkaloids and phenolics from A. chilensis leaves. Copyright © 2017 Elsevier Ltd. All rights reserved.

Neurotoxicity fingerprinting of venoms using on-line microfluidic AChBP profiling.

PubMed

Slagboom, Julien; Otvos, Reka A; Cardoso, Fernanda C; Iyer, Janaki; Visser, Jeroen C; van Doodewaerd, Bjorn R; McCleary, Ryan J R; Niessen, Wilfried M A; Somsen, Govert W; Lewis, Richard J; Kini, R Manjunatha; Smit, August B; Casewell, Nicholas R; Kool, Jeroen

2018-06-15

Venoms from snakes are rich sources of highly active proteins with potent affinity towards a variety of enzymes and receptors. Of the many distinct toxicities caused by envenomation, neurotoxicity plays an important role in the paralysis of prey by snakes as well as by venomous sea snails and insects. In order to improve the analytical discovery component of venom toxicity profiling, this paper describes the implementation of microfluidic high-resolution screening (HRS) to obtain neurotoxicity fingerprints from venoms that facilitates identification of the neurotoxic components of envenomation. To demonstrate this workflow, 47 snake venoms were profiled using the acetylcholine binding protein (AChBP) to mimic the target of neurotoxic proteins, in particular nicotinic acetylcholine receptors (nAChRs). In the microfluidic HRS system, nanoliquid chromatographic (nanoLC) separations were on-line connected to both AChBP profiling and parallel mass spectrometry (MS). For virtually all neurotoxic elapid snake venoms tested, we obtained bioactivity fingerprints showing major and minor bioactive zones containing masses consistent with three-finger toxins (3FTxs), whereas, viperid and colubrid venoms showed little or no detectable bioactivity. Our findings demonstrate that venom interactions with AChBP correlate with the severity of neurotoxicity observed following human envenoming by different snake species. We further, as proof of principle, characterized bioactive venom peptides from a viperid (Daboia russelli) and an elapid (Aspidelaps scutatus scutatus) snake by nanoLC-MS/MS, revealing that different toxin classes interact with the AChBP, and that this binding correlates with the inhibition of α7-nAChR in calcium-flux cell-based assays. The on-line post-column binding assay and subsequent toxin characterization methodologies described here provide a new in vitro analytic platform for rapidly investigating neurotoxic snake venom proteins. Copyright © 2018 The Author

Acetylcholinesterase (AChE) and heat shock proteins (Hsp70) of gypsy moth (Lymantria dispar L.) larvae in response to long-term fluoranthene exposure.

PubMed

Mrdaković, Marija; Ilijin, Larisa; Vlahović, Milena; Matić, Dragana; Gavrilović, Anja; Mrkonja, Aleksandra; Perić-Mataruga, Vesna

2016-09-01

Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in living organisms, thus impairing fitness related traits and influencing their populations. This imposes the need for providing early-warning signals of pollution. Our study aimed to examine changes in the activity of acetylcholinesterase (AChE) and the concentration of heat shock proteins (Hsp70) in homogenates of brain tissues of fifth instar gypsy moth (Lymantria dispar L.) larvae, exposed to the ubiquitous PAH, fluoranthene, supplemented to the rearing diet. Significantly increased activity of AChE in larvae fed on the diets with high fluoranthene concentrations suggests the necessity for elucidation of the role of AChE in these insects when exposed to PAH pollution. Significant induction of Hsp70 in gypsy moth larvae reared on the diets containing low fluoranthene concentrations, indicate that changes in the level of Hsp70 might be useful as an indicator of pollution in this widespread forest species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anti-Alzheimer's disease activity of compounds from the root bark of Morus alba L.

PubMed

Kuk, Eun Bi; Jo, A Ra; Oh, Seo In; Sohn, Hee Sook; Seong, Su Hui; Roy, Anupom; Choi, Jae Sue; Jung, Hyun Ah

2017-03-01

The inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) plays important roles in prevention and treatment of Alzheimer's disease (AD). Among the individual parts of Morus alba L. including root bark, branches, leaves, and fruits, the root bark showed the most potent enzyme inhibitory activities. Therefore, the aim of this study was to evaluate the anti-AD activity of the M. alba root bark and its isolate compounds, including mulberrofuran G (1), albanol B (2), and kuwanon G (3) via inhibition of AChE, BChE, and BACE1. Compounds 1 and 2 showed strong AChE- and BChE-inhibitory activities; 1-3 showed significant BACE1 inhibitory activity. Based on the kinetic study with AChE and BChE, 2 and 3 showed noncompetitive-type inhibition; 1 showed mixed-type inhibition. Moreover, 1-3 showed mixed-type inhibition against BACE1. The molecular docking simulations of 1-3 demonstrated negative binding energies, indicating a high affinity to AChE and BACE1. The hydroxyl group of 1-3 formed hydrogen bond with the amino acid residues located at AChE and BACE1. Consequently, these results indicate that the root bark of M. alba and its active compounds might be promising candidates for preventive and therapeutic agents for AD.

Age Dependency of Inhibition of α7 Nicotinic Receptors and Tonically Active N-Methyl-d-aspartate Receptors by Endogenously Produced Kynurenic Acid in the Brain

PubMed Central

Alkondon, Manickavasagom; Pereira, Edna F. R.; Eisenberg, Howard M.; Kajii, Yasushi; Schwarcz, Robert

2011-01-01

In the mouse hippocampus normal levels of kynurenic acid (KYNA), a neuroactive metabolite synthesized in astrocytes primarily by kynurenine aminotransferase II (KAT II)-catalyzed transamination of l-kynurenine, maintain a degree of tonic inhibition of α7 nicotinic acetylcholine receptors (nAChRs). The present in vitro study was designed to test the hypothesis that α7 nAChR activity decreases when endogenous production of KYNA increases. Incubation (2–7 h) of rat hippocampal slices with kynurenine (200 μM) resulted in continuous de novo synthesis of KYNA. Kynurenine conversion to KYNA was significantly decreased by the KAT II inhibitor (S)-(−)-9-(4-aminopiperazine-1-yl)-8-fluoro-3-methyl-6-oxo-2,3,5,6-tetrahydro-4H-1-oxa-3a-azaphenalene-5carboxylic acid (BFF122) (100 μM) and was more effective in slices from postweaned than preweaned rats. Incubation of slices from postweaned rats with kynurenine inhibited α7 nAChRs and extrasynaptic N-methyl-d-aspartate receptors (NMDARs) on CA1 stratum radiatum interneurons. These effects were attenuated by BFF122 and mimicked by exogenously applied KYNA (200 μM). Exposure of human cerebral cortical slices to kynurenine also inhibited α7 nAChRs. The α7 nAChR sensitivity to KYNA is age-dependent, because neither endogenously produced nor exogenously applied KYNA inhibited α7 nAChRs in slices from preweaned rats. In these slices, kynurenine-derived KYNA also failed to inhibit extrasynaptic NMDARs, which could, however, be inhibited by exogenously applied KYNA. In slices from preweaned and postweaned rats, glutamatergic synaptic currents were not affected by endogenously produced KYNA, but were inhibited by exogenously applied KYNA. These results suggest that in the mature brain α7 nAChRs and extrasynaptic NMDARs are in close apposition to KYNA release sites and, thereby, readily accessible to inhibition by endogenously produced KYNA. PMID:21270133

Effects of hexamethonium, phenothiazines, propranolol and ephedrine on acetylcholinesterase carbamylation by physostigmine, aldicarb and carbaryl: interaction between the active site and the functionally distinct peripheral sites in acetylcholinesterase.

PubMed

Singh, A K; Spassova, D

1998-01-01

Physostigmine, aldicarb and carbaryl were potent inhibitors of acetylcholinesterase (AChE). The physostigmine-inhibited AChE fluoresced at 300 nm excitation and 500 nm emission wavelengths, but the aldicarb and carbaryl inhibited enzyme did not. This suggests that the carbamylated active center is not the fluorescing site in AChE. The fluorescence intensity of physostigmine-inhibited AChE decreased with increasing the substrate (acetylthiocholine) concentration, thus indicating that physostigmine binding to the active site is essential for the development of fluorescence. Thus, the physostigmine-inhibited AChE fluoresces due to the binding of trimethylpyrrolo[2,3-b]indol (TMPI) moiety, formed by the hydrolysis of physostigmine, to a peripheral site in AChE. The fluorescence intensity of the physostigmine-inhibited enzyme decreased when the inhibited-enzyme was dialyzed for either 30 min that poorly reactivated the enzyme or 180 min that fully reactivated the enzyme. This suggests that dialysis dissociates the AChE-TMPI complex much faster than it reactivates the carbamylated AChE. Ephedrine, propranolol and phenothiazines including trifluoparazine (TPZ) caused non-competitive inhibition, while hexamethonium caused an uncompetitive inhibition of AChE activity. TPZ, upon binding with AChE, formed a fluorescent TPZ-enzyme complex. The fluorescence intensity of TPZ-AChE complex was effectively decreased by ephedrine, but not by propranolol or hexamethonium. This indicates that TPZ and ephedrine bind to the same site in AChE which is different from the site/or sites to which propranolol or hexamethonium bind. Hexamethonium protected AChE from inhibition by carbamates and decreased the fluorescence intensity of the physostigmine-inhibited AChE. Phenothiazines and ephedrine did not modulate the enzyme inhibition or the fluorescence intensity of the physostigmine-inhibited AChE. Propranolol and TPZ potentiated the enzyme inhibition and increased the fluorescence intensity

Evaluation of the nature of camel retinal acetylcholinesterase: inhibition by hexamethonium.

PubMed

Alhomida, A S; Kamal, M A; al-Jafari, A A

1997-12-01

Acetylcholinesterase (AChE, EC 3.1.1.7) has been demonstrated in retinas of several species, however, the nature of the interaction of AChE with specific inhibitors are very limited in the literature and the mode of inhibition of camel retinal AChE by hexamethonium has been studied. Hexamethonium reversibly inhibited AChE in a concentration dependent manner, the IC50 value being c. 2.52 mM. The Km for the hydrolysis of acetylthiocholine iodide was found to be 0.087 mM and the Vmax was 0.63 mumol/min/mg protein. Dixon, as well as Lineweaver-Burk, plots and their secondary replots indicated that the nature of the inhibition is of the hyperbolic (partial) mixed type, which is considered to be a partial competitive and non-competitive mixture. The values of Ki(slope) and KI(intercept) from a Lineweaver-Burk plot were estimated as 0.30 mM and 0.17 mM, respectively, while Ki from a Dixon plot was estimated as 0.725 mM. The Ki was greater than KI indicating that hexamethonium has a greater affinity of binding for the active site than the peripheral site of the camel retina AChE.

Study of Inhibition, Reactivation and Aging Processes of Pesticides Using Graphene Nanosheets/Gold Nanoparticles-Based Acetylcholinesterase Biosensor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Lin; Long, Linjuan; Zhang, Weiying

2012-09-10

Organophosphate (OP) and carbamate pesticides exert their toxicity via attacking the hydroxyl moiety of serine in the 'active site' of acetylcholinesterase (AChE). In this paper we developed a stable AChE biosensor based on self-assembling AChE to graphene nanosheet (GN)-gold nanoparticles (AuNPs) nanocomposite electrode for investigation of inhibition, reactivation and aging processes of different pesticides. It is confirmed that pesticides can inhibit AChE in a short time. OPs poisoning is treatable with oximes while carbarmates exposure is insensitive to oximes. The proposed electrochemical approach thus provides a new simple tool for comparison of pesticide sensitivity and guide of therapeutic intervention.

Fetal muscle-type nicotinic acetylcholine receptor activation in TE-671 cells and inhibition of fetal movement in a day 40 pregnant goat model by optical isomers of the piperidine alkaloid coniine.

PubMed

Green, Benedict T; Lee, Stephen T; Welch, Kevin D; Pfister, James A; Panter, Kip E

2013-01-01

Coniine is an optically active toxic piperidine alkaloid and nicotinic acetylcholine receptor (nAChR) agonist found in poison hemlock (Conium maculatum L.). Coniine teratogenicity is hypothesized to be attributable to the binding, activation, and prolonged desensitization of fetal muscle-type nAChR, which results in the complete inhibition of fetal movement. However, pharmacological evidence of coniine actions at fetal muscle-type nAChR is lacking. The present study compared (-)-coniine, (+)-coniine, and nicotine for the ability to inhibit fetal movement in a day 40 pregnant goat model and in TE-671 cells that express fetal muscle-type nAChR. Furthermore, α-conotoxins (CTx) EI and GI were used to antagonize the actions of (+)- and (-)-coniine in TE-671 cells. (-)-Coniine was more effective at eliciting electrical changes in TE-671 cells and inhibiting fetal movement than was (+)-coniine, suggesting stereoselectivity by the receptor. The pyridine alkaloid nicotine did not inhibit fetal movement in a day 40 pregnant goat model, suggesting agonist specificity for the inhibition of fetal movement. Low concentrations of both CTxs potentiated the TE-671 cell response and higher concentrations of CTx EI, and GI antagonized the actions of both coniine enantiomers demonstrating concentration-dependent coagonism and selective antagonism. These results provide pharmacological evidence that the piperidine alkaloid coniine is acting at fetal muscle-type nAChR in a concentration-dependent manner.

Unbinding of fluorinated oxime drug from the AChE gorge in polarizable water: a well-tempered metadynamics study.

PubMed

Pathak, Arup Kumar; Bandyopadhyay, Tusar

2017-02-15

Despite the fact that fluorination makes a drug more lipophilic, the molecular level understanding of protein-fluorinated drug interactions is very poor. Due to their enhanced ability to penetrate the blood brain barrier, they are suitable for reactivation of organophosphorus inactivated acetylcholinesterase (AChE) in the central nervous system. We systematically studied the unbinding of fluorinated obidoxime (FOBI) and non-fluorinated obidoxime (OBI) from the active site gorge of the serine hydrolase AChE in mean field polarizable water by employing all atom molecular dynamics simulations. It is observed that the unbinding process is strongly influenced by cation-π, hydrogen bond (HB) and water bridge interactions. The FOBI drug interacts more strongly with the protein residues than OBI and this is also verified from quantum mechanical calculations. Distinct unbinding pathways for FOBI and OBI are observed as evident from the 1D and 2D potential of mean force of the unbinding profiles. The present study suggests that the FOBI drug is held more firmly in the gorge of AChE in comparison to OBI and may lead to higher reactivation efficiency of the inactivated enzyme.

Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos

PubMed Central

Wheelock, Craig E.; Eder, Kai J.; Werner, Inge; Huang, Huazhang; Jones, Paul D.; Brammell, Benjamin F.; Elskus, Adria A.; Hammock, Bruce D.

2006-01-01

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 μg/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 μg/l), but not a low dose (1.2 μg/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ∼30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was

Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos

USGS Publications Warehouse

Wheelock, C.E.; Eder, K.J.; Werner, I.; Huang, H.; Jones, P.D.; Brammell, B.F.; Elskus, A.A.; Hammock, B.D.

2005-01-01

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 ??g/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 ??g/l), but not a low dose (1.2 ??g/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ???30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was

Inhibition of cholinesterases by stereoisomers of Huperzine-A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saxena, A.; Qian, N.; Kovach, I.M.

1993-05-13

Huperzine-A, a potential drug for the treatment of Alzheimer's disease and possible pretreament for nerve agent toxicity, has recently been characterized as a reversible inhibitor of cholinesterases (Ashani et al., BBRC, 184:719-726, 1992). Long-term incubation of purified cholinesterases with Huperzine-A did not show any chemical modification of Huperzine-A. The dissociation constant, K(I), for fetal bovine serum acetylcholinesterase (FBS AChE) was approximately 20 nM, for Torpedo AChE was 215 nM, and for horse serum butyrylcholinesterase (BChE) was 40 micrometers M. Inhibition studies with the two stereoisomers of Huperzine-A have shown that naturally occurring (-)-Huperzine-A inhibited FBS AChE 35-fold more potently thanmore » (+)-Huperzine-A, with K(I) values of 6.2 nM and 210 nM, respectively. These results are in agreement with those reported previously using crude preparations of rat cortical AChE (McKinney et al., Eur. J. Pharmacol., 203, 303-305, 1991). (-)-Huperzine-A, on the other hand, was 80-fold more potent than (+)-Huperzine-A in inhibiting Torpedo AChE, with K(I), values of 0.25 micrometers M and 22 micrometer M, respectively. No significant differences in K(I) were observed for the two stereoisomers of Huperzine-A with horse serum BChE, indicating the lack of stereoselectivity of this compound for BChE. Molecular modeling studies involving docking of each of the two stereoisomers of Huperzine-A into the active-site gorge of Torpedo AChE also revealed that (-)-Huperzine-A gave a better fit than (+)-Huperzine-A.« less

Gas Chromatography, GC/Mass Analysis and Bioactivity of Essential Oil from Aerial Parts of Ferulago trifida: Antimicrobial, Antioxidant, AChE Inhibitory, General Toxicity, MTT Assay and Larvicidal Activities.

PubMed

Tavakoli, Saeed; Vatandoost, Hassan; Zeidabadinezhad, Reza; Hajiaghaee, Reza; Hadjiakhoondi, Abbas; Abai, Mohammad Reza; Yassa, Narguess

2017-09-01

We aimed to investigate different biological properties of aerial parts essential oil of Ferulago trifida Boiss and larvicidal activity of its volatile oils from all parts of plant. Essential oil was prepared by steam distillation and analyzed by Gas chromatography and GC/Mass. Antioxidant, antimicrobial, cytotoxic effects and AChE inhibitory of the oil were investigated using DPPH, disk diffusion method, MTT assay and Ellman methods. Larvicidal activity of F. trifida essential oil against malaria vector Anopheles stephensi was carried out according to the method described by WHO. In GC and GC/MS analysis, 58 compounds were identified in the aerial parts essential oil, of which E-verbenol (9.66%), isobutyl acetate (25.73%) and E-β-caryophyllene (8.68%) were main compounds. The oil showed (IC 50 = 111.2μg/ml) in DPPH and IC 50 = 21.5 mg/ml in the investigation of AChE inhibitory. Furthermore, the oil demonstrated toxicity with (LD 50 = 1.1μg/ml) in brine shrimp lethality test and with (IC 50 = 22.0, 25.0 and 42.55 μg/ml) on three cancerous cell lines (MCF-7, A-549 and HT-29) respectively. LC 50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of An. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. In antimicrobial activities, essential oil was effective on all specimens except Escherichia coli , Aspergillus niger and Candida albicans. The essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and MTT assay on three cancerous cell lines.

Gas Chromatography, GC/Mass Analysis and Bioactivity of Essential Oil from Aerial Parts of Ferulago trifida: Antimicrobial, Antioxidant, AChE Inhibitory, General Toxicity, MTT Assay and Larvicidal Activities

PubMed Central

Tavakoli, Saeed; Vatandoost, Hassan; Zeidabadinezhad, Reza; Hajiaghaee, Reza; Hadjiakhoondi, Abbas; Abai, Mohammad Reza; Yassa, Narguess

2017-01-01

Background: We aimed to investigate different biological properties of aerial parts essential oil of Ferulago trifida Boiss and larvicidal activity of its volatile oils from all parts of plant. Methods: Essential oil was prepared by steam distillation and analyzed by Gas chromatography and GC/Mass. Antioxidant, antimicrobial, cytotoxic effects and AChE inhibitory of the oil were investigated using DPPH, disk diffusion method, MTT assay and Ellman methods. Larvicidal activity of F. trifida essential oil against malaria vector Anopheles stephensi was carried out according to the method described by WHO. Results: In GC and GC/MS analysis, 58 compounds were identified in the aerial parts essential oil, of which E-verbenol (9.66%), isobutyl acetate (25.73%) and E-β-caryophyllene (8.68%) were main compounds. The oil showed (IC50= 111.2μg/ml) in DPPH and IC50= 21.5 mg/ml in the investigation of AChE inhibitory. Furthermore, the oil demonstrated toxicity with (LD50= 1.1μg/ml) in brine shrimp lethality test and with (IC50= 22.0, 25.0 and 42.55 μg/ml) on three cancerous cell lines (MCF-7, A-549 and HT-29) respectively. LC50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of An. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. In antimicrobial activities, essential oil was effective on all specimens except Escherichia coli, Aspergillus niger and Candida albicans. Conclusion: The essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and MTT assay on three cancerous cell lines. PMID:29322058

Modulatory effects of α7 nAChRs on the immune system and its relevance for CNS disorders.

PubMed

Kalkman, Hans O; Feuerbach, Dominik

2016-07-01

The clinical development of selective alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists has hitherto been focused on disorders characterized by cognitive deficits (e.g., Alzheimer's disease, schizophrenia). However, α7 nAChRs are also widely expressed by cells of the immune system and by cells with a secondary role in pathogen defense. Activation of α7 nAChRs leads to an anti-inflammatory effect. Since sterile inflammation is a frequently observed phenomenon in both psychiatric disorders (e.g., schizophrenia, melancholic and bipolar depression) and neurological disorders (e.g., Alzheimer's disease, Parkinson's disease, and multiple sclerosis), α7 nAChR agonists might show beneficial effects in these central nervous system disorders. In the current review, we summarize information on receptor expression, the intracellular signaling pathways they modulate and reasons for receptor dysfunction. Information from tobacco smoking, vagus nerve stimulation, and cholinesterase inhibition is used to evaluate the therapeutic potential of selective α7 nAChR agonists in these inflammation-related disorders.

Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.

PubMed

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

PubMed

Vulfius, Catherine A; Kasheverov, Igor E; Kryukova, Elena V; Spirova, Ekaterina N; Shelukhina, Irina V; Starkov, Vladislav G; Andreeva, Tatyana V; Faure, Grazyna; Zouridakis, Marios; Tsetlin, Victor I; Utkin, Yuri N

2017-01-01

Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by

Rivastigmine: the advantages of dual inhibition of acetylcholinesterase and butyrylcholinesterase and its role in subcortical vascular dementia and Parkinson's disease dementia.

PubMed

Kandiah, Nagaendran; Pai, Ming-Chyi; Senanarong, Vorapun; Looi, Irene; Ampil, Encarnita; Park, Kyung Won; Karanam, Ananda Krishna; Christopher, Stephen

2017-01-01

Several studies have demonstrated clinical benefits of sustained cholinesterase inhibition with rivastigmine in Alzheimer's disease (AD) and Parkinson's disease dementia (PDD). Unlike donepezil and galantamine that selectively inhibit acetylcholinesterase (AChE; EC 3.1.1.7), rivastigmine is a unique cholinesterase inhibitor with both AChE and butyrylcholinesterase (BuChE; EC 3.1.1.8) inhibitory activity. Rivastigmine is also available as transdermal patch that has been approved by the US Food and Drug Administration for the treatment of mild, moderate, and severe AD as well as mild-to-moderate PDD. In this review, we explore the role of BuChE inhibition in addition to AChE inhibition with rivastigmine in the outcomes of cognition, global function, behavioral symptoms, and activities of daily living. Additionally, we review the evidence supporting the use of dual AChE-BuChE inhibitory activity of rivastigmine as a therapeutic strategy in the treatment of neurological disorders, with a focus on the role of rivastigmine in subcortical dementias such as vascular dementia (VaD) and PDD. Toward this objective, we performed a literature search in PubMed and Ovid with limits to articles published in the English language before June 2016. The available evidence from the literature suggests that the dual inhibition of AChE and BuChE may afford additional therapeutic potential of rivastigmine in subcortical dementias (subcortical VaD and PDD) with benefits on cognition and behavioral symptoms. Rivastigmine was found to specifically benefit executive dysfunction frequently observed in subcortical dementias; however, large randomized clinical studies are warranted to support these observations.

Electrical coupling and release of K+ from endothelial cells co-mediate ACh-induced smooth muscle hyperpolarization in guinea-pig inner ear artery

PubMed Central

Jiang, Zhi-Gen; Nuttall, Alfred L; Zhao, Hui; Dai, Chun-Fu; Guan, Bing-Cai; Si, Jun-Qiang; Yang, Yu-Qin

2005-01-01

The physiological basis of ACh-elicited hyperpolarization in guinea-pig in vitro cochlear spiral modiolar artery (SMA) was investigated by intracellular recording combined with dye labelling of recorded cells and immunocytochemistry. We found the following. (1) The ACh-hyperpolarization was prominent only in cells that had a low resting potential (less negative than −60 mV). ACh-hyperpolarization was reversibly blocked by 4-DAMP, charybdotoxin or BAPTA-AM, but not by Nω-nitro-l-arginine methyl ester, glipizide, indomethacin or 17-octadecynoic acid. (2) Ba2+ (100 μm) and ouabain (1 μm) each attenuated ACh-hyperpolarization by ∼ 30% in smooth muscle cells (SMCs) but had only slight or no inhibition in endothelial cells (ECs). A combination of Ba2+ and 18β-glycyrrhetinic acid near completely blocked the ACh-hyperpolarization in SMCs. (3) High K+ (10 mm) induced a smaller hyperpolarization in ECs than in SMCs, with an amplitude ratio of 0.49: 1. Ba2+ blocked the K+-induced hyperpolarization by ∼ 85% in both cell types, whereas ouabain inhibited K+-hyperpolarization differently in SMCs (19%) and ECs (35%) and increased input resistance. 18β-Glycyrrhetinic acid blocked the high K+-hyperpolarization in ECs only. (4) Weak myoendothelial dye coupling was detected by confocal microscopy in cells recorded with a propidium iodide-containing electrode for longer than 30 min. A sparse plexus of choline acetyltransferase-immunoreactive (ChAT) fibres was observed around the SMA and its up-stream arteries. (5) Evoked excitatory junction potentials (EJP) were partially blocked by 4-DAMP in half of the cells tested. We conclude that ACh-induced hyperpolarization originates from ECs via activation of Ca2+-activated potassium channels, and is independent of the release of NO, cyclo-oxygenase or cytochrome P450 products. ACh-induced hyperpolarization in smooth muscle cells involves two mechanisms: (a) electrical spread of the hyperpolarization from the endothelium, and (b

Electrical coupling and release of K+ from endothelial cells co-mediate ACh-induced smooth muscle hyperpolarization in guinea-pig inner ear artery.

PubMed

Jiang, Zhi-Gen; Nuttall, Alfred L; Zhao, Hui; Dai, Chun-Fu; Guan, Bing-Cai; Si, Jun-Qiang; Yang, Yu-Qin

2005-04-15

The physiological basis of ACh-elicited hyperpolarization in guinea-pig in vitro cochlear spiral modiolar artery (SMA) was investigated by intracellular recording combined with dye labelling of recorded cells and immunocytochemistry. We found the following. (1) The ACh-hyperpolarization was prominent only in cells that had a low resting potential (less negative than -60 mV). ACh-hyperpolarization was reversibly blocked by 4-DAMP, charybdotoxin or BAPTA-AM, but not by N(omega)-nitro-L-arginine methyl ester, glipizide, indomethacin or 17-octadecynoic acid. (2) Ba(2)(+) (100 microm) and ouabain (1 microm) each attenuated ACh-hyperpolarization by approximately 30% in smooth muscle cells (SMCs) but had only slight or no inhibition in endothelial cells (ECs). A combination of Ba(2)(+) and 18beta-glycyrrhetinic acid near completely blocked the ACh-hyperpolarization in SMCs. (3) High K(+) (10 mm) induced a smaller hyperpolarization in ECs than in SMCs, with an amplitude ratio of 0.49 : 1. Ba(2)(+) blocked the K(+)-induced hyperpolarization by approximately 85% in both cell types, whereas ouabain inhibited K(+)-hyperpolarization differently in SMCs (19%) and ECs (35%) and increased input resistance. 18beta-Glycyrrhetinic acid blocked the high K(+)-hyperpolarization in ECs only. (4) Weak myoendothelial dye coupling was detected by confocal microscopy in cells recorded with a propidium iodide-containing electrode for longer than 30 min. A sparse plexus of choline acetyltransferase-immunoreactive (ChAT) fibres was observed around the SMA and its up-stream arteries. (5) Evoked excitatory junction potentials (EJP) were partially blocked by 4-DAMP in half of the cells tested. We conclude that ACh-induced hyperpolarization originates from ECs via activation of Ca(2)(+)-activated potassium channels, and is independent of the release of NO, cyclo-oxygenase or cytochrome P450 products. ACh-induced hyperpolarization in smooth muscle cells involves two mechanisms: (a) electrical spread

Acetylcholinesterase inhibition and anti-Soman efficacy of homologs of physostigmine. (Reannouncement with new availability information)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harris, L.W.; Anderson, D.R.; Pastelak, A.M.

1992-12-31

Inhibition of acetylcholinesterase (AChE) activity by physostigmine (PHY) is reversible due to spontaneous decarbamylation. Physostigmine has been shown to be effective as a pretreatment against potent anticholinesterase poisons (e.g., soman) in experimental animals, yet it is short acting and causes undesirable side effects in mammals. The two-fold purpose of this study was (1) to determine whether extension of the N-substituted alkyl chain (N-SAC) of PHY from N-methyl to N-ethyl (1), N-propyl (2), N-isopropyl (3), N-butyl (4) or N-heptyl (5) affects anti-AChE potency and spontaneous decarbamylation of inhibited AChE of guinea pig blood in vitro and in vivo and (2) tomore » see whether chain extension affects efficacy as pretreatment in poisoning by soman. The in vitro AChE inhibition studies were done using whole blood incubated at 37 deg C for 30 min. All 5 homologs possessed anti-AChE activity with I50s ranging from 1.1 to 27.6 x l0(-7)M; compound III was the least potent in vitro and in vivo. Lengthening of the N-SAC of PHY markedly extended the duration of anti-AChE activity when compared to PHY, but rendered the modified compounds ineffective as pretreatments against soman. These data support the premise that the decrease in decarbamylation rates observed upon extending the N-SAC of PHY is responsible for the loss of effectiveness of pretreatment regimens against soman. Perhaps, these homologs of PHY may have potential use in instances where sustained action of acetylcholine is required at cholinergic junctions because of disease conditions or drug overdosage.... Physostigmine, Nerve agent pretreatment, Soman, Acetylcholinesterase inhibition.« less

Monoamine uptake inhibitors block alpha7-nAChR-mediated cerebral nitrergic neurogenic vasodilation.

PubMed

Long, Cheng; Chen, Mei-Fang; Sarwinski, Susan J; Chen, Po-Yi; Si, Minliang; Hoffer, Barry J; Evans, M Steven; Lee, Tony J F

2006-07-01

We have proposed that activation of cerebral perivascular sympathetic alpha7-nicotinic acetylcholine receptors (alpha7-nAChRs) by nicotinic agonists releases norepinephrine, which then acts on parasympathetic nitrergic nerves, resulting in release of nitric oxide and vasodilation. Using patch-clamp electrophysiology, immunohistochemistry, and in vitro tissue bath myography, we tested this axo-axonal interaction hypothesis further by examining whether blocking norepinephrine reuptake enhanced alpha7-nAChR-mediated cerebral nitrergic neurogenic vasodilation. The results indicated that choline- and nicotine-induced alpha7-nAChR-mediated nitrergic neurogenic relaxation in endothelium-denuded isolated porcine basilar artery rings was enhanced by desipramine and imipramine at lower concentrations (0.03-0.1 microM) but inhibited at higher concentrations (0.3-10 microM). In cultured superior cervical ganglion (SCG) neurons of the pig and rat, choline (0.1-30 mM)-evoked inward currents were reversibly blocked by 1-30 microM mecamylamine, 1-30 microM methyllycaconitine, 10-300 nM alpha-bungarotoxin, and 0.1-10 microM desipramine and imipramine, providing electrophysiological evidence for the presence of similar functional alpha7-nAChRs in cerebral perivascular sympathetic neurons of pigs and rats. In alpha7-nAChR-expressing Xenopus oocytes, choline-elicited inward currents were attenuated by alpha-bungarotoxin, imipramine, and desipramine. These monoamine uptake inhibitors appeared to directly block the alpha7-nAChR, resulting in diminished nicotinic agonist-induced cerebral nitrergic vasodilation. The enhanced nitrergic vasodilation by lower concentrations of monoamine uptake inhibitors is likely due to a greater effect on monoamine uptake than on alpha7-nAChR blockade. These results further support the hypothesis of axo-axonal interaction in nitrergic regulation of cerebral vascular tone.

Endogenous purines modulate K+ -evoked ACh secretion at the mouse neuromuscular junction.

PubMed

Guarracino, Juan F; Cinalli, Alejandro R; Veggetti, Mariela I; Losavio, Adriana S

2018-06-01

At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y 13 and A 1 , A 2A , and A 3 receptors, respectively. To elucidate the action of endogenous purines on K + -dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end-plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K + , the P2Y 13 antagonist N-[2-(methylthio)ethyl]-2-[3,3,3-trifluoropropyl]thio-5'-adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR-C69931MX) increased asynchronous ACh secretion while the A 1 , A 3 , and A 2A antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(±)-dihydropyridine-3,5-, dicarboxylate (MRS-1191), and 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH-58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S-(p-nitrobenzyl)-6-thioinosine provoked a reduction of 10 mM K + -evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K + , endogenous ATP/ADP and adenosine bind to inhibitory P2Y 13 and A 1 and A 3 receptors since AR-C69931MX, DPCPX, and MRS-1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto-5'-nucleotidase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K + , suggesting that more adenosine is needed to activate excitatory A 2A receptors. At high K + concentration, the equilibrative transporters appear to be saturated allowing the accumulation of

Nicotine-Induced Effects on Nicotinic Acetylcholine Receptors (nAChRs), Ca2+ and Brain-Derived Neurotrophic Factor (BDNF) in STC-1 Cells.

PubMed

Qian, Jie; Mummalaneni, Shobha K; Alkahtani, Reem M; Mahavadi, Sunila; Murthy, Karnam S; Grider, John R; Lyall, Vijay

2016-01-01

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and β nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-β-erythroidine, a α4β2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and β4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.

Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward.

PubMed

Henderson, Brandon J; Wall, Teagan R; Henley, Beverley M; Kim, Charlene H; Nichols, Weston A; Moaddel, Ruin; Xiao, Cheng; Lester, Henry A

2016-03-09

Upregulation of β2 subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate β2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of α4* nAChRs, complementing that of chronic nicotine alone, which upregulates α4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (α4)3(β2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(β2)3 nAChRs. Menthol alone also increases the number of α6β2 receptors that exclude the β3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway. Copyright © 2016 the authors 0270-6474/16/362957-18$15.00/0.

Sympathetic α₃β₂-nAChRs mediate cerebral neurogenic nitrergic vasodilation in the swine.

PubMed

Lee, Reggie Hui-Chao; Liu, Yi-Qing; Chen, Po-Yi; Liu, Chin-Hung; Chen, Mei-Fang; Lin, Hung-Wen; Kuo, Jon-Son; Premkumar, Louis S; Lee, Tony Jer-Fu

2011-08-01

The α(7)-nicotinic ACh receptor (α(7)-nAChR) on sympathetic neurons innervating basilar arteries of pigs crossed bred between Landrace and Yorkshire (LY) is known to mediate nicotine-induced, β-amyloid (Aβ)-sensitive nitrergic neurogenic vasodilation. Preliminary studies, however, demonstrated that nicotine-induced cerebral vasodilation in pigs crossbred among Landrace, Yorkshire, and Duroc (LYD) was insensitive to Aβ and α-bungarotoxin (α-BGTX). We investigated nAChR subtype on sympathetic neurons innervating LYD basilar arteries. Nicotine-induced relaxation of porcine isolated basilar arteries was examined by tissue bath myography, inward currents on nAChR-expressing oocytes by two-electrode voltage recording, and mRNA and protein expression in the superior cervical ganglion (SCG) and middle cervical ganglion (MCG) by reverse transcription PCR and Western blotting. Nicotine-induced basilar arterial relaxation was not affected by Aβ, α-BGTX, and α-conotoxin IMI (α(7)-nAChR antagonists), or α-conotoxin AuIB (α(3)β(4)-nAChR antagonist) but was inhibited by tropinone and tropane (α(3)-containing nAChR antagonists) and α-conotoxin MII (selective α(3)β(2)-nAChR antagonist). Nicotine-induced inward currents in α(3)β(2)-nAChR-expressing oocytes were inhibited by α-conotoxin MII but not by α-BGTX, Aβ, or α-conotoxin AuIB. mRNAs of α(3)-, α(7)-, β(2)-, and β(4)-subunits were expressed in both SCGs and MCGs with significantly higher mRNAs of α(3)-, β(2)-, and β(4)-subunits than that of α(7)-subunit. The Aβ-insensitive sympathetic α(3)β(2)-nAChR mediates nicotine-induced cerebral nitrergic neurogenic vasodilation in LYD pigs. The different finding from Aβ-sensitive α(7)-nAChR in basilar arteries of LY pigs may offer a partial explanation for different sensitivities of individuals to Aβ in causing diminished cerebral nitrergic vasodilation in diseases involving Aβ.

Carbachol inhibits basal and forskolin-evoked adult rat striatal acetylcholine release.

PubMed

Login, I S

1997-05-27

Acutely dissociated adult rat striatal cholinergic neurons labeled with [3H]choline were used in a perifusion system to study muscarinic regulation of basal and forskolin-stimulated fractional [3H]acetylcholine ([3H]-ACh) efflux in the absence of synaptic modulation. Carbachol inhibited basal (40% maximal inhibition; IC50 approximately 0.7 microM) and forskolin-evoked release (75% inhibition; IC50 approximately 0.05 microM) in a concentration-dependent manner, and both carbachol actions were abolished with atropine. Thus, activation of striatal muscarinic cholinergic autoreceptors potently inhibits basal and adenylate cyclase-stimulated ACh release. Tonic inhibitory control of cholinergic activity by functional striatal circuitry apparently prevents detection of these important physiological interactions in slices or in situ.

Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons

PubMed Central

Weber, Martin; Motin, Leonid; Gaul, Simon; Beker, Friederike; Fink, Rainer H A; Adams, David J

2004-01-01

The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca2+ concentration ([Ca2+]i) and membrane currents were investigated in neonatal rat intracardiac neurons. In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca2+]I, which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca2+]i transients was 28 μM, close to the estimated clinical EC50 (clinically relevant (half-maximal) effective concentration) of thiopental. In fura-2-loaded neurons, voltage clamped at −60 mV to eliminate any contribution of voltage-gated Ca2+ channels, thiopental (25 μM) simultaneously inhibited nAChR-induced increases in [Ca2+]i and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by ∼ 40% at −120, −80 and −40 mV holding potential, indicating that the inhibition is voltage independent. The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC50 were also shown to inhibit nAChR-induced increases in [Ca2+]i by ∼40%. Thiopental (25 μM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca2+]i, indicating that inhibition of Ca2+ release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. Depolarization-activated Ca2+ channel currents were unaffected in the presence of thiopental (25 μM), pentobarbital (50 μM) and ketamine (10 μM). In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca2+]i transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions. PMID:15644873

Dual inhibition of acetylcholinesterase and butyrylcholinesterase enzymes by allicin

PubMed Central

Kumar, Suresh

2015-01-01

Objectives: The brain of mammals contains two major form of cholinesterase enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The dual inhibition of these enzymes is considered as a promising strategy for the treatment of neurological disorder such as Alzheimer's disease (AD), senile dementia, ataxia, and myasthenia gravis. The present study was undertaken to explore the anticholinesterase inhibition property of allicin. Materials and Methods: An assessment of cholinesterase inhibition was carried out by Ellman's assay. Results: The present study demonstrates allicin, a major ingredient of crushed garlic (Allium sativum L.) inhibited both AChE and BuChE enzymes in a concentration-dependent manner. For allicin, the IC50 concentration was 0.01 mg/mL (61.62 μM) for AChE and 0.05 ± 0.018 mg/mL (308.12 μM) for BuChE enzymes. Conclusions: Allicin shows a potential to ameliorate the decline of cognitive function and memory loss associated with AD by inhibiting cholinesterase enzymes and upregulate the levels of acetylcholine (ACh) in the brain. It can be used as a new lead to target AChE and BuChE to upregulate the level of ACh which will be useful in alleviating the symptoms associated with AD. PMID:26288480

Rivastigmine: the advantages of dual inhibition of acetylcholinesterase and butyrylcholinesterase and its role in subcortical vascular dementia and Parkinson’s disease dementia

PubMed Central

Kandiah, Nagaendran; Pai, Ming-Chyi; Senanarong, Vorapun; Looi, Irene; Ampil, Encarnita; Park, Kyung Won; Karanam, Ananda Krishna; Christopher, Stephen

2017-01-01

Several studies have demonstrated clinical benefits of sustained cholinesterase inhibition with rivastigmine in Alzheimer’s disease (AD) and Parkinson’s disease dementia (PDD). Unlike donepezil and galantamine that selectively inhibit acetylcholinesterase (AChE; EC 3.1.1.7), rivastigmine is a unique cholinesterase inhibitor with both AChE and butyrylcholinesterase (BuChE; EC 3.1.1.8) inhibitory activity. Rivastigmine is also available as transdermal patch that has been approved by the US Food and Drug Administration for the treatment of mild, moderate, and severe AD as well as mild-to-moderate PDD. In this review, we explore the role of BuChE inhibition in addition to AChE inhibition with rivastigmine in the outcomes of cognition, global function, behavioral symptoms, and activities of daily living. Additionally, we review the evidence supporting the use of dual AChE−BuChE inhibitory activity of rivastigmine as a therapeutic strategy in the treatment of neurological disorders, with a focus on the role of rivastigmine in subcortical dementias such as vascular dementia (VaD) and PDD. Toward this objective, we performed a literature search in PubMed and Ovid with limits to articles published in the English language before June 2016. The available evidence from the literature suggests that the dual inhibition of AChE and BuChE may afford additional therapeutic potential of rivastigmine in subcortical dementias (subcortical VaD and PDD) with benefits on cognition and behavioral symptoms. Rivastigmine was found to specifically benefit executive dysfunction frequently observed in subcortical dementias; however, large randomized clinical studies are warranted to support these observations. PMID:28458525

ACh-induced endothelial NO synthase translocation, NO release and vasodilatation in the hamster microcirculation in vivo

PubMed Central

Figueroa, Xavier F; González, Daniel R; Martínez, Agustín D; Durán, Walter N; Boric, Mauricio P

2002-01-01

Studies in cultured cells show that activation of endothelial nitric oxide (NO) synthase (eNOS) requires the dissociation of this enzyme from its inhibitory association with caveolin-1 (Cav-1), and perhaps its translocation from plasma membrane caveolae to other cellular compartments. We investigated the hypothesis that in vivo NO-dependent vasodilatation is associated with the translocation of eNOS from the cell membrane. To this end, we applied ACh topically (10-100 μm for 10 min) to the hamster cheek pouch microcirculation and measured NO production, blood flow and vessel diameter, and assessed subcellular eNOS distribution by Western blotting. Baseline NO production was 54.4 ± 5.2 pmol min−1 (n = 16). ACh increased NO release, caused arteriolar and venular dilatation and elevated microvascular flow. These responses were inhibited by NG-nitro-L-arginine (30 μm). The maximal increase in NO production induced by 10 μm and 100 μm ACh was 45 ± 20 % and 111 ± 33 %, respectively; the corresponding blood flow increases were 50 ± 10 % and 130 ± 24 %, respectively (n = 4-6). Both responses followed a similar time course, although increases in NO preceded flow changes. In non-stimulated tissues, eNOS was distributed mainly in the microsomal fraction. ACh-induced vasodilatation was associated with eNOS translocation to the cytosolic and Golgi-enriched fractions. After 1.5, 3.0 or 6.0 min of application, 10 μm ACh decreased the level of membrane-bound eNOS by -13 ± 4 %, -60 ± 4 % and -19 ± 17 %, respectively; at the same time points, 100 μm ACh reduced microsomal eNOS content by -38 ± 9 %, -61 ± 16 % and -40 ± 18 %, respectively (n = 4-5). In all cases, microsomal Cav-1 content did not change. The close ACh concentration dependence and the concomitance between eNOS subcellular redistribution and NO release support the concept that eNOS translocation from the plasma membrane is part of an activation mechanism that induces NO-dependent vasodilatation in

Curcumin improves episodic memory in cadmium induced memory impairment through inhibition of acetylcholinesterase and adenosine deaminase activities in a rat model.

PubMed

Akinyemi, Ayodele Jacob; Okonkwo, Princess Kamsy; Faboya, Opeyemi Ayodeji; Onikanni, Sunday Amos; Fadaka, Adewale; Olayide, Israel; Akinyemi, Elizabeth Olufisayo; Oboh, Ganiyu

2017-02-01

Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes has been reported to exert cognitive enhancing potential with limited scientific basis. Hence, this study sought to evaluate the effect of curcumin on cerebral cortex acetylcholinesterase (AChE) and adenosine deaminase (ADA) activities in cadmium (Cd)-induced memory impairment in rats. Animals were divided into six groups (n = 6): saline/vehicle, saline/curcumin 12.5 mg/kg, saline/curcumin 25 mg/kg, Cd/vehicle, Cd/curcumin 12.5 mg/kg, and Cd/curcumin 25 mg/kg. Rats received Cd (2.5 mg/kg) and curcumin (12.5 and 25 mg/kg, respectively) by gavage for 7 days. The results of this study revealed that cerebral cortex AChE and ADA activities were increased in Cd-poisoned rats, and curcumin co-treatment reversed these activities to the control levels. Furthermore, Cd intoxication increased the level of lipid peroxidation in cerebral cortex with a concomitant decreased in functional sulfuhydryl (-SH) group and nitric oxide (NO), a potent neurotransmitter and neuromodulatory agent. However, the co-treatment with curcumin at 12.5 and 25 mg/kg, respectively increased the non-enzymatic antioxidant status and NO in cerebral cortex with a decreased in malondialdehyde (MDA) level. Therefore, inhibition of AChE and ADA activities as well as increased antioxidant status by curcumin in Cd-induced memory dysfunction could suggest some possible mechanism of action for their cognitive enhancing properties.

Effect of diet on carboxylesterase activity of tadpoles (Rhinella arenarum) exposed to chlorpyrifos.

PubMed

Attademo, A M; Sanchez-Hernandez, J C; Lajmanovich, R C; Peltzer, P M; Junges, C

2017-01-01

An outdoor microcosm was performed with tadpoles (Rhinella arenarum) exposed to 125μgL -1 chlorpyrifos and fed two types of food, i.e., lettuce (Lactuca sativa) and a formulated commercial pellet. Acetylcholinesterase (AChE) and carboxylesterase (CbE) activities were measured in liver and intestine after 10 days of pesticide exposure. Non-exposed tadpoles fed lettuce had an intestinal AChE activity almost two-fold higher than that of pellet-fed tadpoles. No significant differences were observed, however, in liver AChE activity between diets. Likewise, intestinal CbE activity - measured using two substrates, i.e. 1-naphthyl acetate (1-NA) and 4-nitrophenyl valerate (4-NPV) - was higher in tadpoles fed lettuce than in those fed pellets. However, the diet-dependent response of liver CbE activity was opposite to that in the intestine. Chlorpyrifos caused a significant inhibition of both esterase activities, which was tissue- and diet-specific. The highest inhibition degree was found in the intestinal AChE and CbE activities of lettuce-fed tadpoles (42-78% of controls) compared with pellet-fed tadpoles (<60%). Although chlorpyrifos significantly inhibited liver CbE activity of the group fed lettuce, this effect was not observed in the group fed pellets. In general, intestinal CbE activity was more sensitive to chlorpyrifos inhibition than AChE activity. This finding, together with the high levels of basal CbE activity found in the intestine, may be understood as a detoxification system able to reduce intestinal OP uptake. Moreover, the results of this study suggest that diet is a determinant factor in toxicity testing with tadpoles to assess OP toxicity, because it modulates levels of this potential detoxifying enzyme activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Activation and inhibition of mouse muscle and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

PubMed

Papke, Roger L; Wecker, Lynn; Stitzel, Jerry A

2010-05-01

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric alpha7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-beta-erythroidine as selective antagonists in mouse models of alpha3beta4 and alpha4beta2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal alpha and beta subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse alpha5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse alpha4beta2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity.

Activation and Inhibition of Mouse Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

PubMed Central

Wecker, Lynn; Stitzel, Jerry A.

2010-01-01

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric α7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-β-erythroidine as selective antagonists in mouse models of α3β4 and α4β2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal α and β subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse α5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse α4β2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity. PMID:20100906

The acetylcholinesterase inhibitor BW284c51 is a potent blocker of Torpedo nicotinic AchRs incorporated into the Xenopus oocyte membrane

PubMed Central

Olivera-Bravo, Silvia; Ivorra, Isabel; Morales, Andrés

2004-01-01

This work was aimed to determine if 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51), the most selective acetylcholinesterase inhibitor (AchEI), affects the nicotinic acetylcholine (Ach) receptor (AchR) function. Purified Torpedo nicotinic AchRs were injected into Xenopus laevis oocytes and BW284c51 effects on Ach- and carbamylcholine (Cch)-elicited currents were assessed using the voltage-clamp technique. BW284c51 (up to 1 mM) did not evoke any change in the oocyte membrane conductance. When BW284c51 (10 pM–100 μM) and Ach were coapplied, Ach-evoked currents (IAch) were reversibly inhibited in a concentration-dependent manner (Hill coefficient, 1; IC50, 0.2–0.5 μM for 0.1–1000 μM Ach). Cch-elicited currents showed a similar inhibition by BW284c51. IAch blockade by BW284c51 showed a strong voltage dependence, being only apparent at hyperpolarising potentials. BW284c51 also enhanced IAch desensitisation. BW284c51 changed the Ach concentration-dependence curve of Torpedo AchR response from two-site to single-site kinetics, without noticeably affecting the EC50 value. The BW284c51 blocking effect was highly selective for nicotinic over muscarinic receptors. BW284c51 inhibition potency was stronger than that of tacrine, and similar to that of d-tubocurarine (d-TC). Coapplication of BW284c51 with either tacrine or d-TC revealed synergistic inhibitory effects. Our results indicate that BW284c51 antagonises nicotinic AchRs in a noncompetitive way by blocking the receptor channel, and possibly by other, yet unknown, mechanisms. Therefore, besides acting as a selective AchEI, BW284c51 constitutes a powerful and reversible blocker of nicotinic AchRs that might be used as a valuable tool for understanding their function. PMID:15644872

A mid-infrared flow-through sensor for label-free monitoring of enzyme inhibition.

PubMed

Armenta, S; Tomischko, W; Lendl, B

2008-12-01

Label-free monitoring of acetylcholinesterase (AChE) activity was achieved with a mid-infrared flow-through sensor. The flow-through sensor comprised agarose beads, carrying covalently immobilized AChE, which were placed in a temperature-controlled (37 degrees C) CaF(2) flow cell with an optical path of 60 mum. The sensor was incorporated into a computer-controlled sequential injection (SI) system for automated liquid handling. Different mixtures of enzyme substrate acetylcholine (ACh) and inhibitor (tacrine) were prepared and fed into the flow-through sensor. The flow was stopped as soon as the prepared mixtures reached the sensor. Enzymatic hydrolysis of ACh by AChE was directly monitored as it took place in the flow-through sensor. The inhibition effect of tacrine was calculated from the reaction-induced spectral changes, revealing an important decrease in the activity of AChE, approaching zero when the inhibitor concentration is high enough. The developed mid-infrared flow-through sensor is flexible and can be used to study the inhibitor activity of different target molecules as well as different enzymes.

Acetylcholinesterase Activity and Neurodevelopment in Boys and Girls

PubMed Central

Himes, John H.; Jacobs, David R.; Alexander, Bruce H.; Gunnar, Megan R.

2013-01-01

BACKGROUND: Organophosphate exposures can affect children’s neurodevelopment, possibly due to neurotoxicity induced by acetylcholinesterase (AChE) inhibition, and may affect boys more than girls. We tested the hypothesis that lower AChE activity is associated with lower neurobehavioral development among children living in Ecuadorian floricultural communities. METHODS: In 2008, we examined 307 children (age: 4–9 years; 52% male) and quantified AChE activity and neurodevelopment in 5 domains: attention/executive functioning, language, memory/learning, visuospatial processing, and sensorimotor (NEPSY-II test). Associations were adjusted for demographic and socioeconomic characteristics and height-for-age, flower worker cohabitation, and hemoglobin concentration. RESULTS: Mean ± standard deviation AChE activity was 3.14 ± 0.49 U/mL (similar for both genders). The range of scores among neurodevelopment subtests was 5.9 to 10.7 U (standard deviation: 2.6–4.9 U). Girls had a greater mean attention/executive functioning domain score than boys. In boys only, there were increased odds ratios of low (<9th percentile) neurodevelopment among those in the lowest tertile versus the highest tertile of AChE activity (odds ratios: total neurodevelopment: 5.14 [95% confidence interval (CI): 0.84 to 31.48]; attention/executive functioning domain: 4.55 [95% CI: 1.19 to 17.38], memory/learning domain: 6.03 [95% CI: 1.17 to 31.05]) after adjustment for socioeconomic and demographic factors, height-for-age, and hemoglobin. Within these domains, attention, inhibition and long-term memory subtests were most affected. CONCLUSIONS: Low AChE activity was associated with deficits in neurodevelopment, particularly in attention, inhibition, and memory in boys but not in girls. These critical cognitive skills affect learning and academic performance. Added precautions regarding secondary occupational pesticide exposure would be prudent. PMID:24249815

A positive allosteric modulator of α7 nAChRs augments neuroprotective effects of endogenous nicotinic agonists in cerebral ischaemia

PubMed Central

Kalappa, Bopanna I; Sun, Fen; Johnson, Stephen R; Jin, Kunlin; Uteshev, Victor V

2013-01-01

Background and Purpose Activation of α7 nicotinic acetylcholine receptors (nAChRs) can be neuroprotective. However, endogenous choline and ACh have not been regarded as potent neuroprotective agents because physiological levels of choline/ACh do not produce neuroprotective levels of α7 activation. This limitation may be overcome by the use of type-II positive allosteric modulators (PAMs-II) of α7 nAChRs, such as 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea (PNU-120596). This proof-of-concept study presents a novel neuroprotective paradigm that converts endogenous choline/ACh into potent neuroprotective agents in cerebral ischaemia by inhibiting α7 nAChR desensitization using PNU-120596. Experimental Approach An electrophysiological ex vivo cell injury assay (to quantify the susceptibility of hippocampal neurons to acute injury by complete oxygen and glucose deprivation; COGD) and an in vivo middle cerebral artery occlusion model of ischaemia were used in rats. Key Results Choline (20–200 μM) in the presence, but not absence of 1 μM PNU-120596 significantly delayed anoxic depolarization/injury of hippocampal CA1 pyramidal neurons, but not CA1 stratum radiatum interneurons, subjected to COGD in acute hippocampal slices and these effects were blocked by 20 nM methyllycaconitine, a selective α7 antagonist, thus, activation of α7 nAChRs was required. PNU-120596 alone was ineffective ex vivo. In in vivo experiments, both pre- and post-ischaemia treatments with PNU-120596 (30 mg·kg−1, s.c. and 1 mg·kg−1, i.v., respectively) significantly reduced the cortical/subcortical infarct volume caused by transient focal cerebral ischaemia. PNU-120596 (1 mg·kg−1, i.v., 30 min post-ischaemia) remained neuroprotective in rats subjected to a choline-deficient diet for 14 days prior to experiments. Conclusions and Implications PNU-120596 and possibly other PAMs-II significantly improved neuronal survival in cerebral ischaemia by augmenting

Impacts of oxidative stress on acetylcholinesterase transcription, and activity in embryos of zebrafish (Danio rerio) following Chlorpyrifos exposure.

PubMed

Rodríguez-Fuentes, Gabriela; Rubio-Escalante, Fernando J; Noreña-Barroso, Elsa; Escalante-Herrera, Karla S; Schlenk, Daniel

2015-01-01

Organophosphate pesticides cause irreversible inhibition of AChE which leads to neuronal overstimulation and death. Thus, dogma indicates that the target of OP pesticides is AChE, but many authors postulate that these compounds also disturb cellular redox processes, and change the activities of antioxidant enzymes. Interestingly, it has also been reported that oxidative stress plays also a role in the regulation and activity of AChE. The aims of this study were to determine the effects of the antioxidant, vitamin C (VC), the oxidant, t-butyl hydroperoxide (tBOOH) and the organophosphate Chlorpyrifos (CPF), on AChE gene transcription and activity in zebrafish embryos after 72h exposure. In addition, oxidative stress was evaluated by measuring antioxidant enzymes activities and transcription, and quantification of total glutathione. Apical effects on the development of zebrafish embryos were also measured. With the exception of AChE inhibition and enhanced gene expression, limited effects of CPF on oxidative stress and apical endpoints were found at this developmental stage. Addition of VC had little effect on oxidative stress or AChE, but increased pericardial area and heartbeat rate through an unknown mechanism. TBOOH diminished AChE gene expression and activity, and caused oxidative stress when administered alone. However, in combination with CPF, only reductions in AChE activity were observed with no significant changes in oxidative stress suggesting the adverse apical endpoints in the embryos may have been due to AChE inhibition by CPF rather than oxidative stress. These results give additional evidence to support the role of prooxidants in AChE activity and expression. Copyright © 2015 Elsevier Inc. All rights reserved.

Arisugacins A and B, novel and selective acetylcholinesterase inhibitors from Penicillium sp. FO-4259. I. Screening, taxonomy, fermentation, isolation and biological activity.

PubMed

Kuno, F; Otoguro, K; Shiomi, K; Iwai, Y; Omura, S

1996-08-01

An in vitro screening method for selective acetylcholinesterase (AChE) inhibitors was established. Inhibitory activity of AChE and butyrylcholinesterase (BuChE) was measured and the culture broths of microorganisms that showed selective inhibition against AChE were characterized. By using this method, a strain producing the novel and selective inhibitors of AChE, arisugacins A and B, was picked out among over seven thousand microorganisms tested. Arisugacins were obtained as white powders from the culture broth together with three known compounds, territrems B and C and cyclopenin that also showed selective inhibition against AChE. Arisugacins and territrems are members of the meroterpenoid compounds. They showed potent inhibitory activities against AChE with IC50 values in range of 1.0 approximately 25.8 nM. Furthermore, they showed greater than 2,000-fold more potent inhibition against AChE than BuChE.

Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward

PubMed Central

Henderson, Brandon J.; Wall, Teagan R.; Henley, Beverley M.; Kim, Charlene H.; Nichols, Weston A.; Moaddel, Ruin; Xiao, Cheng

2016-01-01

Upregulation of β2 subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate β2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of α4* nAChRs, complementing that of chronic nicotine alone, which upregulates α4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (α4)3(β2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(β2)3 nAChRs. Menthol alone also increases the number of α6β2 receptors that exclude the β3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway. SIGNIFICANCE STATEMENT Menthol, the most popular flavorant for tobacco products, has been considered simply a benign flavor additive. However, as we show here

Avoidance behaviour response and esterase inhibition in the earthworm, Lumbricus terrestris, after exposure to chlorpyrifos.

PubMed

Martínez Morcillo, S; Yela, J L; Capowiez, Y; Mazzia, C; Rault, M; Sanchez-Hernandez, Juan C

2013-05-01

The avoidance response of earthworms to polluted soils has been standardised using a simple and low-cost test, which facilitates soil toxicity screening. In this study, the avoidance response of Lumbricus terrestris was quantified in chlorpyrifos-spiked soils, depending on the pesticide concentration and exposure duration. The inhibition of acetylcholinesterase (AChE) and carboxylesterase (CbE) activities was also determined as indirect measures of pesticide bioavailability. The effects of different chlorpyrifos concentrations were examined in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyrifos. A modification of the test involved a pre-exposure step (24, 48 or 72 h) in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to avoid the contaminated soils. However, the esterase activities showed that all earthworms were exposed to chlorpyrifos. Acetylcholinesterase activity did not change in earthworms in the standardised behavioural test (0.58 ± 0.20 U/mg protein, mean ± SD; n = 72), whereas the CbE activity was significantly inhibited (62-87 % inhibition) in earthworms exposed to 3 and 15 mg/kg. In the modified test, earthworms had greatly inhibited AChE activity (0.088 ± 0.034 U/mg protein, n = 72), which was supported by reactivation of the inhibited enzyme activity in the presence of pralidoxime (2-PAM). Similarly, the CbE activity was significantly inhibited in earthworms with all treatments. This study suggests that the avoidance behaviour test for organophosphorus-contaminated soils could be supported by specific biomarkers to facilitate a better understanding of pesticide exposure and toxicity during this test.

AT–1001: a high-affinity α3β4 nAChR ligand with novel nicotine-suppressive pharmacology

PubMed Central

Cippitelli, Andrea; Wu, Jinhua; Gaiolini, Kelly A; Mercatelli, Daniela; Schoch, Jennifer; Gorman, Michelle; Ramirez, Alejandra; Ciccocioppo, Roberto; Khroyan, Taline V; Yasuda, Dennis; Zaveri, Nurulain T; Pascual, Conrado; Xie, Xinmin (Simon); Toll, Lawrence

2015-01-01

Background and Purpose The α3β4 subtype of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediating nicotine reinforcement processes. AT-1001 has been recently described as a high-affinity and selective α3β4 nAChR antagonist that blocks nicotine self-administration in rats. The aim of this study was to investigate the mechanism of action underlying the nicotine-suppressive effects of AT-1001. Experimental Approach Effects of AT-1001 were determined using in vitro assays and rat models of nicotine addiction, and compared with varenicline. Key Results AT-1001 and its analogue AT-1012 were functionally selective as antagonists for α3β4 over α4β2 nAChRs, but not to the same extent as the binding selectivity, and had partial agonist activity at α3β4 nAChRs. In contrast, varenicline was a partial agonist at α4β2, a weak agonist at α3β4 and inhibited α4β2 at a much lower concentration than it inhibited α3β4 nAChRs. AT-1001 and varenicline also had very different in vivo properties. Firstly, AT-1001 did not exhibit reinforcing properties per se while varenicline was self-administered. Secondly, systemic treatment with AT-1001 did not induce reinstatement of nicotine seeking but rather attenuated reinstatement induced by varenicline, as well as nicotine. Finally, unlike varenicline, AT-1001 selectively blocked nicotine self-administration without altering alcohol lever pressing as assessed in an operant co-administration paradigm. Conclusions and Implications These findings describe a more complex AT-1001 in vitro profile than previously appreciated and provide further support for the potential of AT-1001 and congeners as clinically useful compounds for smoking cessation, with a mechanism of action distinct from currently available medications. PMID:25440006

Comparison of oxime reactivation and aging of nerve agent-inhibited monkey and human acetylcholinesterases.

PubMed

Luo, Chunyuan; Tong, Min; Maxwell, Donald M; Saxena, Ashima

2008-09-25

Non-human primates are valuable animal models that are used for the evaluation of nerve agent toxicity as well as antidotes and results from animal experiments are extrapolated to humans. It has been demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited acetylcholinesterase (AChE). If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the aging and reactivation of human and different monkey (Rhesus, Cynomolgus, and African Green) AChEs inhibited by GF, GD, and VR. The oximes examined include the traditional oxime 2-PAM, two H-oximes HI-6 and HLo-7, and the new candidate oxime MMB4. Results indicate that oxime reactivation of all three monkey AChEs was very similar to human AChE. The maximum difference in the second-order reactivation rate constant between human and three monkey AChEs or between AChEs from different monkey species was 5-fold. Aging rate constants of GF-, GD-, and VR-inhibited monkey AChEs were very similar to human AChE except for GF-inhibited monkey AChEs, which aged 2-3 times faster than the human enzyme. The results of this study suggest that all three monkey species are suitable animal models for nerve agent antidote evaluation since monkey AChEs possess similar biochemical/pharmacological properties to human AChE.

ACh-induced hyperpolarization and decreased resistance in mammalian type II vestibular hair cells.

PubMed

Poppi, Lauren A; Tabatabaee, Hessam; Drury, Hannah R; Jobling, Phillip; Callister, Robert J; Migliaccio, Americo A; Jordan, Paivi M; Holt, Joseph C; Rabbitt, Richard D; Lim, Rebecca; Brichta, Alan M

2018-01-01

In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9 -/- ) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9 -/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted

Effects of structural modifications on the metal binding, anti-amyloid activity, and cholinesterase inhibitory activity of chalcones.

PubMed

Fosso, Marina Y; LeVine, Harry; Green, Keith D; Tsodikov, Oleg V; Garneau-Tsodikova, Sylvie

2015-09-28

As the number of individuals affected with Alzheimer's disease (AD) increases and the availability of drugs for AD treatment remains limited, the need to develop effective therapeutics for AD becomes more and more pressing. Strategies currently pursued include inhibiting acetylcholinesterase (AChE) and targeting amyloid-β (Aβ) peptides and metal-Aβ complexes. This work presents the design, synthesis, and biochemical evaluation of a series of chalcones, and assesses the relationship between their structures and their ability to bind metal ions and/or Aβ species, and inhibit AChE/BChE activity. Several chalcones were found to exhibit potent disaggregation of pre-formed N-biotinyl Aβ1-42 (bioAβ42) aggregates in vitro in the absence and presence of Cu(2+)/Zn(2+), while others were effective at inhibiting the action of AChE.

Acute toxicity of organophosphorus compounds in guinea pigs is sex- and age-dependent and cannot be solely accounted for by acetylcholinesterase inhibition.

PubMed

Fawcett, William P; Aracava, Yasco; Adler, Michael; Pereira, Edna F R; Albuquerque, Edson X

2009-02-01

This study was designed to test the hypothesis that the acute toxicity of the nerve agents S-[2-(diisopropylamino)ethyl]-O-ethyl methylphosphonothioate (VX), O-pinacolyl methylphosphonofluoridate (soman), and O-isopropyl methylphosphonofluoridate (sarin) in guinea pigs is age- and sex-dependent and cannot be fully accounted for by the irreversible inhibition of acetylcholinesterase (AChE). The subcutaneous doses of nerve agents needed to decrease 24-h survival of guinea pigs by 50% (LD(50) values) were estimated by probit analysis. In all animal groups, the rank order of LD(50) values was sarin > soman > VX. The LD(50) value of soman was not influenced by sex or age of the animals. In contrast, the LD(50) values of VX and sarin were lower in adult male than in age-matched female or younger guinea pigs. A colorimetric assay was used to determine the concentrations of nerve agents that inhibit in vitro 50% of AChE activity (IC(50) values) in guinea pig brain extracts, plasma, red blood cells, and whole blood. A positive correlation between LD(50) values and IC(50) values for AChE inhibition would support the hypothesis that AChE inhibition is a major determinant of the acute toxicity of the nerve agents. However, such a positive correlation was found only between LD(50) values and IC(50) values for AChE inhibition in brain extracts from neonatal and prepubertal guinea pigs. These results demonstrate for the first time that the lethal potencies of some nerve agents in guinea pigs are age- and sex-dependent. They also support the contention that mechanisms other than AChE inhibition contribute to the lethality of nerve agents.

Inhibition of acetylcholinesterase activity in the central nervous system of the red swamp crayfish, Procambarus clarkii, by mercury, cadmium, and lead

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devi, M.; Fingerman, M.

The toxicological, physiological and biochemical responses of aquatic crustaceans to heavy metals have been reported by several investigators. Levels of glucose, lactic acid, sodium, potassium, aspartate aminotransferase and alanine aminotransferase in the blood of the crab Scylla serrata increased, while glycogen levels in hepatopancreas and muscle decreased after a four-week exposure to mercuric chloride. In fiddler crab, Uca pugilator, enzyme activity was observed to decrease in the hepatopancreas but increased in abdominal muscle after 48 hr cadmium exposure. In the red swamp crayfish, Procambarus clarkii, exposed for 96 hr to cadmium, glutahione (GSH) level and GSH S-transferase activity deceased inmore » the midgut. In crayfish Astacus astacus exposed to sublethal concentrations of lead and cadmium, oxidative enzyme (succine dehydrogenase and NADPH-cytochrome P450 reductase) activities in gills and hepatopancrease decreased. Acetylcholinesterase (AChE) inhibition by organophosphates and organocarbamates in various crustaceans has bee reported. In vivo cadmium exposure caused increases in esterase activities, but mercury exposure decreases these activities in the hepatopancreas of the shrimp Callianassa tyrrhena. The freshwater crab, Barytelphusa guerini, exposed to 0.6 ppm cadmium showed reduced oxygen consumption throughout the experiment whereas AChE activity increased after 4 days but decreased after 15 days. The authors wanted to determine the effects of cadmium, lead and mercury on AChE activity in central nervous tissue of Procambarus clarkii. This enzyme has the potential for serving both as a biochemical indicator of toxic stress and a sensitive parameter for testing water for the presence of toxicants. These three biologically silent metals have, according to Schweinsberg and Karsa great toxicological significance to humans because their use is widespread. 14 refs., 4 figs.« less

Insecticidal and acetylcholine esterase inhibition activity of Apiaceae plant essential oils and their constituents against adults of German cockroach (Blattella germanica).

PubMed

Yeom, Hwa-Jeong; Kang, Jae Soon; Kim, Gil-Hah; Park, Il-Kwon

2012-07-25

We evaluated the insecticidal and acetylcholine esterase (AChE) inhibition activity of 11 Apiaceae plant essential oils and their constituents in adult male and female Blattella germanica. Of the 11 Apiaceae plant essential oils tested, dill (Anethum graveolens), carvi (Carum carvi), and cumin (Cuminum cyminum) demonstrated >90% fumigant toxicity against adult male German cockroaches at a concentration of 5 mg/filter paper. In a contact toxicity test, dill (Anethum graveolens), carvi (Carum carvi), cumin (Cuminum cyminum), and ajowan (Trachyspermum ammi) produced strong insecticidal activity against adult male and female German cockroaches. Among the test compounds, (S)-(+)-carvone, 1,8-cineole, trans-dihydrocarvone, cuminaldehyde, trans-anethole, p-cymene, and γ-terpinene demonstrated strong fumigant toxicity against adult male and female B. germanica. In a contact toxicity test, carveol, cuminaldehyde, (S)-(+)-carvone, trans-anethole, thymol, and p-cymene showed strong contact toxicity against adult male and female B. germanica. IC(50) values of α-pinene, carvacrol, and dihydrocarvone against female AChE were 0.28, 0.17, and 0.78 mg/mL, respectively. The toxicity of the blends of constituents identified in 4 active oils indicated that carvone, cuminaldehyde, and thymol were major contributors to the fumigant activity or contact toxicity of the artificial blend.

Syntheses, cholinesterases inhibition, and molecular docking studies of pyrido[2,3-b]pyrazine derivatives.

PubMed

Hameed, Abdul; Zehra, Syeda T; Shah, Syed J A; Khan, Khalid M; Alharthy, Rima D; Furtmann, Norbert; Bajorath, Jürgen; Tahir, Muhammad N; Iqbal, Jamshed

2015-11-01

Cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), have a role in cholinergic deficit which evidently leads to Alzheimer's disease (AD). Inhibition of cholinesterases with small molecules is an attractive strategy in AD therapy. This study demonstrates synthesis of pyrido[2,3-b]pyrazines (6a-6q) series, their inhibitory activities against both cholinesterases, AChE and BChE, and molecular docking studies. The bioactivities data of pyrido[2,3-b]pyrazines showed 3-(3'-nitrophenyl)pyrido[2,3-b]pyrazine 6n a potent dual inhibitor among the series against both AChE and BChE with IC50 values of 0.466 ± 0.121 and 1.89 ± 0.05 μm, respectively. The analogues 3-(3'-methylphenyl)pyrido[2,3-b]pyrazine 6c and 3-(3'-fluorophenyl)pyrido[2,3-b]pyrazine 6f were found to be selective inhibition for BChE with IC50 values of 0.583 ± 0.052 μm and AChE with IC50 value of 0.899 ± 0.10 μm, respectively. Molecular docking studies of the active compounds suggested the putative binding modes with cholinesterases. The potent compounds among the series could potentially serves as good leads for the development of new cholinesterase inhibitors. © 2015 John Wiley & Sons A/S.

Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release

PubMed Central

Acevedo-Rodriguez, Alexandra; Zhang, Lifen; Zhou, Fuwen; Gong, Suzhen; Gu, Howard; De Biasi, Mariella; Zhou, Fu-Ming; 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 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. PMID:25237305

Diverse inhibitory actions of quaternary ammonium cholinesterase inhibitors on Torpedo nicotinic ACh receptors transplanted to Xenopus oocytes

PubMed Central

Olivera-Bravo, Silvia; Ivorra, Isabel; Morales, Andrés

2007-01-01

Background and purpose: This work was aimed at comparing and analysing the effects and mechanisms of action of the quaternary ammonium cholinesterase inhibitors (QChEIs) BW284c51, decamethonium and edrophonium, on nicotinic ACh receptor (nAChR) function. Experimental approach: nAChRs purified from Torpedo electroplax were transplanted to oocytes and currents elicited by ACh (IACh) either alone or in presence of these QChEIs were recorded. Key results: None of the QChEIs, by itself, elicited changes in membrane conductance; however, when co-applied with ACh, all of them decreased IACh in a concentration-dependent way. The mechanisms of nAChR inhibition were different for these QChEIs. BW284c51 blockade was non-competitive and voltage-dependent, although it also affected the nH of the dose-response curve. By contrast, decamethonium and edrophonium inhibition, at –60 mV, was apparently competitive and did not modify either desensitisation or nH. Decamethonium effects were voltage-independent and washed out slowly after its removal; by contrast, edrophonium blockade had strong voltage dependence and its effects disappeared quickly after its withdrawal. Analysis of the voltage-dependent blockade indicated that BW284c51 bound to a shallow site into the channel pore, whereas edrophonium bound to a deeper locus. Accordingly, additive inhibitory effects on IACh were found among any pairs of these QChEIs. Conclusions and implications: The tested QChEIs bound to the nAChR at several and different loci, which might account for their complex inhibitory behaviour, acting both as allosteric effectors and, in the case of BW284c51 and edrophonium, as open channel blockers. PMID:17572698

Anticholinesterase and Antityrosinase Activities of Ten Piper Species from Malaysia

PubMed Central

Salleh, Wan Mohd Nuzul Hakimi Wan; Hashim, Nur Athirah; Ahmad, Farediah; Heng Yen, Khong

2014-01-01

Purpose: The aim of this study was to investigate acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and antityrosinase activities of extracts from ten Piper species namely; P. caninum, P. lanatum, P. abbreviatum, P. aborescens, P. porphyrophyllum, P. erecticaule, P. ribesioides, P. miniatum, P. stylosum, and P. majusculum. Methods: Anticholinesterase and antityrosinase activities were evaluated against in vitro Ellman spectroscopy method and mushroom tyrosinase, respectively. Results: The EtOAc extract of P. erecticaule showed the highest AChE and BChE inhibitory with 22.9% and 70.9% inhibition, respectively. In antityrosinase activity, all extracts of P. porphyrophyllum showed the highest inhibitory effects against mushroom tyrosinase, compared to standard, kojic acid. Conclusion: This study showed that P. erecticaule and P. porphyrophyllum have potential AChE/BChE and tyrosinase inhibition activities. The respective extracts can be explored further for the development of novel lead as AChE/BChE and tyrosinase inhibitors in therapeutic management of Alzheimer’s disease. PMID:25671185

Gymnopilins, a product of a hallucinogenic mushroom, inhibit the nicotinic acetylcholine receptor.

PubMed

Kayano, Tomohiko; Kitamura, Naoki; Miyazaki, Shunsuke; Ichiyanagi, Tsuyoshi; Shimomura, Norihiro; Shibuya, Izumi; Aimi, Tadanori

2014-04-01

Gymnopilins are substances produced in fruiting bodies of the hallucinogenic mushroom, Gymnopilus junonius. Although, only a few biological effects of gymnopilins on animal tissues have been reported, it is believed that gymnopilins are a key factor of the G. junonius poisoning. In the present study, we found that gymnopilins inhibited ACh-evoked responses in neuronal cell line, PC12 cell, and determine the underlying mechanism. Gymnopilins were purified from wild fruiting bodies of G. junonius collected in Japan. Ca(2+)-imaging revealed that gymnopilins reduced the amplitude of ACh-evoked [Ca(2+)]i rises by about 50% and abolished the ACh responses remaining in the presence of atropine. Gymnopilins greatly reduced the amplitude of [Ca(2+)]i rises evoked by nicotinic ACh receptor agonists, 1,1-Dimethyl-4-phenylpiperazinium iodide (DMPP) and nicotine. In the whole-cell voltage clamp recording, gymnopilins inhibited the DMPP-evoked currents, but did not affect the voltage-gated Ca(2+) channel currents. These results indicate that gymnopilins directly act on nicotinic ACh receptors and inhibit their activity. This biological action of gymnopilins may be one of the causes of the G. junonius poisoning. Copyright © 2014 Elsevier Ltd. All rights reserved.

Design, synthesis and preliminary structure-activity relationship investigation of nitrogen-containing chalcone derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors: a further study based on Flavokawain B Mannich base derivatives.

PubMed

Liu, Haoran; Fan, Haoqun; Gao, Xiaohui; Huang, Xueqing; Liu, Xianjun; Liu, Linbo; Zhou, Chao; Tang, Jingjing; Wang, Qiuan; Liu, Wukun

2016-08-01

In order to study the structure-activity relationship of Flavokawain B Mannich-based derivatives as acetylcholinesterase (AChE) inhibitors in our recent investigation, 20 new nitrogen-containing chalcone derivatives (4 a-8d) were designed, synthesized, and evaluated for AChE inhibitory activity in vitro. The results suggested that amino alkyl side chain of chalcone dramatically influenced the inhibitory activity against AChE. Among them, compound 6c revealed the strongest AChE inhibitory activity (IC50 value: 0.85 μmol/L) and the highest selectivity against AChE over BuChE (ratio: 35.79). Enzyme kinetic study showed that the inhibition mechanism of compound 6c against AChE was a mixed-type inhibition. The molecular docking assay showed that this compound can both bind with the catalytic site and the peripheral site of AChE.

The activity of detoxifying enzymes in the infective juveniles of Heterorhabditis bacteriophora strains: Purification and characterization of two acetylcholinesterases.

PubMed

Mohamed, Magda A; Mahdy, El-Sayed M E; Ghazy, Abd-El-Hady M; Ibrahim, Nihal M; El-Mezayen, Hatem A; Ghanem, Manal M E

2016-02-01

The infectivity and detoxifying enzyme activities including glutathione-S-transferase (GST), acetylcholinesterase (AChE) and carboxylesterase (CaE) are investigated in the infective juveniles (IJs) of six different strains of Heterorhabditis bacteriophora as a biocontrol agent against insect pests. The specific activities ranged from 10.8-29.8 and 50-220units/mg protein for GST and AChE, respectively; and from 24.7-129 and 22.6-77.3units/mg protein for CaE as estimated by P-nitrophenyl and α-naphthyl acetates, respectively. H. bacteriophora EM2 strain has the highest infectivity and the highest enzymatic activities as well. AChE is the predominant detoxifying enzyme that might imply its major role in the detoxification of insecticide(s). The isoenzyme pattern demonstrated two major slow-moving isoforms in all EPN strains examined. Purification of two AChE isoforms, AChEAII and AChEBI, from H. bacteriophora EM2 strain is performed by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and chromatography on DEAE-Sepharose. AChEAII and AChEBII have specific activities of 1207 and 1560unit/mg protein, native molecular weights of 180 and 68kDa, and are found in dimeric and monomeric forms, respectively. Both isoforms showed optimum activity at pH8.5 and 35°C. AChEBI exhibited higher thermal stability and higher activation energy than AChEAII. The enzymatic activities of purified AChEs are completely inhibited by Hg(+2) and Ni(+2) and greatly enhanced by Mn(+2). The substrate specificity, the relative efficiency of substrates hydrolysis, substrate inhibition and inhibition by BW284C51, but not by iso-OMPA, clearly indicated that they are true AChEs; their properties are compared with those recorded for insects as target hosts for H. bacteriophora EM2. Copyright © 2015 Elsevier Inc. All rights reserved.

Tricyclic antidepressants inhibit hippocampal α7* and α9α10 nicotinic acetylcholine receptors by different mechanisms.

PubMed

Arias, Hugo R; Vázquez-Gómez, Elizabeth; Hernández-Abrego, Andy; Gallino, Sofía; Feuerbach, Dominik; Ortells, Marcelo O; Elgoyhen, Ana Belén; García-Colunga, Jesús

2018-07-01

The activity of tricyclic antidepressants (TCAs) at α7 and α9α10 nicotinic acetylcholine receptors (AChRs) as well as at hippocampal α7-containing (i.e., α7*) AChRs is determined by using Ca 2+ influx and electrophysiological recordings. To determine the inhibitory mechanisms, additional functional tests and molecular docking experiments are performed. The results established that TCAs (a) inhibit Ca 2+ influx in GH3-α7 cells with the following potency (IC 50 in μM) rank: amitriptyline (2.7 ± 0.3) > doxepin (5.9 ± 1.1) ∼ imipramine (6.6 ± 1.0). Interestingly, imipramine inhibits hippocampal α7* AChRs (42.2 ± 8.5 μM) in a noncompetitive and voltage-dependent manner, whereas it inhibits α9α10 AChRs (0.53 ± 0.05 μM) in a competitive and voltage-independent manner, and (b) inhibit [ 3 H]imipramine binding to resting α7 AChRs with the following affinity rank (IC 50 in μM): imipramine (1.6 ± 0.2) > amitriptyline (2.4 ± 0.3) > doxepin (4.9 ± 0.6), whereas imipramine's affinity was no significantly different to that for the desensitized state. The molecular docking and functional results support the notion that imipramine noncompetitively inhibits α7 AChRs by interacting with two overlapping luminal sites, whereas it competitively inhibits α9α10 AChRs by interacting with the orthosteric sites. Collectively our data indicate that TCAs inhibit α7, α9α10, and hippocampal α7* AChRs at clinically relevant concentrations and by different mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.

In vitro and ex vivo anticholinesterase activities of Erythrina velutina leaf extracts.

PubMed

Santos, Wanderson Praxedes; da Silva Carvalho, Ana Carla; dos Santos Estevam, Charles; Santana, Antônio Euzébio Goulart; Marçal, Rosilene Moretti

2012-07-01

Erythrina velutina (EV) Willd (Fabaceae-Faboideae) is a medicinal tree that is commonly used in Brazil for the treatment of several central nervous system disorders. The anticholinesterase activity of EV is described in this work. Concentration-response curves (0-1.6 mg/mL) for EV leaf aqueous extract (AE) and alkaloid-rich extracts (AKEs) were performed in vitro. Cholinesterase inhibition was examined in mouse brains, as the cholinesterase source, and in pure acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE). Mice were treated with AE or AKE (100, 200, and 400 mg/kg, p.o.) and their brains were used for the measurement of cholinesterase activity (CA) ex vivo. CA was inhibited by AE (IC(50) = 0.57 [0.43-0.75] mg/mL) and AKE (IC(50) = 0.52 [0.39-0.70] mg/mL) in brain homogenates in a concentration-dependent manner. The ex vivo experiments indicated that AE (400 mg/kg, p < 0.05, 32.2 ± 3.9% of inhibition) and AKE (all doses: p < 0.05-p < 0.001, 29.6 ± 3.2% as the maximum inhibition) significantly inhibited CA in the central nervous system after oral administration. AE and AKE inhibited AChE and BuChE activities in a concentration-dependent manner (AE: IC(50AChE) = 0.56 [0.38-0.81] mg/mL, IC(50BuChE) = 2.95 [1.51-5.76] mg/mL, AKE: IC(50AChE) = 0.87 [0.60-12.5] mg/mL, IC(50BuChE) = 2.67 [0.87-8.11] mg/mL). These data indicated that AE and AKE crossed the blood-brain barrier to inhibit CA in the brain. AE and AKE also exhibited a dual inhibitory action on acetyl- and BuChE.

Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity.

PubMed

Miller, K W; Schamber, R; Osmanagaoglu, O; Ray, B

1998-06-01

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle.

PubMed

Franco, L; Bruzzone, S; Song, P; Guida, L; Zocchi, E; Walseth, T F; Crimi, E; Usai, C; De Flora, A; Brusasco, V

2001-01-01

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.

Assessing joint toxicity of four organophosphate and carbamate insecticides in common carp (Cyprinus carpio) using acetylcholinesterase activity as an endpoint.

PubMed

Wang, Yanhua; Chen, Chen; Zhao, Xueping; Wang, Qiang; Qian, Yongzhong

2015-07-01

Mixtures of organophosphate (OP) and carbamate (CB) pesticides are commonly detected in freshwater ecosystems. These pesticides inhibit the activity of acetylcholinesterase (AChE) and have potential to interfere with behaviors that may be essential for the survival of species. Although the effects of individual anticholinesterase insecticides on aquatic species have been studied for decades, the neurotoxicity of mixtures is still poorly understood. In the present study, brain AChE inhibition in carp (Cyprinus carpio) exposed to a series of concentrations of the organophosphates (malathion and triazophos) as well as the carbamates (fenobucarb and carbosulfan) was measured. In equitoxic mixtures, the observed AChE activity inhibition of the malathion plus triazophos, and triazophos plus carbosulfan mixtures, was synergism. In equivalent concentration mixtures, the combination of malathion plus fenobucarb mixture conformed to synergism, while the observed AChE activity inhibition of the remaining pairings was less than additive. Single pesticide risk assessments are likely to underestimate the impacts of these insecticides on carps in aquatic environment where mixtures occur. Moreover, mixtures of pesticides that have been commonly reported in aquatic ecosystems may pose a more important challenge than previously anticipated. Copyright © 2014 Elsevier Inc. All rights reserved.

Anticholinesterase inhibitory activity of quaternary alkaloids from Tinospora crispa.

PubMed

Yusoff, Mashitah; Hamid, Hazrulrizawati; Houghton, Peter

2014-01-20

Quaternary alkaloids are the major alkaloids isolated from Tinospora species. A previous study pointed to the necessary presence of quaternary nitrogens for strong acetylcholinesterase (AChE) inhibitory activity in such alkaloids. Repeated column chromatography of the vine of Tinospora crispa extract led to the isolation of one new protoberberine alkaloid, 4,13-dihydroxy-2,8,9-trimethoxydibenzo[a,g]quinolizinium (1), along with six known alkaloids-dihydrodiscretamine (2), columbamine (3), magnoflorine (4), N-formylannonaine (5), N-formylnornuciferine (6), and N-trans-feruloyltyramine (7). The seven compounds were isolated and structurally elucidated by spectroscopic analysis. Two known alkaloids, namely, dihydrodiscretamine and columbamine are reported for the first time for this plant. The compounds were tested for AChE inhibitory activity using Ellman's method. In the AChE inhibition assay, only columbamine (3) showed strong activity with IC50 48.1 µM. The structure-activity relationships derived from these results suggest that the quaternary nitrogen in the skeleton has some effect, but that a high degree of methoxylation is more important for acetylcholinesterase inhibition.

Pharmacological and immunochemical characterization of α2* nicotinic acetylcholine receptors (nAChRs) in mouse brain

PubMed Central

Whiteaker, Paul; Wilking, Jennifer A; Brown, Robert WB; Brennan, Robert J; Collins, Allan C; Lindstrom, Jon M; Boulter, Jim

2009-01-01

Aim: α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of α2* nAChRs in these mouse brain regions. Methods: α2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing (α2*) nAChRs in these two regions. Results: [125I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that α2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8±1.7 and 92±26 fmol̇mg-1 protein, respectively). Pharmacological characterization using the β2-selective drug A85380 to inhibit [125I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2* nAChRs. Protocols were established to immunoprecipitate β2 and β4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from β2- and β4-null mutant mice, and efficacy was good (>90% of β2* and >80% of β4* nAChRs were routinely recovered). Conclusion: Immunoprecipitation experiments indicated that interpeduncular nucleus α2* nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits. PMID:19498420

Acetylcholinesterase and neuropathy target esterase activities in 11 cases of symptomatic flight crew members after fume events.

PubMed

Heutelbeck, Astrid R R; Bornemann, Catherine; Lange, Martina; Seeckts, Anke; Müller, Michael M

2016-01-01

In modern aviation, so-called fume events such as exposure to an unknown mixture of chemicals introduced into the aircraft cabin with bleed air drawn off at the engines may occur. Human exposure may result in (neuro)toxic symptoms described as so-called "aerotoxic syndrome." Currently, among other agents organophosphates (OP) are regarded as a likely cause of the observed adverse effects. After fume events 11 flight crew members (9 female/2 male; ages 23-58 yr) were admitted for a medical examination within 5 d post exposure. Individual acetylcholinesterase (AChE) and neuropathy target esterase (NTE) activities were determined. Anamnesis and clinical findings confirmed prominent symptoms of an intoxication, including headache, cognitive difficulties, and neurological disorders, among others. Patient AChE activities ranged from 37 to 50 U/g hemoglobin (reference values: 26.7-50.9 U/g hemoglobin). Ten individuals showed NTE activities ranging from 3.14 to 6.3 nmol phenyl valerate/(min × mg protein) (reference values: 3.01-24), with one patient exhibiting low NTE activity of 1.4. Biochemical effect monitoring was applied to encompass a broad range of AChE-inhibiting compounds such as OP, carbamates, and isocyanates, or to detect inhibition of NTE. The measured AChE activities indicated a subordinate contribution of OP or related compounds to the observed symptoms. All noted NTE activities were clustered at low levels. Our data suggest a likely inhibition of NTE activities in patients after fume events, which warrants further investigation. The observed symptoms may be linked to known chemical compounds in fume events, and it is not possible to infer a direct correlation between manifestations and AChE -inhibiting compounds at this time.

Acetylcholinesterase inhibition and locomotor function after motor-sensory cortex impact injury.

PubMed

Holschneider, Daniel P; Guo, Yumei; Roch, Margareth; Norman, Keith M; Scremin, Oscar U

2011-09-01

Traumatic brain injury (TBI) induces transient or persistent dysfunction of gait and balance. Enhancement of cholinergic transmission has been reported to accelerate recovery of cognitive function after TBI, but the effects of this intervention on locomotor activity remain largely unexplored. The hypothesis that enhancement of cholinergic function by inhibition of acetylcholinesterase (AChE) improves locomotion following TBI was tested in Sprague-Dawley male rats after a unilateral controlled cortical impact (CCI) injury of the motor-sensory cortex. Locomotion was tested by time to fall on the constant speed and accelerating Rotarod, placement errors and time to cross while walking through a horizontal ladder, activity monitoring in the home cages, and rearing behavior. Assessments were performed the 1st and 2nd day and the 1st, 2nd, and 3rd week after TBI. The AChE inhibitor physostigmine hemisulfate (PHY) was administered continuously via osmotic minipumps implanted subcutaneously at the rates of 1.6-12.8 μmol/kg/day. All measures of locomotion were impaired by TBI and recovered to initial levels between 1 and 3 weeks post-TBI, with the exception of the maximum speed achievable on the accelerating Rotarod, as well as rearing in the open field. PHY improved performance in the accelerating Rotarod at 1.6 and 3.2 μmol/kg/day (AChE activity 95 and 78% of control, respectively), however, higher doses induced progressive deterioration. No effect or worsening of outcomes was observed at all PHY doses for home cage activity, rearing, and horizontal ladder walking. Potential benefits of cholinesterase inhibition on locomotor function have to be weighed against the evidence of the narrow range of useful doses.

Muscarinic Long-Term Enhancement of Tonic and Phasic GABAA Inhibition in Rat CA1 Pyramidal Neurons

PubMed Central

Domínguez, Soledad; Fernández de Sevilla, David; Buño, Washington

2016-01-01

Acetylcholine (ACh) regulates network operation in the hippocampus by controlling excitation and inhibition in rat CA1 pyramidal neurons (PCs), the latter through gamma-aminobutyric acid type-A receptors (GABAARs). Although, the enhancing effects of ACh on GABAARs have been reported (Dominguez et al., 2014, 2015), its role in regulating tonic GABAA inhibition has not been explored in depth. Therefore, we aimed at determining the effects of the activation of ACh receptors on responses mediated by synaptic and extrasynaptic GABAARs. Here, we show that under blockade of ionotropic glutamate receptors ACh, acting through muscarinic type 1 receptors, paired with post-synaptic depolarization induced a long-term enhancement of tonic GABAA currents (tGABAA) and puff-evoked GABAA currents (pGABAA). ACh combined with depolarization also potentiated IPSCs (i.e., phasic inhibition) in the same PCs, without signs of interactions of synaptic responses with pGABAA and tGABAA, suggesting the contribution of two different GABAA receptor pools. The long-term enhancement of GABAA currents and IPSCs reduced the excitability of PCs, possibly regulating plasticity and learning in behaving animals. PMID:27833531

M1/M2 muscarinic receptor selectivity using potassium (K/sup +/)-stimulated release of (/sup 3/H)-dopamine (DA) and (/sup 14/C)-acetyl-choline (ACH) in striatum

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeHaven, D.L.; Steranka, L.R.

Raiteri et al have suggested that muscarinic receptor subtypes can be differentiated in striatal synaptosomes by the release of DA (M1) or ACh (M2). The authors attempted to replicate this finding and to characterize responses of selective and non-selective cholinergic agonists and antagonists using K+-stimulated release of transmitters from rat striatal slices. The non-selective agonists ACh, carbachol and oxotremorine stimulated release of (/sup 3/H)-DA and inhibited release of (/sup 14/C)-ACh with EC50 values of 10.6, 9.2 and 4.2 ..mu..M (DA) and 1.2, 0.77 and 0.43 ..mu..M (ACh), respectively. The M1 agonist McN-A-343-11 selectively inhibited release of DA with an EC50more » value of 4.8 ..mu..M. Pilocarpine was ineffective in this system. The M1 antagonist pirenzepine reversed the effects of 10/sup -4/ M carbachol on release with an eight-fold selectivity for release of (/sup 3/H)-DA (IC50 = 0.77 ..mu..M) vs (/sup 14/C)-ACh (IC50 = 6.3 ..mu..M). These results suggest that although this system can determine relative subtype selectivities, the results obtained in this assay do not always correlate with those obtained from phosphatidyl inositol turnover or adenylate cyclase activity.« less

9-Substituted acridine derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors possessing antioxidant activity for Alzheimer's disease treatment.

PubMed

Makhaeva, Galina F; Lushchekina, Sofya V; Boltneva, Natalia P; Serebryakova, Olga G; Rudakova, Elena V; Ustyugov, Alexey A; Bachurin, Sergey O; Shchepochkin, Alexander V; Chupakhin, Oleg N; Charushin, Valery N; Richardson, Rudy J

2017-11-01

We investigated the inhibitory activity of 4 groups of novel acridine derivatives against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) using the methods of enzyme kinetics and molecular docking. Antioxidant activity of the compounds was determined using the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ) radical decolorization assay as their ability to scavenge free radicals. Analysis of the esterase profiles and antiradical activities of the acridine derivatives showed that 9-aryl(heteroaryl)-N-methyl-9,10-dihydroacridines have a high radical-scavenging activity but low potency as AChE and BChE inhibitors, whereas 9-aryl(heteroaryl)-N-methyl-acridinium tetrafluoroborates effectively inhibit cholinesterases but do not exhibit antiradical activity. In contrast, a group of derivatives of 9-heterocyclic amino-N-methyl-9,10-dihydroacridine has been found that combine effective inhibition of AChE and BChE with rather high radical-scavenging activity. The results of molecular docking well explain the observed features in the efficacy, selectivity, and mechanism of cholinesterase inhibition by the acridine derivatives. Thus, in a series of acridine derivatives we have found compounds possessing dual properties of effective and selective cholinesterase inhibition together with free radical scavenging, which makes promising the use of the acridine scaffold to create multifunctional drugs for the therapy of neurodegenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Non-quantal release of acetylcholine in rat atrial myocardium is inhibited by noradrenaline.

PubMed

Borodinova, Anastasia A; Abramochkin, Denis V; Sukhova, Galina S

2013-12-01

In the mammalian myocardium, ACh, which is the main neurotransmitter of cardiac parasympathetic postganglionic fibres, can be released via both quantal (vesicular) and non-quantal (non-vesicular) mechanisms of secretion. Non-quantal release is continuous and independent of vagus activity and exocytosis of ACh-containing vesicles. During the incubation of myocardium in the presence of acetylcholinesterase (AChE) inhibitors, non-quantal ACh release leads to accumulation of ACh in the myocardium and cholinergic effects, which are proportional to the intensity of non-quantal secretion. The aim of the present study was to reveal whether non-quantal release of ACh can be modulated by another major cardioregulator, noradrenaline, or whether it represents uncontrolled leakage of ACh from cholinergic fibres. Cholinergic changes of electrical activity induced by the AChE inhibitor paraoxon (5 × 10(-6) M) in isolated rat right atrial preparations were determined by means of a standard microlectrode technique and used as a measure of the intensity of non-quantal release. Noradrenaline (10(-7) and 10(-6) M) substantially suppressed, but did not abolish, effects of paraoxon via stimulation of α-adrenoceptors, because all experiments were conducted in the presence of the β-blocker propranolol (5 × 10(-6) M). A blocker of ganglionic transmission, hexamethonium bromide (10(-4) M), failed to alter the inhibitory effect of noradrenaline, indicating that only non-quantal ACh release is suppressed by this neurotransmitter. The effects of noradrenaline could be reduced by the α2-antagonist yohimbine (10(-6) M). However, both the α1-agonist phenylephrine (10(-6) M) and the α2-agonist clonidine (10(-6) M) significantly inhibited the cholinergic effects of paraoxon, indicating the possible involvement of both α-adrenoceptor subtypes in mediation of the adrenergic inhibition of non-quantal ACh release. Thus, cardiac non-quantal ACh release can be negatively regulated by

Zingiberis Siccatum Rhizoma, the active component of the Kampo formula Daikenchuto, induces anti-inflammatory actions through α7 nicotinic acetylcholine receptor activation.

PubMed

Endo, M; Hori, M; Mihara, T; Ozaki, H; Oikawa, T; Odaguchi, H; Hanawa, T

2017-12-01

We previously reported that Daikenchuto (DKT), a gastrointestinal prokinetic Japanese herbal (Kampo) medicine used for the treatment of postoperative ileus (POI), has characteristic potent anti-inflammatory activity. This effect may be partly mediated by the activation of α7 nicotinic acetylcholine receptor (nAChR). In this study, we identified the specific herbs in DKT that induce anti-inflammatory action. The herbal components of DKT were individually administered orally to each mouse four times before and after intestinal manipulation (IM) was carried out on the distal ileum. The anti-inflammatory activity of each crude drug was subsequently evaluated using immunohistochemical analyses of relevant molecules. Treatment with Zingiberis Siccatum Rhizoma (ZSR) but not the other components inhibited the infiltration of cluster of differentiation 68 (CD68)-positive macrophages as effectively as DKT treatment. Selective α7nAChR antagonists, such as methyllycaconitine citrate, or transient receptor potential ankyrin 1 (TRPA1) antagonists, such as HC-030031, significantly inhibited the amelioration of macrophage infiltration by ZSR. The inhibition of macrophage infiltration by ZSR was abolished in both α7nAChR and 5-hydroxytryptamine 4 receptor (5-HT 4 R) knockout mice. Daikenchuto-induced anti-inflammatory activity, which was mediated by inhibiting macrophage infiltration in POI, is dependent on the effects of ZSR. Zingiberis Siccatum Rhizoma activates TRPA1 channels possibly in enterochromaffin (EC) cells to release 5-HT, which stimulates 5-HT 4 R in the myenteric plexus neurons to release ACh, which in turn activates α7nAChR on macrophages to inhibit inflammation in POI. © 2017 John Wiley & Sons Ltd.

Cholinesterase Inhibitory Activity of Some semi-Rigid Spiro Heterocycles: POM Analyses and Crystalline Structure of Pharmacophore Site.

PubMed

Hadda, Taibi Ben; Talhi, Oualid; Silva, Artur S M; Senol, Fatma Sezer; Orhan, Ilkay Erdogan; Rauf, Abdur; Mabkhot, Yahia N; Bachari, Khaldoun; Warad, Ismail; Farghaly, Thoraya A; Althagafi, Ismail I; Mubarak, Mohammad S

2018-01-01

Cholinesterase family consists of two sister enzymes; acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which hydrolyze acetylcholine. Since deficit of acetylcholine has been evidenced in patients of Alzheimer's disease (AD), cholinesterase inhibitors are currently the most prescribed drugs for the treatment of AD. our aim in this article was to investigate the inhibitory potential of five known compounds (2-6) with spiro skeleton against AChE and BChE using ELISA microplate assays. In addition to their ChE inhibitory effect, their physico-chemical properties were also calculated. Moreover, the present work aims at investigating the charge/geometrical effect of a hypothetical pharmacophore or bidentate site in a bioactive group, on the inhibition efficiency of spiro compounds 2-6 by using Petra/Osiris/ molinspiration (POM) and X-ray analyses. In the present study, five compounds (2-6) with spiro skeleton have been synthesized and tested in vitro for their inhibitory potential against AChE and BChE using ELISA microtiter plate assays at 25 µg/mL. Results revealed that three of the spiro compounds tested exert more than 50% inhibition against one of cholinesterases. Compound 5 displayed 68.73 ± 4.73% of inhibition toward AChE, whereas compound 6 showed 56.17 ± 0.83% of inhibition toward BChE; these two previously synthesized compounds have been the most active hits. Our data obtained from screening of compounds 2-6 against the two cholinesterases indicate that three of these show good potential to selectively inhibit AChE or BChE. Spiro compounds 2, 5, and 6 exhibited the most potent activity of the series against AChE or BChE with inhibition values in the range 55-70%. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

AOP description: Acetylcholinesterase inhibition

EPA Science Inventory

This adverse outcome pathway (AOP) leverages existing knowledge in the open literature to describe the linkage between inhibition of acetylcholinesterase (AChE) and the subsequent mortality resulting from impacts at cholinergic receptors. The AOP takes a chemical category approa...

Inhibition of acetylcholinesterase by two genistein derivatives: kinetic analysis, molecular docking and molecular dynamics simulation.

PubMed

Fang, Jiansong; Wu, Ping; Yang, Ranyao; Gao, Li; Li, Chao; Wang, Dongmei; Wu, Song; Liu, Ai-Lin; Du, Guan-Hua

2014-12-01

In this study two genistein derivatives (G1 and G2) are reported as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and differences in the inhibition of AChE are described. Although they differ in structure by a single methyl group, the inhibitory effect of G1 (IC50=264 nmol/L) on AChE was 80 times stronger than that of G2 (IC50=21,210 nmol/L). Enzyme-kinetic analysis, molecular docking and molecular dynamics (MD) simulations were conducted to better understand the molecular basis for this difference. The results obtained by kinetic analysis demonstrated that G1 can interact with both the catalytic active site and peripheral anionic site of AChE. The predicted binding free energies of two complexes calculated by the molecular mechanics/generalized born surface area (MM/GBSA) method were consistent with the experimental data. The analysis of the individual energy terms suggested that a difference between the net electrostatic contributions (ΔE ele+ΔG GB) was responsible for the binding affinities of these two inhibitors. Additionally, analysis of the molecular mechanics and MM/GBSA free energy decomposition revealed that the difference between G1 and G2 originated from interactions with Tyr124, Glu292, Val294 and Phe338 of AChE. In conclusion, the results reveal significant differences at the molecular level in the mechanism of inhibition of AChE by these structurally related compounds.

Distinct Effect of Benzalkonium Chloride on the Esterase and Aryl Acylamidase Activities of Butyrylcholinesterase.

PubMed

Jaganathan; Boopathy

2000-08-01

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) from vertebrates, other than their predominant acylcholine hydrolase (esterase) activity, display a genuine aryl acylamidase activity (AAA) capable of hydrolyzing the synthetic substrate o-nitroacetanilide to o-nitroaniline. This AAA activity is strongly inhibited by classical cholinesterase (ChE) inhibitors. In the present study, benzalkonium chloride (BAC), a cationic detergent widely used as a preservative in pharmaceutical preparations, has been shown to distinctly modulate the esterase and AAA activities of BChEs. The detergent BAC was able to inhibit the esterase activity of human serum and horse serum BChEs and AChEs from electric eel and human erythrocyte. The remarkable property of BAC was its ability to profoundly activate the AAA activity of human serum and horse serum BChEs but not the AAA activity of AChEs. Thus BAC seem to preferentially activate the AAA activity of BChEs alone. Results of the study using the ChE active site-specific inhibitor diisopropyl phosphorofluoridate indicated that BAC binds to the active site of ChEs. Furthermore, studies using a structural homolog of BAC indicated that the alkyl group of BAC is essential not only for its interaction with ChEs but also for its distinct effect on the esterase and AAA activities of BChEs. This is the first report of a compound that inhibits the esterase activity, while simultaneously activating the AAA activity, of BChEs. Copyright 2000 Academic Press.

N-(4-Trifluoromethylphenyl)amide group of the synthetic histamine receptor agonist inhibits nicotinic acetylcholine receptor-mediated catecholamine secretion.

PubMed

Kim, Dong-Chan; Park, Yong-Soo; Jun, Dong-Jae; Hur, Eun-Mi; Kim, Sun-Hee; Choi, Bo-Hwa; Kim, Kyong-Tai

2006-02-28

The therapeutic targeting of nicotinic receptors requires the identification of drugs that selectively activate or inhibit a limited range of nicotine acetylcholine receptors (nAChRs). In this study, we identified N-(4-trifluoromethylphenyl)amide group of the synthetic histamine receptor ligands, histamine-trifluoromethyltoluide, that act as potent inhibitors of nAChRs in bovine adrenal chromaffin cells. Catecholamine secretion induced by the nAChRs agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), was significantly inhibited by histamine-trifluoromethyltoluide. Real time carbon-fiber amperometry confirmed the ability of histamine-trifluoromethyltoluide to inhibit DMPP-induced exocytosis in single chromaffin cells. We also found that histamine-trifluoromethyltoluide inhibited DMPP-induced [Ca(2+)](i) and [Na(+)](i) increases, as well as DMPP-induced inward currents in the absence of extracellular calcium. Histamine-trifluoromethyltoluide had no effect on [(3)H]nicotine binding or on calcium increases induced by high K(+), bradykinin, veratridine, histamine, and benzoylbenzoyl ATP. Among the synthetic histamine receptor ligands, clobenpropit exhibited similarity. In addition, 4'-nitroacetanilide also significantly attenuated nAChR-mediated catecholamine secretion. In conclusion, the N-(4-trifluoromethylphenyl)amide group of the histamine-trifluoromethyltoluide might be the critical moiety in the inhibition of nAChR-mediated CA secretion.

Chitooligosaccharides suppress the level of protein expression and acetylcholinesterase activity induced by Abeta25-35 in PC12 cells.

PubMed

Lee, Sang-Hoon; Park, Jin-Sook; Kim, Se-Kwon; Ahn, Chang-Bum; Je, Jae-Young

2009-02-01

Clinical applications of acetylcholinesterase (AChE) inhibitors are widespread in Alzheimer's sufferers in order to activate central cholinergic system and alleviate cognitive deficits by inhibiting the hydrolysis of acetylcholine. In this study, six kinds of chitooligosaccharides (COSs) with different molecular weight and degree of deacetylation were examined for their inhibitory effects against AChE. The 90-COSs exhibited potent AChE inhibitory activities compared to 50-COSs, while 90-MMWCOS (1000-5000 Da) in the 90-COSs showed the highest activity. Cell culture experiment revealed that 90-MMWCOS suppressed the level of AChE protein expression and AChE activity induced by Abeta(25-35) in PC12 cell lines.

Phytochemical Constituents, ChEs and Urease Inhibitions, Antiproliferative and Antioxidant Properties of Elaeagnus umbellata Thunb.

PubMed

Ozen, Tevfik; Yenigun, Semiha; Altun, Muhammed; Demirtas, Ibrahim

2017-01-01

Due to the common ethnopharmacological used or scientifically examined biochemical properties, Elaeagnaceae family, Elaeagnus umbellate (Thunb.) (EU, Guz yemisi) was worth investigating. In this investigation, we revealed antioxidant, antiproliferative and enzyme inhibition activities of the water, methanol, ethanol, acetone, ethyl acetate and hexane extracts of EU as well as the contents of their phenolic, flavonoid, anthocyanin, ascorbic acid, lycopene and β- carotene. The antioxidant activity was screened by total antioxidant (phosphomolybdenum), inhibition of linoleic acid peroxidation, reducing power, 2-deoxyribose degradation assay, H2O2 scavenging and metal chelating activities of the samples were tested in vitro. Additionally, the scavenging activities of the extracts were determined against 1,1-diphenyl-2-picrylhydrazyl (DPPH˙), 2,2-azino-bis(3-ethylbenzothiazloine-6-sulfonicacid (ABTS˙+), superoxide anion and peroxide radicals. The samples were determined for their inhibitory activities against urease, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In vitro, antiproliferative activities of six different extracts were tested using the xCELLigence system against HeLa and HT29 cell lines. The antioxidant activities of the extracts were found higher than standard antioxidants. The water extracts of fruit and leaf showed the best antioxidant activity. In inhibition assays of urease, AChE and BuChE, all extracts exhibited remarkable inhibition potential. Ethyl acetate extracts, especially, showed better inhibition capacity. It was found that the antioxidant activities of the extracts presented consistently with their chemical contents. The antiproliferative activities of leaf extracts were more effective than the fruit extracts. The chromatographic methods were applied to the different solvents to analyses phenolic secondery metabolites. It was found that fumaric acid, 4- hydroxybenzoic acid, rutin and quercetin-3-�

Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

PubMed Central

Essono, Sosthène; Mondielli, Grégoire; Lamourette, Patricia; Boquet, Didier; Grassi, Jacques; Marchot, Pascale

2013-01-01

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis. PMID:24146971

Oximes: Inhibitors of Human Recombinant Acetylcholinesterase. A Structure-Activity Relationship (SAR) Study

PubMed Central

Sepsova, Vendula; Karasova, Jana Zdarova; Korabecny, Jan; Dolezal, Rafael; Zemek, Filip; Bennion, Brian J.; Kuca, Kamil

2013-01-01

Acetylcholinesterase (AChE) reactivators were developed for the treatment of organophosphate intoxication. Standard care involves the use of anticonvulsants (e.g., diazepam), parasympatolytics (e.g., atropine) and oximes that restore AChE activity. However, oximes also bind to the active site of AChE, simultaneously acting as reversible inhibitors. The goal of the present study is to determine how oxime structure influences the inhibition of human recombinant AChE (hrAChE). Therefore, 24 structurally different oximes were tested and the results compared to the previous eel AChE (EeAChE) experiments. Structural factors that were tested included the number of pyridinium rings, the length and structural features of the linker, and the number and position of the oxime group on the pyridinium ring. PMID:23959117

Neurologically Potent Molecules from Crataegus oxyacantha; Isolation, Anticholinesterase Inhibition, and Molecular Docking

PubMed Central

Ali, Mumtaz; Muhammad, Sultan; Shah, Muhammad R.; Khan, Ajmal; Rashid, Umer; Farooq, Umar; Ullah, Farhat; Sadiq, Abdul; Ayaz, Muhammad; Ali, Majid; Ahmad, Manzoor; Latif, Abdul

2017-01-01

Crataegus oxyacantha is an important herbal supplement and famous for its antioxidant potential. The antioxidant in combination with anticholinesterase activity can be considered as an important target in the management of Alzheimer’s disease. The compounds isolated from C. oxyacantha were evaluated for cholinesterases inhibitory activity using Ellman’s assay with Galantamine as standard drug. Total of nine (1–9) compounds were isolated. Compounds 1 and 2 were isolated for the first time from natural source. Important natural products like β-Sitosterol-3-O-β-D-Glucopyranoside (3), lupeol (4), β-sitosterol (5), betulin (6), betulinic acid (7), oleanolic acid (8), and chrysin (9) have also been isolated from C. oxyacantha. Overall, all the compounds exhibited an overwhelming acetylcholinesterase (AChE) inhibition potential in the range 5.22–44.47 μM. The compound 3 was prominent AChE inhibitor with IC50 value of 5.22 μM. Likewise, all the compounds were also potent in butyrylcholinesterase (BChE) inhibitions with IC50s of up to 0.55–15.36 μM. All the compounds, except 3, were selective toward BChE. Mechanism of the inhibition of both the enzymes were further studied by docking procedures using Genetic Optimization for Ligand Docking suit v5.4.1. Furthermore, computational blood brain barrier prediction of the isolated compounds suggest that these are BBB+. PMID:28638340

Studies on the interaction of BDE-47 and BDE-209 with acetylcholinesterase (AChE) based on the neurotoxicity through fluorescence, UV-vis spectra, and molecular docking.

PubMed

Wang, Shutao; Wu, Chuan; Liu, Zhisheng; You, Hong

2018-05-01

The neurotoxicity of polybrominated diphenyl ethers (PBDEs) has been of concern. Acetylcholinesterase (AChE) is a critical enzyme in the central and peripheral nervous system related to neurotoxicity. The interaction between BDE-47, BDE-209, and AChE was investigated through fluorescence and UV-vis spectra combined with molecular docking. Both BDE-47 and BDE-209 bound with AChE and changed the microenvironment of some amino acid residues, resulting in a change of AChE conformation. Hydrophobic interaction is the main binding force between BDE-47, BDE-209, and AChE, and electrostatic interaction exists according to the thermodynamic parameters of the interaction between them. A hydrophobic interaction of BDE-47-AChE and BDE-209-AChE has been confirmed through molecular docking to dominate the binding force. The binding constants of BDE-47-AChE and BDE-209-AChE were 4.2 × 10 4 and 4.1 × 10 4  L/mol, respectively, and the lowest binding energies of BDE-47-AChE and BDE-209-AChE were -7.8 and -5.9 kJ/mol, respectively. BDE-47 is more likely to bind with AChE than BED-209. Copyright © 2018 Elsevier B.V. All rights reserved.

Reactivation of organophosphate-inhibited human acetylcholinesterase by isonitrosoacetone (MINA): a kinetic analysis.

PubMed

Worek, Franz; Thiermann, Horst

2011-11-15

Treatment of poisoning by highly toxic organophosphorus compounds (OP) with atropine and an acetylcholinesterase (AChE) reactivator (oxime) is of limited effectiveness in case of different nerve agents and pesticides. One challenge is the reactivation of OP-inhibited brain AChE which shows inadequate success with charged pyridinium oximes. Recent studies with high doses of the tertiary oxime isonitrosoacetone (MINA) indicated a beneficial effect on central and peripheral AChE and on survival in nerve agent poisoned guinea pigs. Now, an in vitro study was performed to determine the reactivation kinetics of MINA with tabun-, sarin-, cyclosarin-, VX- and paraoxon-inhibited human AChE. MINA showed an exceptionally low affinity to inhibited AChE but, with the exception of tabun-inhibited AChE, a moderate to high reactivity. In comparison to the pyridinium oximes obidoxime, 2-PAM and HI-6 the affinity and reactivity of MINA was in most cases lower and in relation to the most effective reactivators, the second order reactivation constant of MINA was 500 to 3400-fold lower. Hence, high in vivo MINA concentrations would be necessary to achieve at least partial reactivation. This assumption corresponds to in vivo data showing a dose-dependent effect on reactivation and survival in animals. In view, of the toxic potential of MINA in animals human studies would be necessary to determine the tolerability and pharmacokinetics of MINA in order to enable a proper assessment of the value of this oxime as an antidote in OP poisoning. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Anticonvulsants Based on the α-Substituted Amide Group Pharmacophore Bind to and Inhibit Function of Neuronal Nicotinic Acetylcholine Receptors.

PubMed

Krivoshein, Arcadius V

2016-03-16

Although the antiepileptic properties of α-substituted lactams, acetamides, and cyclic imides have been known for over 60 years, the mechanism by which they act remains unclear. I report here that these compounds bind to the nicotinic acetylcholine receptor (nAChR) and inhibit its function. Using transient kinetic measurements with functionally active, nondesensitized receptors, I have discovered that (i) α-substituted lactams and cyclic imides are noncompetitive inhibitors of heteromeric subtypes (such as α4β2 and α3β4) of neuronal nAChRs and (ii) the binding affinity of these compounds toward the nAChR correlates with their potency in preventing maximal electroshock (MES)-induced convulsions in mice. Based on the hypothesis that α-substituted amide group is the essential pharmacophore of these drugs, I found and tested a simple compound, 2-phenylbutyramide. This compound indeed inhibits nAChR and shows good anticonvulsant activity in mice. Molecular docking simulations suggest that α-substituted lactams, acetamides, and cyclic imides bind to the same sites on the extracellular domain of the receptor. These new findings indicate that inhibition of brain nAChRs may play an important role in the action of these antiepileptic drugs, a role that has not been previously recognized.

Coconut (Cocos nucifera) Ethanolic Leaf Extract Reduces Amyloid-β (1-42) Aggregation and Paralysis Prevalence in Transgenic Caenorhabditis elegans Independently of Free Radical Scavenging and Acetylcholinesterase Inhibition

PubMed Central

Manalo, Rafael Vincent; Silvestre, Maries Ann; Barbosa, Aza Lea Anne; Medina, Paul Mark

2017-01-01

Virgin coconut oil (VCO) has been the subject of several studies which have aimed to alleviate Alzheimer’s disease (AD) pathology, focusing on in vitro antioxidant and acetylcholinesterase (AChE) inhibitory activities. Here, we studied an underutilized and lesser-valued part of the coconut tree, specifically the leaves, using in vitro and in vivo approaches. Coconut leaf extract (CLE) was screened for antioxidant and AChE inhibitory properties in vitro and therapeutic effects in two strains of transgenic Caenorhabditis elegans expressing amyloid-β1–42 (Aβ1-42) in muscle cells. CLE demonstrated free radical scavenging activity with an EC50 that is 79-fold less compared to ascorbic acid, and an AChE inhibitory activity that is 131-fold less compared to Rivastigmine. Surprisingly, in spite of its low antioxidant activity and AChE inhibition, CLE reduced Aβ deposits by 30.31% in CL2006 in a dose-independent manner, and reduced the percentage of paralyzed nematodes at the lowest concentration of CLE (159.38 μg/mL), compared to dH2O/vehicle (control). Phytochemical analysis detected glycosides, anthocyanins, and hydrolyzable tannins in CLE, some of which are known to be anti-amyloidogenic. Taken together, these findings suggest that CLE metabolites alternatively decrease AB1–42 aggregation and paralysis prevalence independently of free radical scavenging and AChE inhibition, and this warrants further investigation on the bioactive compounds of CLE. PMID:28536360

Coconut (Cocos nucifera) Ethanolic Leaf Extract Reduces Amyloid-β (1-42) Aggregation and Paralysis Prevalence in Transgenic Caenorhabditis elegans Independently of Free Radical Scavenging and Acetylcholinesterase Inhibition.

PubMed

Manalo, Rafael Vincent; Silvestre, Maries Ann; Barbosa, Aza Lea Anne; Medina, Paul Mark

2017-04-21

Virgin coconut oil (VCO) has been the subject of several studies which have aimed to alleviate Alzheimer's disease (AD) pathology, focusing on in vitro antioxidant and acetylcholinesterase (AChE) inhibitory activities. Here, we studied an underutilized and lesser-valued part of the coconut tree, specifically the leaves, using in vitro and in vivo approaches. Coconut leaf extract (CLE) was screened for antioxidant and AChE inhibitory properties in vitro and therapeutic effects in two strains of transgenic Caenorhabditis elegans expressing amyloid-β 1-42 (Aβ 1-42 ) in muscle cells. CLE demonstrated free radical scavenging activity with an EC 50 that is 79-fold less compared to ascorbic acid, and an AChE inhibitory activity that is 131-fold less compared to Rivastigmine. Surprisingly, in spite of its low antioxidant activity and AChE inhibition, CLE reduced Aβ deposits by 30.31% in CL2006 in a dose-independent manner, and reduced the percentage of paralyzed nematodes at the lowest concentration of CLE (159.38 μg/mL), compared to dH₂O/vehicle (control). Phytochemical analysis detected glycosides, anthocyanins, and hydrolyzable tannins in CLE, some of which are known to be anti-amyloidogenic. Taken together, these findings suggest that CLE metabolites alternatively decrease AB 1-42 aggregation and paralysis prevalence independently of free radical scavenging and AChE inhibition, and this warrants further investigation on the bioactive compounds of CLE.

Highly Sensitive and Selective Immuno-capture/Electrochemical Assay of Acetylcholinesterase Activity in Red Blood Cells: A Biomarker of Exposure to Organophosphorus Pesticides and Nerve Agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Aiqiong; Du, Dan; Lin, Yuehe

Acetylcholinesterase (AChE) enzyme activity in red blood cells (RBCs) is a useful biomarker for biomonitoring of exposures to organophosphorus (OP) pesticides and chemical nerve agents. In this paper, we reported a new method for AChE activity assay based on selective immuno-capture of AChE from biological samples followed by enzyme activity assay of captured AChE using a disposable electrochemical sensor. The electrochemical sensor is based on multiwalled carbon nanotubes-gold nanocomposites (MWCNTs-Au) modified screen printed carbon electrode (SPCE). Upon the completion of immunoreaction, the target AChE (including active and inhibited) is captured onto the electrode surface and followed by an electrochemical detectionmore » of enzymatic activity in the presence of acetylthiocholine. A linear response is obtained over standard AChE concentration range from 0.1 to 10 nM. To demonstrate the capability of this new biomonitoring method, AChE solutions dosed with different concentration of paraoxon were used to validate the new AChE assay method. AChE inhibition in OP dosed solutions was proportional to its concentration from 0.2 to 50 nM. The new AChE activity assay method for biomonitoring of OP exposure was further validated with in-vitro paraoxon-dosed RBC samples. The established electrochemical sensing platform for AChE activity assay not only avoids the problem of overlapping substrate specificity with esterases by using selective antibody, but also eliminates potential interference from other electroactive species in biological samples. It offers a new approach for sensitive, selective, and rapid AChE activity assay for biomonitoring of exposures to OPs.« less

Insights into cholinesterase inhibitory and antioxidant activities of five Juniperus species.

PubMed

Orhan, Nilufer; Orhan, Ilkay Erdogan; Ergun, Fatma

2011-09-01

In vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory and antioxidant activities of the aqueous and ethanol extracts of the leaves, ripe fruits, and unripe fruits of Juniperus communis ssp. nana, Juniperus oxycedrus ssp. oxycedrus, Juniperus sabina, Juniperus foetidissima, and Juniperus excelsa were investigated in the present study. Cholinesterase inhibition of the extracts was screened using ELISA microplate reader. Antioxidant activity of the extracts was tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging, ferrous ion-chelating, and ferric-reducing antioxidant power (FRAP) assays. Total phenol and flavonoid contents of the extracts were determined spectrophotometrically. The extracts had low or no inhibition towards AChE, whereas the leaf aqueous extract of J. foetidissima showed the highest BChE inhibition (93.94 ± 0.01%). The leaf extracts usually exerted higher antioxidant activity. We herein describe the first study on anticholinesterase and antioxidant activity by the methods of ferrous ion-chelating, superoxide radical scavenging, and ferric-reducing antioxidant power (FRAP) assays of the mentioned Juniperus species. Copyright © 2011 Elsevier Ltd. All rights reserved.

α7nAchR/NMDAR coupling affects NMDAR function and object recognition.

PubMed

Li, Shupeng; Nai, Qiang; Lipina, Tatiana V; Roder, John C; Liu, Fang

2013-12-20

The α7 nicotinic acetylcholine receptor (nAchR) and NMDA glutamate receptor (NMDAR) are both ligand-gated ion channels permeable to Ca2+ and Na+. Previous studies have demonstrated functional modulation of NMDARs by nAchRs, although the molecular mechanism remains largely unknown. We have previously reported that α7nAchR forms a protein complex with the NMDAR through a protein-protein interaction. We also developed an interfering peptide that is able to disrupt the α7nAchR-NMDAR complex and blocks cue-induced reinstatement of nicotine-seeking in rat models of relapse. In the present study, we investigated whether the α7nAchR-NMDAR interaction is responsible for the functional modulation of NMDAR by α7nAchR using both electrophysiological and behavioral tests. We have found that activation of α7nAchR upregulates NMDAR-mediated whole cell currents and LTP of mEPSC in cultured hippocampal neurons, which can be abolished by the interfering peptide that disrupts the α7nAchR-NMDAR interaction. Moreover, administration of the interfering peptide in mice impairs novel object recognition but not Morris water maze performance. Our results suggest that α7nAchR/NMDAR coupling may selectively affect some aspects of learning and memory.

Anti-cholinesterase activity of the standardized extract of Syzygium aromaticum L.

PubMed

Dalai, Manoj K; Bhadra, Santanu; Chaudhary, Sushil K; Bandyopadhyay, Arun; Mukherjee, Pulok K

2014-04-01

Clove (Syzygium aromaticum) is a well-known culinary spice with strong aroma; contains a high amount of oil known as clove oil. The major phyto-constituent of the clove oil is eugenol. Clove and its oil possess various medicinal uses in indigenous medicine as an antiseptic, anti-oxidant, analgesic and neuroprotective properties. Thus, it draws much attention among researchers from pharmaceutical, food and cosmetic industries. The aim of the present study was to determine the anti-cholinesterase activity of the methanol extract of clove, its oil and eugenol. In vitro anti-cholinesterase activity of S. aromaticum was performed by a thin layer chromatography bio autography, 96 well micro titer plate and kinetic methods. Reverse phase high performance liquid chromatography (RP-HPLC) analysis was carried out to identify the biomarker compound eugenol in clove oil. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition study revealed that eugenol possess better inhibition of the enzymes than extract and oil. Clove extract, its oil and eugenol showed better inhibition of AChE than BChE. Polyphenolic compound eugenol was detected through RP-HPLC analysis. The content of eugenol in essential oil was found to be 0.5 μg/ml. Kinetic analysis of the cholinesterase inhibition study of the extract; clove oil and eugenol have shown that they possess mixed type of inhibition for AChE and non-competitive type of inhibition for BChE. These results might be useful in explaining the effect of clove as anti-cholinesterase agent for the management of cognitive ailments like Alzheimer's disease.

Characterization of monoclonal antibodies that strongly inhibit Electrophorus electricus acetylcholinesterase.

PubMed

Remy, M H; Frobert, Y; Grassi, J

1995-08-01

In this study, we describe three different monoclonal antibodies (mAbs Elec-403, Elec-408, and Elec-410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies (Elec-403 and Elec-410), recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec-403 was antagonized by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these low-molecular-mass inhibitors. The third mAb (Elec-408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec-403, Elec-410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent Ki values less than 0.1 nM. Elec-403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o-nitrophenyl acetate) and had the characteristics of a non-competitive process. Elec-403 and Elec-410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec-408 has not been localized, but it may correspond to a new regulatory site on AChE.

Menthol Binding and Inhibition of α7-Nicotinic Acetylcholine Receptors

PubMed Central

Ashoor, Abrar; Nordman, Jacob C.; Veltri, Daniel; Yang, Keun-Hang Susan; Al Kury, Lina; Shuba, Yaroslav; Mahgoub, Mohamed; Howarth, Frank C.; Sadek, Bassem; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

2013-01-01

Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner. PMID:23935840

Acetylcholinesterases of Rhipicephalus (Boophilus) microplus – Multiple gene expression presents an opportune model system for elucidation of multiple functions of AChEs.

USDA-ARS?s Scientific Manuscript database

Acetylcholinesterase (AChE) is a key neural enzyme of both vertebrates and invertebrates, and is the biochemical target of organophosphate and carbamate pesticides for invertebrates, as well as vertebrate nerve agents, e.g., soman, tabun, VX, and others. AChE inhibitors are also key drugs among thos...

Muscarinic ACh Receptors Contribute to Aversive Olfactory Learning in Drosophila.

PubMed

Silva, Bryon; Molina-Fernández, Claudia; Ugalde, María Beatriz; Tognarelli, Eduardo I; Angel, Cristian; Campusano, Jorge M

2015-01-01

The most studied form of associative learning in Drosophila consists in pairing an odorant, the conditioned stimulus (CS), with an unconditioned stimulus (US). The timely arrival of the CS and US information to a specific Drosophila brain association region, the mushroom bodies (MB), can induce new olfactory memories. Thus, the MB is considered a coincidence detector. It has been shown that olfactory information is conveyed to the MB through cholinergic inputs that activate acetylcholine (ACh) receptors, while the US is encoded by biogenic amine (BA) systems. In recent years, we have advanced our understanding on the specific neural BA pathways and receptors involved in olfactory learning and memory. However, little information exists on the contribution of cholinergic receptors to this process. Here we evaluate for the first time the proposition that, as in mammals, muscarinic ACh receptors (mAChRs) contribute to memory formation in Drosophila. Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae. This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs. These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila.

Muscarinic ACh Receptors Contribute to Aversive Olfactory Learning in Drosophila

PubMed Central

Silva, Bryon; Molina-Fernández, Claudia; Ugalde, María Beatriz; Tognarelli, Eduardo I.; Angel, Cristian; Campusano, Jorge M.

2015-01-01

The most studied form of associative learning in Drosophila consists in pairing an odorant, the conditioned stimulus (CS), with an unconditioned stimulus (US). The timely arrival of the CS and US information to a specific Drosophila brain association region, the mushroom bodies (MB), can induce new olfactory memories. Thus, the MB is considered a coincidence detector. It has been shown that olfactory information is conveyed to the MB through cholinergic inputs that activate acetylcholine (ACh) receptors, while the US is encoded by biogenic amine (BA) systems. In recent years, we have advanced our understanding on the specific neural BA pathways and receptors involved in olfactory learning and memory. However, little information exists on the contribution of cholinergic receptors to this process. Here we evaluate for the first time the proposition that, as in mammals, muscarinic ACh receptors (mAChRs) contribute to memory formation in Drosophila. Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae. This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs. These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila. PMID:26380118

Bisquaternary pyridinium oximes: Comparison of in-vitro reactivation potency of compounds bearing aliphatic linkers and heteroaromatic linkers for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase

PubMed Central

Bharate, Sandip B.; Guo, Lilu; Reeves, Tony E.; Cerasoli, Douglas M.; Thompson, Charles M.

2009-01-01

Oxime reactivators are the drugs of choice for the post-treatment of OP (organophosphorus) intoxication and used widely for mechanistic and kinetic studies of OP-inhibited cholinesterases. The purpose of the present study was to evaluate new oxime compounds to reactivate acetylcholinesterase (AChE) inhibited by the OP paraoxon. Several new bisquaternary pyridinium oximes with heterocyclic linkers along with some known bisquaternary pyridinium oximes bearing aliphatic linkers were synthesized and evaluated for their in vitro reactivation potency against paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE). Results herein indicate that most of the compounds are better reactivators of EeAChE than of rHuAChE. The reactivation potency of two different classes of compounds with varying linker chains was compared and observed that the structure of the connecting chain is an important factor for the activity of the reactivators. At a higher concentration (10−3 M), compounds bearing aliphatic linker showed better reactivation than compounds with heterocyclic linkers. Interestingly, oximes with a heterocyclic linker inhibited AChE at higher concentration (10−3 M), whereas their ability to reactivate was increased at lower concentrations (10−4 M and 10−5 M). Compounds bearing either a thiophene linker 26, 46 or a furan linker 31 showed 59%, 49% and 52% reactivation of EeAChE, respectively, at 10−5 M. These compounds showed 14%, 6% and 15% reactivation of rHuAChE at 10−4 M. Amongst newly synthesized analogs with heterocyclic linkers (26–35 and 45–46), compound 31, bearing furan linker chain, was found to be the most effective reactivator with a kr 0.042 min−1, which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed a kr 0.0041 min−1 that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE. PMID:20005727

Rosemary tea consumption results to anxiolytic- and anti-depressant-like behavior of adult male mice and inhibits all cerebral area and liver cholinesterase activity; phytochemical investigation and in silico studies.

PubMed

Ferlemi, Anastasia-Varvara; Katsikoudi, Antigoni; Kontogianni, Vassiliki G; Kellici, Tahsin F; Iatrou, Grigoris; Lamari, Fotini N; Tzakos, Andreas G; Margarity, Marigoula

2015-07-25

Our aim was to investigate the possible effects of regular drinking of Rosmarinus officinalis L. leaf infusion on behavior and on AChE activity of mice. Rosemary tea (2% w/w) phytochemical profile was investigated through LC/DAD/ESI-MS(n). Adult male mice were randomly divided into two groups: "Rosemary-treated" that received orally the rosemary tea for 4weeks and "control" that received drinking water. The effects of regular drinking of rosemary tea on behavioral parameters were assessed by passive avoidance, elevated plus maze and forced swimming tests. Moreover, its effects on cerebral and liver cholinesterase (ChE) isoforms activity were examined colorimetricaly. Phytochemical analysis revealed the presence of diterpenes, flavonoids and hydroxycinnamic derivatives in rosemary tea; the major compounds were quantitatively determined. Its consumption rigorously affected anxiety/fear and depression-like behavior of mice, though memory/learning was unaffected. ChE isoforms activity was significantly decreased in brain and liver of "rosemary treated" mice. In order to explain the tissue ChE inhibition, principal component analysis, pharmacophore alignment and molecular docking were used to explore a possible relationship between main identified compounds of rosemary tea, i.e. rosmarinic acid, luteolin-7-O-glucuronide, caffeic acid and known AChE inhibitors. Results revealed potential common pharmacophores of the phenolic components with the inhibitors. Our findings suggest that rosemary tea administration exerts anxiolytic and antidepressant effects on mice and inhibits ChE activity; its main phytochemicals may function in a similar way as inhibitors. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons.

PubMed

Juárez, E H; Ochoa-Cortés, F; Miranda-Morales, M; Espinosa-Luna, R; Montaño, L M; Barajas-López, C

2014-01-01

The three most common Cys-loop receptors expressed by myenteric neurons are nACh, 5-HT3 and GABAA . To investigate the function of these proteins researchers have used channel inhibitors such as hexamethonium (antagonist of nACh receptors), ondansetron (antagonist of 5-HT3 receptors), picrotoxin and bicuculline (both antagonists of GABAA receptors). The aim of this study was to investigate the specificity of these inhibitors on Cys-loop receptors of primary cultured neurons obtained from the guinea-pig small intestine. The whole-cell configuration of the patch clamp techniques was used to record membrane currents induced by ACh (IACh ), 5-HT (I5-HT ) and GABA (IGABA ) in the absence and the presence of various concentrations of hexamethonium, ondansetron, picrotoxin or bicuculline. The three Cys-loop receptors present in enteric neurons are expressed independently and they do not cross-desensitized. Hexamethonium inhibited IACh without affecting I5-HT and IGABA . Ondansetron inhibited I5-HT and also IACh but did not affect IGABA . Picrotoxin and bicuculline inhibited I5-HT , IACh and IGABA with different potency, being the lowest potency on 5-HT3 receptors. All these inhibitory effects were concentration dependent and reversible. Our observations showed that except for hexamethonium, all other inhibitors used here show different degrees of selectivity, which has to be considered when these antagonists are used in experimental studies aimed to investigate the functions of these receptors. In particular, in tissues expressing nACh receptors because these are the targets of all other inhibitors used here. The low potency of picrotoxin and bicuculline to inhibit 5-HT3 receptors suggests that these receptors are heteromeric proteins. © 2013 John Wiley & Sons Ltd.

Structure-activity relationship investigation of tertiary amine derivatives of cinnamic acid as acetylcholinesterase and butyrylcholinesterase inhibitors: compared with that of phenylpropionic acid, sorbic acid and hexanoic acid.

PubMed

Gao, Xiaohui; Tang, Jingjing; Liu, Haoran; Liu, Linbo; Kang, Lu; Chen, Wen

2018-12-01

In the present investigation, 48 new tertiary amine derivatives of cinnamic acid, phenylpropionic acid, sorbic acid and hexanoic acid (4d-6g, 10d-12g, 16d-18g and 22d-24g) were designed, synthesized and evaluated for the effect on AChE and BChE in vitro. The results revealed that the alteration of aminoalkyl types and substituted positions markedly influences the effects in inhibiting AChE. Almost of all cinnamic acid derivatives had the most potent inhibitory activity than that of other acid derivatives with the same aminoalkyl side chain. Unsaturated bond and benzene ring in cinnamic acid scaffold seems important for the inhibitory activity against AChE. Among them, compound 6g revealed the most potent AChE inhibitory activity (IC 50 value: 3.64 µmol/L) and highest selectivity over BChE (ratio: 28.6). Enzyme kinetic study showed that it present a mixed-type inhibition against AChE. The molecular docking study suggested that it can bind with the catalytic site and peripheral site of AChE.

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction

PubMed Central

Ghazanfari, Nazanin; Morsch, Marco; Reddel, Stephen W; Liang, Simon X; Phillips, William D

2014-01-01

Muscle-specific kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse-labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β-subunit-Y390 was reduced at endplates. In contrast, endplate staining for β-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold. PMID:24860174

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.

PubMed

Ghazanfari, Nazanin; Morsch, Marco; Reddel, Stephen W; Liang, Simon X; Phillips, William D

2014-07-01

Muscle-specific kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse-labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β-subunit-Y390 was reduced at endplates. In contrast, endplate staining for β-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

Anticholinesterase, antioxidant activity and phytochemical investigation into aqueous extracts from five species of Agrimonia genus.

PubMed

Kubínová, Renata; Švajdlenka, Emil; Jankovská, Dagmar

2016-01-01

Aqueous extracts of aerial flowering parts of five Agrimonia species (Rosaceae): Agrimonia coreana Nakai, Agrimonia japonica (Miq.) Koidz, Agrimonia procera Wallr., Agrimonia eupatoria L. and Agrimonia leucantha Kunze were investigated on their antioxidant activity, measured using five different methods; the best was the extract from A. procera with IC50 values from 6 to 29 μg/mL. All the extracts displayed inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) at the tested concentration of 100 μg/mL. We found the highest inhibition of cholinesterase in the extract of A. japonica with inhibition 70.4% for AChE and 79.8% for BuChE. These findings are statistically significant in comparison with those of other extracts (p < 0.001). The phytochemical analyses showed that the antioxidant activity of Agrimonia extracts can be affected especially by hexahydroxydiphenoyl (HHDP)-glucose and quercetin glycosides, and inhibition of cholinesterases by apigenin, luteolin and quercetin glycosides.

Negative inotropic effect of carbachol and interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein in mouse isolated atrium--a novel methodological trial.

PubMed

Okada, Muneyoshi; Noma, Chihiro; Yamawaki, Hideyuki; Hara, Yukio

2013-01-01

Interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein was examined by an immunoprecipitation-Western blotting system in mouse isolated atrium. The carbachol-induced negative inotropic action in indomethacin-pretreated mouse atrium was significantly inhibited by a K.ACh channel blocker, tertiapin or atropine. Kir3.1 K.ACh channel (Kir3.1) was immunoprecipitated with a mouse anti-Kir3.1 antibody. Coprecipitating Gβ with Kir3.1, detected by Western blotting, was significantly augmented by carbachol. Atropine, but not tertiapin, significantly inhibited the carbachol-induced coprecipitating Gβ with Kir3.1. The data indicate that immunoprecipitation with Kir3.1 and Western blotting of Gβ system is a useful method for assessing interaction between K.ACh channel and GTP binding protein in mouse atrium.

Protection by pyridostigmine bromide of marmoset hemi-diaphragm acetylcholinesterase activity after soman exposure.

PubMed

Haigh, Julian R; Adler, Michael; Apland, James P; Deshpande, Sharad S; Barham, Charles B; Desmond, Patrick; Koplovitz, Irwin; Lenz, David E; Gordon, Richard K

2010-09-06

Pyridostigmine bromide (PB) was approved by the U.S. Food and Drug Administration (FDA) in 2003 as a pretreatment in humans against the lethal effects of the irreversible nerve agent soman (GD). Organophosphate (OP) chemical warfare agents such as GD exert their toxic effects by inhibiting acetylcholinesterase (AChE) from terminating the action of acetylcholine at postsynaptic sites in cholinergic nerve terminals (including crucial peripheral muscle such as diaphragm). As part of the post-marketing approval of PB, the FDA required (under 21CFR314, the "two animal rule") the study of a non-human primate model (the common marmoset Callithrix jacchus jacchus) to demonstrate increased survival against lethal GD poisoning, and protection of physiological hemi-diaphragm function after PB pretreatment and subsequent GD exposure. Marmosets (male and female) were placed in the following experimental groups: (i) control (saline pretreatment only), (ii) low dose PB (12.5 microg/kg), or (iii) high dose (39.5 microg/kg) PB. Thirty minutes after the PB dose, animals were challenged with either saline (control) or soman (GD, 45 microg/kg), followed 1 min later by atropine (2mg/kg) and 2-PAM (25mg/kg). After a further 16 min, animals were euthanized and the complete diaphragm removed; the right hemi-diaphragm was frozen immediately at -80 degrees C, and the left hemi-diaphragm was placed in a tissue bath for 4h (to allow for decarbamylation to occur), then frozen. AChE activities were determined using the automated WRAIR cholinesterase assay. Blood samples were collected for AChE activities prior to PB, before GD challenge, and after sacrifice. RBC-AChE was inhibited by approximately 18% and 50% at the low and high doses of PB, respectively, compared to control (baseline) activity. In the absence of PB pretreatment, the inhibition of RBC-AChE by GD was 98%. The recovery of hemi-diaphragm AChE activity after the 4h wash period (decarbamylation) was approximately 8% and 17%, at the

Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herkert, N.M.; Schulz, S.; Wille, T.

2011-05-15

Standard treatment of organophosphorus (OP) poisoning includes administration of an antimuscarinic (e.g., atropine) and of an oxime-based reactivator. However, successful oxime treatment in soman poisoning is limited due to rapid aging of phosphylated acetylcholinesterase (AChE). Hence, the inability of standard treatment procedures to counteract the effects of soman poisoning resulted in the search for alternative strategies. Recently, results of an in vivo guinea pig study indicated a therapeutic effect of physostigmine given after soman. The present study was performed to investigate a possible pre- and post-treatment effect of physostigmine on soman-inhibited human AChE given at different time intervals before ormore » after perfusion with soman by using a well-established dynamically working in vitro model for real-time analysis of erythrocyte and muscle AChE. The major findings were that prophylactic physostigmine prevented complete inhibition of AChE by soman and resulted in partial spontaneous recovery of the enzyme by decarbamylation. Physostigmine given as post-treatment resulted in a time-dependent reduction of the protection from soman inhibition and recovery of AChE. Hence, these date indicate that physostigmine given after soman does not protect AChE from irreversible inhibition by the OP and that the observed therapeutic effect of physostigmine in nerve agent poisoning in vivo is probably due to other factors.« less

Single channel properties of human α3 AChRs: impact of β2, β4 and α5 subunits

PubMed Central

Nelson, Mark E; Lindstrom, Jon

1999-01-01

We performed single channel analysis on human α3 acetylcholine receptors (AChRs) in Xenopus oocytes and native AChRs from the human neuroblastoma cell line IMR-32. α3 AChRs exhibit channel properties that reflect subunit composition.α3β2 AChR open times were 0.71 ± 0.14 and 3.5 ± 0.4 ms with a predominant conductance of 26 pS. α3β4 AChRs had open times of 1.4 ± 0.2 and 6.5 ± 0.8 ms and a predominant conductance of 31 pS. Burst times were 0.82 ± 0.12 and 5.3 ± 0.7 ms for α3β2 and 1.7 ± 0.1 and 16 ± 1 ms for α3β4. Desensitization was faster for AChRs with the β2 subunit than for those with the β4 subunit.One open time for α3α5β2 AChRs (5.5 ± 0.3 ms) was different from those of α3β2 AChRs. For α3α5β4 AChRs, an additional conductance, open time and burst time (36 pS, 22 ± 3 ms and 43 ± 4 ms, respectively) were different from those for α3β4 AChRs.α3 AChRs were inhibited by hexamethonium or mecamylamine. The rate constants for block of α3β4 by hexamethonium and of α3β2 by mecamylamine were 1.2 × 107 and 4.6 × 107 M−1 s−1, respectively.AChRs from IMR-32 cells had a predominant conductance of 32 pS and open times of 1.5 ± 0.3 and 9.6 ± 1.2 ms. These properties were most similar to those of α3β4 AChRs expressed in oocytes. Antibodies revealed that 5 ± 2% of IMR-32 α3 AChRs contained α5 subunits and 6 ± 2% contained β2 subunits. IMR-32 α3 AChRs are primarily α3β4 AChRs. PMID:10200416

Drimane Sesquiterpenoids Noncompetitively Inhibit Human α4β2 Nicotinic Acetylcholine Receptors with Higher Potency Compared to Human α3β4 and α7 Subtypes.

PubMed

Arias, Hugo R; Feuerbach, Dominik; Schmidt, Bernd; Heydenreich, Matthias; Paz, Cristian; Ortells, Marcelo O

2018-04-27

The drimane sesquiterpenoids drimenin, cinnamolide, dendocarbin A, and polygodial were purified from the Canelo tree ( Drimys winteri) and chemically characterized by spectroscopic methods. The pharmacological activity of these natural compounds were determined on hα4β2, hα3β4, and hα7 nicotinic acetylcholine receptors (AChRs) by Ca 2+ influx measurements. The results established that drimane sesquiterpenoids inhibit AChRs with the following selectivity: hα4β2 > hα3β4 > hα7. In the case of hα4β2 AChRs, the following potency rank order was determined (IC 50 's in μM): drimenin (0.97 ± 0.35) > cinnamolide (1.57 ± 0.36) > polygodial (62.5 ± 19.9) ≫ dendocarbin A (no activity). To determine putative structural features underlying the differences in inhibitory potency at hα4β2 AChRs, additional structure-activity relationship and molecular docking experiments were performed. The Ca 2+ influx and structural results supported a noncompetitive mechanism of inhibition, where drimenin interacted with luminal and nonluminal (TMD-β2 intrasubunit) sites. The structure-activity relationship results, i.e., the lower the ligand polarity, the higher the inhibitory potency, supported the nonluminal interaction. Ligand binding to both sites might inhibit the hα4β2 AChR by a cooperative mechanism, as shown experimentally ( n H > 1). Drimenin could be used as a molecular scaffold for the development of more potent inhibitors with higher selectivity for the hα4β2 AChR.

Biomonitoring of blood cholinesterases and acylpeptide hydrolase activities in rural inhabitants exposed to pesticides in the Coquimbo Region of Chile

PubMed Central

Ramírez-Santana, Muriel; Farías-Gómez, Cristián; Zúñiga-Venegas, Liliana; Sandoval, Rodrigo; Roeleveld, Nel; Van der Velden, Koos; Scheepers, Paul T. J.

2018-01-01

In Chile, agriculture is a relevant economic activity and is concomitant with the use of pesticides to improve the yields. Acute intoxications of agricultural workers occur with some frequency and they must be reported to the surveillance system of the Ministry of Health. However the impacts of chronic and environmental pesticide exposure have been less studied. Among pesticides frequently used in Chile for insects control are organophosphates (OP) and carbamates (CB). They are inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In this study we determined the pattern of both biomarkers activity in three populations with different type of chronic exposure to OP/CB: environmentally exposed (EE), occupationally exposed (OE) and a reference group (RG) without exposure. Besides this, we also measured the activity of acylpeptide hydrolase (APEH), an enzyme involved in relevant functions in the central synapses that is also expressed in erythrocytes and previously reported to be highly inhibited by some OP. A baseline measurement was done in both exposure groups and then a second measurement was done during the spraying season. The RG was measured only once at any time of the year. Our results indicate that people under chronic OP/CB exposure showed an adaptive response through an increase of basal BChE activity. During the spray season only BChE activity was decreased in the EE and OE groups (p<0.05 and p<0.01, respectively) and the higher magnitude of BChE inhibition was observed in the EE group. The analysis of the frequencies of inhibition above 30% (biological tolerance limit declared by Chilean legislation) indicated that BChE was most frequently inhibited in the EE group (53% of the individuals displayed inhibition) and AChE in the OE group (55% of the individuals displayed AChE inhibition). APEH activity showed the highest frequency of inhibition in the EE group independent of its magnitude (64%). Our results demonstrate that the rural

Blockade of Neuronal α7-nAChR by α-Conotoxin ImI Explained by Computational Scanning and Energy Calculations

PubMed Central

Yu, Rilei; Craik, David J.; Kaas, Quentin

2011-01-01

α-Conotoxins potently inhibit isoforms of nicotinic acetylcholine receptors (nAChRs), which are essential for neuronal and neuromuscular transmission. They are also used as neurochemical tools to study nAChR physiology and are being evaluated as drug leads to treat various neuronal disorders. A number of experimental studies have been performed to investigate the structure-activity relationships of conotoxin/nAChR complexes. However, the structural determinants of their binding interactions are still ambiguous in the absence of experimental structures of conotoxin-receptor complexes. In this study, the binding modes of α-conotoxin ImI to the α7-nAChR, currently the best-studied system experimentally, were investigated using comparative modeling and molecular dynamics simulations. The structures of more than 30 single point mutants of either the conotoxin or the receptor were modeled and analyzed. The models were used to explain qualitatively the change of affinities measured experimentally, including some nAChR positions located outside the binding site. Mutational energies were calculated using different methods that combine a conformational refinement procedure (minimization with a distance dependent dielectric constant or explicit water, or molecular dynamics using five restraint strategies) and a binding energy function (MM-GB/SA or MM-PB/SA). The protocol using explicit water energy minimization and MM-GB/SA gave the best correlations with experimental binding affinities, with an R2 value of 0.74. The van der Waals and non-polar desolvation components were found to be the main driving force for binding of the conotoxin to the nAChR. The electrostatic component was responsible for the selectivity of the various ImI mutants. Overall, this study provides novel insights into the binding mechanism of α-conotoxins to nAChRs and the methodological developments reported here open avenues for computational scanning studies of a rapidly expanding range of wild

nAChRs-ERK1/2-Egr-1 signaling participates in the developmental toxicity of nicotine by epigenetically down-regulating placental 11β-HSD2.

PubMed

Zhou, Jin; Liu, Fulin; Yu, Luting; Xu, Dan; Li, Bin; Zhang, Guohui; Huang, Wen; Li, Lu; Zhang, Yuanzhen; Zhang, Wei; Wang, Hui

2018-04-01

Impaired placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity which inactivates maternal glucocorticoids is associated with poor fetal growth and a higher risk of chronic diseases in adulthood. This study aimed to elucidate the epigenetically regulatory mechanism of nicotine on placental 11β-HSD2 expression. Pregnant Wistar rats were administered 1.0 mg/kg nicotine subcutaneously twice a day from gestational day 9 to 20. The results showed that prenatal nicotine exposure increased corticosterone levels in the placenta and fetal serum, disrupted placental morphology and endocrine function, and reduced fetal bodyweight. Meanwhile, histone modification abnormalities (decreased acetylation and increased di-methylation of histone 3 Lysine 9) on the HSD11B2 promoter and lower-expression of 11β-HSD2 were observed. Furthermore, the expression of nicotinic acetylcholine receptor (nAChR) α4/β2, the phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and Ets-like protein-1 (Elk-1), and the expression of early growth response-1 (Egr-1) were increased in the nicotine groups. In human BeWo cells, nicotine decreased 11β-HSD2 expression, increased nAChRα9 expression, and activated ERK1/2/Elk-1/Egr-1 signaling in the concentration (0.1-10 μM)-dependent manner. Antagonism of nAChRs, inhibition of ERK1/2 and Egr-1 knockdown by siRNA were able to block/abrogate the effects of nicotine on histone modification and expression of 11β-HSD2. Taken together, nicotine can impair placental structure and function, and induce fetal developmental toxicity. The underlying mechanism involves histone modifications and down-regulation of 11β-HSD2 through nAChRs/ERK1/2/Elk-1/Egr-1 signaling, which increases active glucocorticoids levels in the placenta and fetus, and eventually inhibits the fetal development. Copyright © 2018 Elsevier Inc. All rights reserved.

In Silico Pharmacophore Model for Tabun-Inhibites Acetylcholinesterase Reactivators: A study of Their Stereoelectronic Properties

DTIC Science & Technology

2009-01-01

Army Institute of Research, 503 Robert Grant AVenue, SilVer Spring, Maryland 20910, and Center for AdVanced Studies and Department of Toxicology ...Department of Toxicology , Faculty of Military Health Sciences. Chem. Res. Toxicol. XXXX, , 000 A 10.1021/tx900192u  XXXX American Chemical Society...GA-inhibited AChE derived from theoretical stereoelectronic and three-dimensional (3D) quantitative struc- ture-activity relationship ( QSAR

Inhibition properties of propolis extracts to some clinically important enzymes.

PubMed

Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

2016-01-01

The present study was conducted to envisage inhibition effects of propolis on the crucial enzymes, urease, xanthine oxidase (XO) and acetylcholinesterase (AChE). Some of the antioxidant properties of the propolis samples were determined using the total phenolic content (TPE) and total flavonoids in the eight different ethanolic propolis extracts (EPE) samples. Inhibition values of the enzymes were expressed as inhibition concentration (IC 50 ; mg/mL or μg/mL) causing 50% inhibition of the enzymes with donepezil, acetohydroxamic acid and allopurinol as reference inhibitors. All the propolis extracts exhibited variable inhibition effects on these enzymes, but the higher the phenolic contents the lower the inhibitions values (IC 50 = 0.074 to 1.560 mg/mL). IC 50 values of the P5 propolis sample having the highest TPE, obtained from Zonguldak, for AChE, urease and XO were 0.081 ± 0.009, 0.080 ± 0.006 and 0.074 ± 0.011 μg/mL, respectively. The EPE proved to be a good source of inhibitor agents that can be used as natural inhibitors to serve human health.

The antiparasitic clioquinol induces apoptosis in leukemia and myeloma cells by inhibiting histone deacetylase activity.

PubMed

Cao, Biyin; Li, Jie; Zhu, Jingyu; Shen, Mingyun; Han, Kunkun; Zhang, Zubin; Yu, Yang; Wang, Yali; Wu, Depei; Chen, Suning; Sun, Aining; Tang, Xiaowen; Zhao, Yun; Qiao, Chunhua; Hou, Tingjun; Mao, Xinliang

2013-11-22

The antiparasitic clioquinol (CQ) represents a class of novel anticancer drugs by interfering with proteasome activity. In the present study, we found that CQ induced blood cancer cell apoptosis by inhibiting histone deacetylases (HDACs). CQ accumulated the acetylation levels of several key proteins including histone H3 (H3), p53, HSP90, and α-tubulin. In the mechanistic study, CQ was found to down-regulate HDAC1, -3, -4, and -5 in both myeloma and leukemia cells. Computer modeling analysis revealed that CQ was well docked into the active pocket of the enzyme, where the oxygen and nitrogen atoms in CQ formed stable coordinate bonds with the zinc ion, and the hydroxyl group from CQ formed an effective hydrogen bond with Asp-267. Moreover, co-treatment with CQ and zinc/copper chloride led to decreased Ac-H3. Furthermore, CQ inhibited the activity of Class I and IIa HDACs in the cell-free assays, demonstrating that CQ interfered with HDAC activity. By inhibiting HDAC activity, CQ induced expression of p21, p27, and p53, cell cycle arrest at G1 phase, and cell apoptosis. This study suggested that the HDAC enzymes are targets of CQ, which provided a novel insight into the molecular mechanism of CQ in the treatment of hematological malignancies.

In vitro ability of currently available oximes to reactivate organophosphate pesticide-inhibited human acetylcholinesterase and butyrylcholinesterase.

PubMed

Jun, Daniel; Musilova, Lucie; Musilek, Kamil; Kuca, Kamil

2011-01-01

We have in vitro tested the ability of common, commercially available, cholinesterase reactivators (pralidoxime, obidoxime, methoxime, trimedoxime and HI-6) to reactivate human acetylcholinesterase (AChE), inhibited by five structurally different organophosphate pesticides and inhibitors (paraoxon, dichlorvos, DFP, leptophos-oxon and methamidophos). We also tested reactivation of human butyrylcholinesterase (BChE) with the aim of finding a potent oxime, suitable to serve as a "pseudocatalytic" bioscavenger in combination with this enzyme. Such a combination could allow an increase of prophylactic and therapeutic efficacy of the administered enzyme. According to our results, the best broad-spectrum AChE reactivators were trimedoxime and obidoxime in the case of paraoxon, leptophos-oxon, and methamidophos-inhibited AChE. Methamidophos and leptophos-oxon were quite easily reactivatable by all tested reactivators. In the case of methamidophos-inhibited AChE, the lower oxime concentration (10(-5) M) had higher reactivation ability than the 10(-4) M concentration. Therefore, we evaluated the reactivation ability of obidoxime in a concentration range of 10(-3)-10(-7) M. The reactivation of methamidophos-inhibited AChE with different obidoxime concentrations resulted in a bell shaped curve with maximum reactivation at 10(-5) M. In the case of BChE, no reactivator exceeded 15% reactivation ability and therefore none of the oximes can be recommended as a candidate for "pseudocatalytic" bioscavengers with BChE.

A Novel Direct Factor Xa Inhibitory Peptide with Anti-Platelet Aggregation Activity from Agkistrodon acutus Venom Hydrolysates.

PubMed

Chen, Meimei; Ye, Xiaohui; Ming, Xin; Chen, Yahui; Wang, Ying; Su, Xingli; Su, Wen; Kong, Yi

2015-06-02

Snake venom is a natural substance that contains numerous bioactive proteins and peptides, nearly all of which have been identified over the last several decades. In this study, we subjected snake venom to enzymatic hydrolysis to identify previously unreported bioactive peptides. The novel peptide ACH-11 with the sequence LTFPRIVFVLG was identified with both FXa inhibition and anti-platelet aggregation activities. ACH-11 inhibited the catalytic function of FXa towards its substrate S-2222 via a mixed model with a Ki value of 9.02 μM and inhibited platelet aggregation induced by ADP and U46619 in a dose-dependent manner. Furthermore, ACH-11 exhibited potent antithrombotic activity in vivo. It reduced paralysis and death in an acute pulmonary thrombosis model by 90% and attenuated thrombosis weight in an arterio-venous shunt thrombosis model by 57.91%, both at a dose of 3 mg/kg. Additionally, a tail cutting bleeding time assay revealed that ACH-11 did not prolong bleeding time in mice at a dose of 3 mg/kg. Together, our results reveal that ACH-11 is a novel antithrombotic peptide exhibiting both FXa inhibition and anti-platelet aggregation activities, with a low bleeding risk. We believe that it could be a candidate or lead compound for new antithrombotic drug development.

Myasthenia Gravis and the Tops and Bottoms of AChRs Antigenic Structure of the MIR and Specific Immunosuppression of EAMG Using AChR Cytoplasmic Domains

PubMed Central

Lindstrom, Jon; Luo, Jie; Kuryatov, Alexander

2009-01-01

The main immunogenic region (MIR), against which half or more of the autoantibodies to acetylcholine receptors (AChRs) in myasthenia gravis (MG) or experimental autoimmune MG (EAMG) are directed, is located at the extracellular end of α1 subunits. Rat monoclonal antibodies (mAbs) to the MIR efficiently compete with MG patient autoantibodies for binding to human muscle AChRs. Antibodies bound to the MIR do not interfere with cholinergic ligand binding or AChR function, but target complement and trigger antigenic modulation. Rat mAbs to the MIR also bind to human ganglionic AChR α3 subunits, but MG patient antibodies do not. By making chimeras of α1 subunits with α7 subunits or ACh binding protein, the structure of the MIR and its functional effects are being investigated. Many mAbs to the MIR bind only to the native conformation of α1 subunits because they bind to sequences that are adjacent only in the native structure. The MIR epitopes recognized by these mAbs are not recognized by most patient antibodies whose epitopes must be nearby. The presence of the MIR epitopes in α1/α7 chimeras greatly promotes AChR expression and sensitivity to activation. EAMG can be suppressed by treatment with denatured, bacterially expressed mixtures of extracellular and cytoplasmic domains of human α1, β1, γ, δ, and ε subunits. A mixture of only the cytoplasmic domains not only avoids the potential liability of provoking formation antibodies to pathologically significant epitopes on the extracellular surface, but also potently suppresses the development of EAMG. PMID:18567851

Small Quaternary Inhibitors K298 and K524: Cholinesterases Inhibition, Absorption, Brain Distribution, and Toxicity.

PubMed

Karasova, Jana Zdarova; Hroch, Milos; Musilek, Kamil; Kuca, Kamil

2016-02-01

Inhibitors of acetylcholinesterase (AChE) may be used in the treatment of various cholinergic deficits, among them being myasthenia gravis (MG). This paper describes the first in vivo data for promising small quaternary inhibitors (K298 and K524): acute toxicity study, cholinesterase inhibition, absorption, and blood-brain barrier penetration. The newly prepared AChE inhibitors (bis-quinolinium and quinolinium compounds) possess a positive charge in the molecule which ensures that anti-AChE action is restricted to peripheral effect. HPLC-MS was used for determination of real plasma and brain concentration in the pharmacokinetic part of the study, and standard non-compartmental analysis was performed. The maximum plasma concentrations were attained at 30 min (K298; 928.76 ± 115.20 ng/ml) and 39 min (K524; 812.40 ± 54.96 ng/ml) after i.m. Both compounds are in fact able to target the central nervous system. It seems that the difference in the CNS distribution profile depends on an active efflux system. The K524 brain concentration was actively decreased to below an effective level; in contrast, K298 progressively accumulated in brain tissue. Peripheral AChE inhibitors are still first-line treatment in the mild forms of MG. Commonly prescribed carbamates have many severe side effects related to AChE carbamylation. The search for new treatment strategies is still important. Unlike carbamates, these new compounds target AChE via apparent π-π or π-cationic interaction aside at the AChE catalytic site.

Effects of intralipid and caffeic acid phenethyl ester on neurotoxicity, oxidative stress, and acetylcholinesterase activity in acute chlorpyriphos intoxication

PubMed Central

Ozkan, Umit; Osun, Arif; Basarslan, Kagan; Senol, Serkan; Kaplan, Ibrahim; Alp, Harun

2014-01-01

Chlorpyriphos is one of the most widely used organophosphate (OP) insecticide in agriculture with potential toxicity. Current post-exposure treatments consist of anti-cholinergic drugs and oxime compounds. We studied the effects of intralipid and caffeic acid phenethyl ester (CAPE) on chlorpyriphos toxicity to compose an alternative or supportive treatment for OP poisoning. Methods: Forty-nine rats were randomly divided into seven groups. Chlorpyriphos was administered for toxicity. Intralipid (IL) and CAPE administered immediately after chlorpyriphos. Serum acetylcholinesterase (AChE) level, total oxidant status (TOS), total antioxidant response (TAR), and histologic examination of cerebellum and brain tissue with Hematoxylin-Eosin and immunohistochemical dyes were examined. Results: Serum enzym levels showed that chlorpyriphos and CAPE inhibited AChE while IL alone had no effect, chlorpyriphos and CAPE intensifies the inhibition effect. Significant difference at AChE levels between the chlorpyriphos+IL and chlorpyriphos+CAPE verified that IL has a protective effect on AChE inhibition. TAR levels were significantly increased in all groups except chlorpyriphos group, TOS levels revealed that CAPE and IL decrease the amount of oxidative stress. Histologic examination revealed that neuronal degeneration was slightly decreased at chlorpyriphos+IL group, but CAPE had a significant effect on protection of neuronal degeneration. Conclusion: The results of this study gave us three key points. 1) AChE activity is important for diagnosis of OP intoxication but it has no value for determining the neuro-degeneration. 2) CAPE inhibits AChE activity and may increase the muscarinic-nicotinic hyperactivation. Therefore it should not be used for treatment of OP intoxication. 3) IL decreases the severity of neurodegeneration and symptoms of OP intoxication and it can be used as a supportive agent. PMID:24955152

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

PubMed Central

Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

Nicotine-Induced Airway Smooth Muscle Cell Proliferation Involves TRPC6-Dependent Calcium Influx Via α7 nAChR.

PubMed

Hong, Wei; Peng, Gongyong; Hao, Binwei; Liao, Baoling; Zhao, Zhuxiang; Zhou, Yumin; Peng, Fang; Ye, Xiuqin; Huang, Lingmei; Zheng, Mengning; Pu, Jinding; Liang, Chunxiao; Yi, Erkang; Peng, Huanhuan; Li, Bing; Ran, Pixin

2017-01-01

The proliferation of human bronchial smooth muscle cells (HBSMCs) is a key pathophysiological component of airway remodeling in chronic obstructive pulmonary disease (COPD) for which pharmacotherapy is limited, and only slight improvements in survival have been achieved in recent decades. Cigarette smoke is a well-recognized risk factor for COPD; however, the pathogenesis of cigarette smoke-induced COPD remains incompletely understood. This study aimed to investigate the mechanisms by which nicotine affects HBSMC proliferation. Cell viability was assessed with a CCK-8 assay. Proliferation was measured by cell counting and EdU immunostaining. Fluorescence calcium imaging was performed to measure intracellular Ca2+ concentration ([Ca2+]i). The results showed that nicotine promotes HBSMC proliferation, which is accompanied by elevated store-operated calcium entry (SOCE), receptor-operated calcium entry (ROCE) and basal [Ca2+]i in HBSMCs. Moreover, we also confirmed that canonical transient receptor potential protein 6 (TRPC6) and α7 nicotinic acetylcholine receptor (α7 nAChR) are involved in nicotine-induced upregulation of cell proliferation. Furthermore, we verified that activation of the PI3K/Akt signaling pathway plays a pivotal role in nicotine-enhanced proliferation and calcium influx in HBSMCs. Inhibition of α7 nAChR significantly decreased Akt phosphorylation levels, and LY294002 inhibited the protein expression levels of TRPC6. Herein, these data provide compelling evidence that calcium entry via the α7 nAChR-PI3K/Akt-TRPC6 signaling pathway plays an important role in the physiological regulation of airway smooth muscle cell proliferation, representing an important target for augmenting airway remodeling. © 2017 The Author(s). Published by S. Karger AG, Basel.

Diagnosis of anticholinesterase poisoning in birds: Effects of environmental temperature and underfeeding on cholinesterase activity

USGS Publications Warehouse

Rattner, B.A.

1982-01-01

Brain cholinesterase (ChE) activity has been used extensively to monitor exposure to organophosphorus (OP) and carbamate (CB) insecticides in wild birds. A series of factorial experiments was conducted to assess the extent to which noncontaminant-related environmental conditions might affect brain ChE activity and thereby confound the diagnosis of OP and CB intoxication. Underfeeding (restricting intake to 50% of control for 21 d or fasting for 1-3 d) or exposure to elevated temperature (36 + 1?C for 1 d) caused only slight reductions (10-17%) in brain AChE activity in adult male Japanese quail (Coturnix coturnix japonica). This degree of 'reduction' in brain AChE activity is considerably less than the 50% 'inhibition' criterion employed in the diagnosis of insecticide-induced mortality, but nevertheless approaches the 20% 'inhibition' level used as a conservative estimate of sublethal exposure to a known insecticide application.

Inhibitory activities of major anthraquinones and other constituents from Cassia obtusifolia against β-secretase and cholinesterases.

PubMed

Jung, Hyun Ah; Ali, Md Yousof; Jung, Hee Jin; Jeong, Hyong Oh; Chung, Hae Young; Choi, Jae Sue

2016-09-15

Semen Cassiae has been traditionally used as an herbal remedy for liver, eye, and acute inflammatory diseases. Recent pharmacological reports have indicated that Cassiae semen has neuroprotective effects, attributable to its anti-inflammatory actions, in ischemic stroke and Alzheimer's disease (AD) models. The basic goal of this study was to evaluate the anti-AD activities of C. obtusifolia and its major constituents. Previously, the extract of C. obtusifolia seeds, was reported to have memory enhancing properties and anti-AD activity to ameliorate amyloid β-induced synaptic dysfunction. However, the responsible components of C. obtusifolia seeds in an AD are currently still unknown. In this study, we investigated the inhibitory effects of C. obtusifolia and its constituents against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) enzyme activity. In vitro cholinesterase enzyme assays by using AChE, BChE, and BACE1 were performed. We also scrutinized the potentials of Cassiae semen active component as BACE1 inhibitors via enzyme kinetics and molecular docking simulation. In vitro enzyme assays demonstrated that C. obtusifolia and its major constituents have promising inhibitory potential against AChE, BChE, and BACE1. All Cassiae semen constituents exhibited potent inhibitory activities against AChE and BACE1 with IC50 values of 6.29-109µg/mL and 0.94-190µg/mL, whereas alaternin, questin, and toralactone gentiobioside exhibited significant inhibitory activities against BChE with IC50 values of 113.10-137.74µg/mL. Kinetic study revealed that alaternin noncompetitively inhibited, whereas cassiaside and emodin showed mixed-type inhibition against BACE1. Furthermore, molecular docking simulation results demonstrated that hydroxyl group of alaternin and emodin tightly interacted with the active site residues of BACE1 and their relevant binding energies (-6.62 and -6.89kcal

Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity.

PubMed

Robles-Mendoza, Cecilia; Zúñiga-Lagunes, Sebastian R; Ponce de León-Hill, Claudia A; Hernández-Soto, Jesús; Vanegas-Pérez, Cecilia

2011-10-01

The axolotl Ambystoma mexicanum is a neotenic salamander considered a good biological model due to its ability to regenerate limbs, tail, brain and heart cells. Nevertheless, severe reduction of A. mexicanum wild populations in the lacustrine area of Xochimilco, the natural habitat of the axolotl, could be related to several environmental pressures as the presence of organophosphate pesticides (OPPs), intensively applied in agricultural activities in Xochimilco. Thus the aim of this study was to evaluate the effect of environmentally realistic chlorpyrifos (CPF) concentrations, a OPP commonly used in this zone, on esterases activity (acetylcholinesterase and carboxylesterase) and bioconcentration of CPF and to relate them with the motor activity of A. mexicanum juveniles. Axolotls were exposed 48 h to 0.05 and 0.1mg CPF/L, and the responses were evaluated at the end of the CPF exposure. Results suggest that CPF is bioconcentrated into axolotls and that the CPF internal concentrations are related with the observed inhibition activity of AChE (>50%) and CbE (≈ 50%). CPF concentration responsible of the inhibition of the 50% of AChE activity (IC50) was estimated in 0.04 mg CPF/L; however IC50 for CbE activity was not possible to calculate since inhibition levels were lower than 50%, results that suggest a higher resistance of CbE enzymatic activity to CPF. However, motor activity was a more sensitive endpoint to CPF poisoning since time that axolotls spent active and walking, frequency and speed of swimming, frequency of prey attack were reduced >90% of control groups. The motor activity alterations in the axolotl could be related with the registered esterases inhibition. Thus important alterations on axolotls were identified even at short time and low concentrations of CPF exposure. Also, it was possible to link biochemical responses as esterases activity with higher levels of biological organization as behavior. This study provides tools for the regulation of the

Anti-listeria activity of poly(lactic acid)/sawdust particle biocomposite film impregnated with pediocin PA-1/AcH and its use in raw sliced pork.

PubMed

Woraprayote, Weerapong; Kingcha, Yutthana; Amonphanpokin, Pannawit; Kruenate, Jittiporn; Zendo, Takeshi; Sonomoto, Kenji; Benjakul, Soottawat; Visessanguan, Wonnop

2013-10-15

A novel poly(lactic acid) (PLA)/sawdust particle (SP) biocomposite film with anti-listeria activity was developed by incorporation of pediocin PA-1/AcH (Ped) using diffusion coating method. Sawdust particle played an important role in embedding pediocin into the hydrophobic PLA film. The anti-listeria activity of the PLA/SP biocomposite film incorporated with Ped (PLA/SP+Ped) was detected, while no activity against the tested pathogen was observed for the control PLA films (without SP and/or Ped). Dry-heat treatment of film before coating with Ped resulted in the highest Ped adsorption (11.63 ± 3.07 μg protein/cm(2)) and the highest anti-listeria activity. A model study of PLA/SP+Ped as a food-contact antimicrobial packaging on raw sliced pork suggests a potential inhibition of Listeria monocytogenes (99% of total listerial population) on raw sliced pork during the chilled storage. This study supports the feasibility of using PLA/SP+Ped film to reduce the initial load of L. monocytogenes on the surface of raw pork. © 2013.

In Vitro Ability of Currently Available Oximes to Reactivate Organophosphate Pesticide-Inhibited Human Acetylcholinesterase and Butyrylcholinesterase

PubMed Central

Jun, Daniel; Musilova, Lucie; Musilek, Kamil; Kuca, Kamil

2011-01-01

We have in vitro tested the ability of common, commercially available, cholinesterase reactivators (pralidoxime, obidoxime, methoxime, trimedoxime and HI-6) to reactivate human acetylcholinesterase (AChE), inhibited by five structurally different organophosphate pesticides and inhibitors (paraoxon, dichlorvos, DFP, leptophos-oxon and methamidophos). We also tested reactivation of human butyrylcholinesterase (BChE) with the aim of finding a potent oxime, suitable to serve as a “pseudocatalytic” bioscavenger in combination with this enzyme. Such a combination could allow an increase of prophylactic and therapeutic efficacy of the administered enzyme. According to our results, the best broad-spectrum AChE reactivators were trimedoxime and obidoxime in the case of paraoxon, leptophos-oxon, and methamidophos-inhibited AChE. Methamidophos and leptophos-oxon were quite easily reactivatable by all tested reactivators. In the case of methamidophos-inhibited AChE, the lower oxime concentration (10−5 M) had higher reactivation ability than the 10−4 M concentration. Therefore, we evaluated the reactivation ability of obidoxime in a concentration range of 10−3–10−7 M. The reactivation of methamidophos-inhibited AChE with different obidoxime concentrations resulted in a bell shaped curve with maximum reactivation at 10−5 M. In the case of BChE, no reactivator exceeded 15% reactivation ability and therefore none of the oximes can be recommended as a candidate for “pseudocatalytic” bioscavengers with BChE. PMID:21673941

Canonical and Novel Non-Canonical Cholinergic Agonists Inhibit ATP-Induced Release of Monocytic Interleukin-1β via Different Combinations of Nicotinic Acetylcholine Receptor Subunits α7, α9 and α10

PubMed Central

Zakrzewicz, Anna; Richter, Katrin; Agné, Alisa; Wilker, Sigrid; Siebers, Kathrin; Fink, Bijan; Krasteva-Christ, Gabriela; Althaus, Mike; Padberg, Winfried; Hone, Arik J.; McIntosh, J. Michael; Grau, Veronika

2017-01-01

Recently, we discovered a cholinergic mechanism that inhibits the adenosine triphosphate (ATP)-dependent release of interleukin-1β (IL-1β) by human monocytes via nicotinic acetylcholine receptors (nAChRs) composed of α7, α9 and/or α10 subunits. Furthermore, we identified phosphocholine (PC) and dipalmitoylphosphatidylcholine (DPPC) as novel nicotinic agonists that elicit metabotropic activity at monocytic nAChR. Interestingly, PC does not provoke ion channel responses at conventional nAChRs composed of subunits α9 and α10. The purpose of this study is to determine the composition of nAChRs necessary for nicotinic signaling in monocytic cells and to test the hypothesis that common metabolites of phosphatidylcholines, lysophosphatidylcholine (LPC) and glycerophosphocholine (G-PC), function as nAChR agonists. In peripheral blood mononuclear cells from nAChR gene-deficient mice, we demonstrated that inhibition of ATP-dependent release of IL-1β by acetylcholine (ACh), nicotine and PC depends on subunits α7, α9 and α10. Using a panel of nAChR antagonists and siRNA technology, we confirmed the involvement of these subunits in the control of IL-1β release in the human monocytic cell line U937. Furthermore, we showed that LPC (C16:0) and G-PC efficiently inhibit ATP-dependent release of IL-1β. Of note, the inhibitory effects mediated by LPC and G-PC depend on nAChR subunits α9 and α10, but only to a small degree on α7. In Xenopus laevis oocytes heterologously expressing different combinations of human α7, α9 or α10 subunits, ACh induced canonical ion channel activity, whereas LPC, G-PC and PC did not. In conclusion, we demonstrate that canonical nicotinic agonists and PC elicit metabotropic nAChR activity in monocytes via interaction of nAChR subunits α7, α9 and α10. For the metabotropic signaling of LPC and G-PC, nAChR subunits α9 and α10 are needed, whereas α7 is virtually dispensable. Furthermore, molecules bearing a PC group in general seem to

Understanding the molecular mechanism of aryl acylamidase activity of acetylcholinesterase - An in silico study.

PubMed

Chinnadurai, Raj Kumar; Saravanaraman, Ponne; Boopathy, Rathanam

2015-08-15

Acetylcholinesterase (AChE) exhibits two different activities, namely esterase and aryl acylamidase (AAA). Unlike esterase, AAA activity of AChE is inhibited by the active site inhibitors while remaining unaffected by the peripheral anionic site inhibitors. This differential inhibitory pattern of active and peripheral anionic site inhibitors on the AAA activity remains unanswered. To answer this, we investigated the mechanism of binding and trafficking of AAA substrates using in silico tools. Molecular docking of serotonin and AAA substrates (o-nitroacetanilide, and o-nitrotrifluoroacetanilide,) onto AChE shows that these compounds bind at the side door of AChE. Thus, we conceived that the AAA substrates prefer the side door to reach the active site for their catalysis. Further, steered molecular dynamics simulations show that the force required for binding and trafficking of the AAA substrate through the side door is comparatively lesser than their dissociation (900kJ/mol/nm). Among the two substrates, o-nitrotrifluoroacetanilide required lesser force (380kJ/mol/nm) than o-nitroacetanilide the (550kJ/mol/nm) for its binding, thus validating o-nitrotrifluoroacetanilide as a better substrate. With these observations, we resolve that the AAA activity of AChE is mediated through its side door. Therefore, binding of PAS inhibitors at the main door of AChE remain ineffective against AAA activity. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro acetylcholinesterase activity of peptide derivatives isolated from two species of Leguminosae.

PubMed

Alves, Clayton Q; Lima, Luciano S; David, Jorge M; Lima, Marcos V B; David, Juceni P; Lima, Fernanda W M; Pedroza, Kelly C M C; Queiroz, Luciano P

2013-07-01

Cratylia mollis Martius ex Benth. and Cenostigma macrophyllum Tul. (Leguminosae) are both endemic Brazilian plants and they are used by the natives as medicinal plants, and the leaves of C. mollis are also employed as forage for cattle during the dry season of region. Isolation of the compounds responsible for the acetylcholinesterase (AChE) inhibition from the CHCl3 active extract. Two peptidic compounds were isolated by chromatographic techniques from the CHCl3 extract of the leaves of C. mollis and C. macrophyllum. They were identified by spectrometric data analysis (MS and NMR) and they were subjected to AChE inhibition employing Ellman's test. The peptides were identified as N-benzoylphenylalaninoyl-phenlyalaninolacetate (aurentiamide acetate) (1) and N-benzoylphenylalaninyl-N-benzoylphenylalaninate (2). Both peptides 1 and 2 exhibit AChE inhibition, with IC50 values equal to 111.34 µM and 137.6 µM, respectively. Compound 1 (aurentiamide acetate) has rarely been isolated from the Leguminosae family, and N-benzoylphenylalaninyl-N-benzoylphenylalaninate (2) is a compound that has never previously been isolated from this family. Compound 1 is shown to be a potent inhibitor of AChE, with IC50 values similar to the physostigmine control (141.51 µM).

Antioxidant and cholinesterase inhibitory activity of a new peptide from Ziziphus jujuba fruits.

PubMed

Zare-Zardini, Hadi; Tolueinia, Behnaz; Hashemi, Azam; Ebrahimi, Leila; Fesahat, Farzaneh

2013-11-01

Antioxidant agents and cholinesterase inhibitors are the foremost drugs for the treatment of Alzheimer's disease (AD). In this study, a new peptide from Ziziphus jujuba fruits was investigated for its inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes as well as antioxidant activity. This peptide was introduced as a new peptide and named Snakin-Z. The Snakin-Z displayed considerable cholinesterase inhibition against AChE and BChE. The half maximal inhibitory concentration (IC50) values of Snakin-Z against AChE and BChE are 0.58 ± 0.08 and 0.72 ± 0.085 mg/mL, respectively. This peptide has 80% enzyme inhibitory activity on AChE and BChE at 1.5 mg/mL. The Snakin-Z also had the high antioxidant activity (IC50 = 0.75 ± 0.09 mg/mL). Thus, it is suggested that Snakin-Z may be beneficial in the treatment of AD. However, more detailed researches are still required as in vivo testing its anticholinesterase and antioxidant activities.

Selectivity of coronaridine congeners at nicotinic acetylcholine receptors and inhibitory activity on mouse medial habenula.

PubMed

Arias, Hugo R; Jin, Xiaotao; Feuerbach, Dominik; Drenan, Ryan M

2017-11-01

The inhibitory activity of coronaridine congeners on human (h) α4β2 and α7 nicotinic acetylcholine receptors (AChRs) is determined by Ca 2+ influx assays, whereas their effects on neurons in the ventral inferior (VI) aspect of the mouse medial habenula (MHb) are determined by patch-clamp recordings. The Ca 2+ influx results clearly establish that coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to hα4β2 and hα7 subtypes, and with the following potency sequence, for hα4β2: (±)-18-methoxycoronaridine [(±)-18-MC]>(+)-catharanthine>(±)-18-methylaminocoronaridine [(±)-18-MAC] ∼ (±)-18-hydroxycoronaridine [(±)-18-HC]; and for hα7: (+)-catharanthine>(±)-18-MC>(±)-18-HC>(±)-18-MAC. Interestingly, the inhibitory potency of (+)-catharanthine (27±4μM) and (±)-18-MC (28±6μM) on MHb (VI) neurons was lower than that observed on hα3β4 AChRs, suggesting that these compounds inhibit a variety of endogenous α3β4* AChRs. In addition, the interaction of bupropion with (-)-ibogaine sites on hα3β4 AChRs is tested by [ 3 H]ibogaine competition binding experiments. The results indicate that bupropion binds to ibogaine sites at desensitized hα3β4 AChRs with 2-fold higher affinity than at resting receptors, suggesting that these compounds share the same binding sites. In conclusion, coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to other AChRs, by interacting with the bupropion (luminal) site. Coronaridine congeners also inhibit α3β4*AChRs expressed in MHb (VI) neurons, supporting the notion that these receptors are important endogenous targets for their anti-addictive activities. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selectivity of coronaridine congeners at nicotinic acetylcholine receptors and inhibitory activity on mouse medial habenula

PubMed Central

Arias, Hugo R.; Jin, Xiaotao; Feuerbach, Dominik; Drenan, Ryan M.

2018-01-01

The inhibitory activity of coronaridine congeners on human (h) α4β2 and α7 nicotinic acetylcholine receptors (AChRs) is determined by Ca2+ influx assays, whereas their effects on neurons in the ventral inferior (VI) aspect of the mouse medial habenula (MHb) are determined by patch-clamp recordings. The Ca2+ influx results clearly establish that coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to hα4β2 and hα7 subtypes, and with the following potency sequence, for hα4β2: (±)-18-methoxycoronaridine [(±)-18-MC] > (+)-catharanthine > (±)-18-methylaminocoronaridine [(±)-18-MAC] ∼ (±)-18-hydroxycoronaridine [(±)-18-HC]; and for hα7: (+)-catharanthine > (±)-18-MC > (±)-18-HC > (±)-18-MAC. Interestingly, the inhibitory potency of (+)-catharanthine (27 ± 4 μM) and (±)-18-MC (28 ± 6 μM) on MHb (VI) neurons was lower than that observed on hα3β4 AChRs, suggesting that these compounds inhibit a variety of endogenous α3β4* AChRs. In addition, the interaction of bupropion with (−)-ibogaine sites on hα3β4 AChRs is tested by [3H]ibogaine competition binding experiments. The results indicate that bupropion binds to ibogaine sites at desensitized hα3β4 AChRs with 2-fold higher affinity than at resting receptors, suggesting that these compounds share the same binding sites. In conclusion, coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to other AChRs, by interacting with the bupropion (luminal) site. Coronaridine congeners also inhibit α3β4*AChRs expressed in MHb (VI) neurons, supporting the notion that these receptors are important endogenous targets for their anti-addictive activities. PMID:29042244

High aryl acylamidase activity associated with cobra venom acetylcholinesterase: biological significance.

PubMed

Rajesh, Ramanna V; Layer, Paul G; Boopathy, Rathanam

2009-01-01

Investigation of the non-classical functions of cholinesterases (ChEs) has been the subject of interest in the past three decades. One of which is aryl acylamidase (AAA) activity associated with ChEs, but characterized in in vitro, as an enzyme, splitting the artificial substrate o-nitroacetanilide with unknown physiological function. In the present study, we have compared levels of AAA activity of AChE from different sources like goat brain, electric eel organ and from venoms of different snakes. Remarkably cobra venom showed the highest AAA activity and also high AAA/AChE ratio. Both serotonergenic and cholinergic inhibitors inhibited the cobra venom AAA activity in a concentration dependent manner, which also underlines the association of AAA with AChE of cobra venom. The study becomes interesting because of i) the cobra venom AChE exists in monomeric globular forms; ii) in Alzheimer's disease too the most abundant forms of cholinesterases are monomeric globular forms, thought to be involved in the pathogenesis of Alzheimer's disease; iii) the effect of Alzheimer's disease drugs on the AAA activity of cobra venom, indicated that AAA activity of cobra venom was more sensitive than AChE and iv) Huperzine and Tacrine showed more pronounced effect on AAA. Thus, this study elucidates the high AAA associated with cobra venom AChE may serve as one of the prominent activity to test the pharmacological effect of AD drugs, as other sources were found to have lower activity.

2-Arylbenzofurans from Artocarpus lakoocha and methyl ether analogs with potent cholinesterase inhibitory activity.

PubMed

Namdaung, Umalee; Athipornchai, Anan; Khammee, Thongchai; Kuno, Mayuso; Suksamrarn, Sunit

2018-01-01

In vitro screening for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of the Artocarpus lakoocha root-bark extracts revealed interesting results. Bioassay-guided fractionation resulted in the isolation of two new (1 and 2) and six known 2-arylbenzofurans 3-8, along with one stilbenoid 9 and one flavonoid 10. The structures of the isolated compounds were elucidated by UV, IR, 1D- and 2D-NMR and MS spectroscopic data analysis. Compounds 4, 6 and 7 exhibited more potent AChE inhibitory activity (IC 50  = 0.87-1.10 μM) than the reference drug, galantamine. Compounds 4, 8 and 9 displayed greater BChE inhibition than the standard drug. The preferential inhibition of BChE over AChE indicated that 4 also showed a promising dual AChE and BChE inhibitor. The synthetic mono-methylated analogs 4a-c and 6a-b were found to be good BChE inhibitors with IC 50 values ranging between 0.31 and 1.11 μM. Based on the docking studies, compounds 4 and 6 are well-fitted in the catalytic triad of AChE. Compounds 4 and 6 showed different binding orientations on BChE, and the most potent BChE inhibitor 4 occupied dual binding to both CAS and PAS more efficiently. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Reactivation of organophosphate-inhibited human, Cynomolgus monkey, swine and guinea pig acetylcholinesterase by MMB-4: a modified kinetic approach.

PubMed

Worek, Franz; Wille, Timo; Aurbek, Nadine; Eyer, Peter; Thiermann, Horst

2010-12-15

Treatment of poisoning by highly toxic organophosphorus compounds (OP, nerve agents) is a continuous challenge. Standard treatment with atropine and a clinically used oxime, obidoxime or pralidoxime is inadequate against various nerve agents. For ethical reasons testing of oxime efficacy has to be performed in animals. Now, it was tempting to investigate the reactivation kinetics of MMB-4, a candidate oxime to replace pralidoxime, with nerve agent-inhibited acetylcholinesterase (AChE) from human and animal origin in order to provide a kinetic basis for the proper assessment of in vivo data. By applying a modified kinetic approach, allowing the use of necessary high MMB-4 concentrations, it was possible to determine the reactivation constants with sarin-, cyclosarin-, VX-, VR- and tabun-inhibited AChE. MMB-4 exhibited a high reactivity and low affinity towards OP-inhibited AChE, except of tabun-inhibited enzyme where MMB-4 had an extremely low reactivity. Species differences between human and animal AChE were low (Cynomolgus) to moderate (swine, guinea pig). Due to the high reactivity of MMB-4 a rapid reactivation of inhibited AChE can be anticipated at adequate oxime concentrations which are substantially higher compared to HI-6. Additional studies are necessary to determine the in vivo toxicity, tolerability and pharmacokinetics of MMB-4 in humans in order to enable a proper assessment of the value of this oxime as an antidote against nerve agent poisoning. Copyright © 2010 Elsevier Inc. All rights reserved.

Activation and modulation of human α4β2 nicotinic acetylcholine receptors by the neonicotinoids clothianidin and imidacloprid

PubMed Central

Li, Ping; Ann, Jason; Akk, Gustav

2013-01-01

Neonicotinoids are synthetic, nicotine-derived insecticides used for agricultural and household pest control. While highly effective at activating insect nicotinic receptors, many neonicotinoids are also capable of directly activating and/or modulating the activation of vertebrate nicotinic receptors. In this study, we have investigated the actions of the neonicotinoids clothianidin (CTD) and imidacloprid (IMI) on human neuronal α4β2 nicotinic acetylcholine receptors. The data demonstrate that the compounds are weak agonists of the human receptors with relative peak currents of 1–4 % of the response to 1 mM acetylcholine (ACh). Coapplication of IMI strongly inhibited currents elicited by ACh. From Schild plot analysis, we estimate that the affinity of IMI to the human α4β2 receptor is 18 µM. The application of low concentrations of CTD potentiated responses to low concentrations of ACh, suggesting that receptors occupied by one ACh and one CTD molecule have a higher gating efficacy than receptors with one ACh bound. Interestingly, subunit stoichiometry affected inhibition by CTD, with (α4)2(β2)3 receptors significantly more strongly inhibited than the (α4)3(β2)2 receptors. PMID:21538459

Cholinesterases inhibition and molecular modeling studies of piperidyl-thienyl and 2-pyrazoline derivatives of chalcones.

PubMed

Shah, Muhammad Shakil; Khan, Shafi Ullah; Ejaz, Syeda Abida; Afridi, Saifullah; Rizvi, Syed Umar Farooq; Najam-Ul-Haq, Muhammad; Iqbal, Jamshed

2017-01-22

Super-activation of cholinesterases (acetylcholinesterase and butyrylcholinesterase) are linked to various neurological problems most precisely Alzheimer's disease (AD), which leads to senile dementia. Therefore, cholinesterases (AChE & BChE) inhibition are considered as a promising strategy for the treatment of Alzheimer's disease. FDA approved drugs for the treatment of AD, belong to a group of cholinesterase inhibitors. However, none of them is able to combat or completely abrogate the disease progression. Herein, we report a series of newly synthesized chalcone derivatives with anti-AD potential. For this purpose, a series of piperidyl-thienyl and 2-pyrazoline derivatives of chalcones were tested for their cholinesterases (AChE & BChE) inhibitory activity. All compounds were found as selective inhibitor of AChE. In piperidyl chalcones derivatives compound 1e having IC 50 of 0.16 ± 0.008 μM and 2m in 2-pyrazoline chalcones with IC 50 of 0.13 ± 0.006 μM, were found to be the most potent inhibitors of AChE, exhibiting ≈142 and ≈ 173-fold greater inhibitory potential compared to the reference inhibitor i.e., Neostigmine (IC 50  ± SEM = 22.2 ± 3.2 μM). Molecular docking studies of most potent inhibitors were carried out to investigate the binding interactions inside the active site. Molecular docking study revealed that potent compounds and co-crystalized ligand had same binding orientation within the active site of target enzyme. Most of these compounds are selective inhibitors of AChE with a potential use against progressive neurodegenerative disorder and age related problems. Copyright © 2016 Elsevier Inc. All rights reserved.

Cholinesterase activities and behavioral changes in Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed to glufosinate ammonium herbicide.

PubMed

Peltzer, Paola M; Junges, Celina M; Attademo, Andrés M; Bassó, Agustín; Grenón, Paula; Lajmanovich, Rafael C

2013-09-01

In this study, amphibian tadpoles of Hypsiboas pulchellus were exposed to herbicide Liberty®, which contains glufosinate ammonium (GLA), for 48 h to the following concentrations: 0 (control), 3.55, 4.74, 6.32, 8.43, 11.25, 15, 20, 26.6, and 35.5 mg GLA L(-1). Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities, as well as swimming capabilities (swimming speed and mean distance) were measured in tadpoles whose concentrations displayed survival rates > 85 %. Our results reveal that sublethal concentrations of GLA significantly inhibited both AChE and BChE activities in tadpoles with respect to the control, showing a concentration-dependent inhibitory effect. The highest inhibition percentages of AChE (50.86%) and BChE (53.02%) were registered in tadpoles exposed to 15 mg GLA L(-1). At this concentration, a significant increase of the swimming speed and mean distance were found in exposed tadpoles with respect to the control, as well as a negative and significant correlation between swimming speed and BChE activity, thus suggesting that this enzyme inhibition is related to an increase in swimming speed. Therefore, exposure of tadpoles to GLA in the wild at concentrations similar to those tested here may have adverse consequences at population level because neurotransmission and swimming performance are essential for tadpole performance and survival.

Plasma B-esterase activities in European raptors.

PubMed

Roy, Claudie; Grolleau, Gérard; Chamoulaud, Serge; Rivière, Jean-Louis

2005-01-01

B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and

The Antiparasitic Clioquinol Induces Apoptosis in Leukemia and Myeloma Cells by Inhibiting Histone Deacetylase Activity*

PubMed Central

Cao, Biyin; Li, Jie; Zhu, Jingyu; Shen, Mingyun; Han, Kunkun; Zhang, Zubin; Yu, Yang; Wang, Yali; Wu, Depei; Chen, Suning; Sun, Aining; Tang, Xiaowen; Zhao, Yun; Qiao, Chunhua; Hou, Tingjun; Mao, Xinliang

2013-01-01

The antiparasitic clioquinol (CQ) represents a class of novel anticancer drugs by interfering with proteasome activity. In the present study, we found that CQ induced blood cancer cell apoptosis by inhibiting histone deacetylases (HDACs). CQ accumulated the acetylation levels of several key proteins including histone H3 (H3), p53, HSP90, and α-tubulin. In the mechanistic study, CQ was found to down-regulate HDAC1, -3, -4, and -5 in both myeloma and leukemia cells. Computer modeling analysis revealed that CQ was well docked into the active pocket of the enzyme, where the oxygen and nitrogen atoms in CQ formed stable coordinate bonds with the zinc ion, and the hydroxyl group from CQ formed an effective hydrogen bond with Asp-267. Moreover, co-treatment with CQ and zinc/copper chloride led to decreased Ac-H3. Furthermore, CQ inhibited the activity of Class I and IIa HDACs in the cell-free assays, demonstrating that CQ interfered with HDAC activity. By inhibiting HDAC activity, CQ induced expression of p21, p27, and p53, cell cycle arrest at G1 phase, and cell apoptosis. This study suggested that the HDAC enzymes are targets of CQ, which provided a novel insight into the molecular mechanism of CQ in the treatment of hematological malignancies. PMID:24114842

Assisted inhibition effect of acetylcholinesterase with n-octylphosphonic acid and application in high sensitive detection of organophosphorous pesticides by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry.

PubMed

Cai, Tingting; Zhang, Li; Wang, Haoyang; Zhang, Jing; Guo, Yinlong

2011-11-14

A simple and practical approach to improve the sensitivity of acetylcholinesterase (AChE)-inhibited method has been developed for monitoring organophosphorous (OP) pesticide residues. In this work, matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to detect AChE activity. Due to its good salt-tolerance and low sample consumption, MALDI-FTMS facilitates rapid and high-throughput screening of OP pesticides. Here we describe a new method to obtain low detection limits via employing external reagents. Among candidate compounds, n-octylphosphonic acid (n-Octyl-PA) displays assistant effect to enhance AChE inhibition by OP pesticides. In presence of n-Octyl-PA, the percentages of AChE inhibition still kept correlation with OP pesticide concentrations. The detection limits were improved significantly even by 10(2)-10(3) folds in comparison with conventional enzyme-inhibited methods. Different detection limits of OP pesticides with different toxicities were as low as 0.005 μg L(-1) for high toxic pesticides and 0.05 μg L(-1) for low toxic pesticides. Besides, the reliability of results from this method to analyze cowpea samples had been demonstrated by liquid-chromatography tandem mass spectrometry (LC-MS/MS). The application of this commercial available assistant agent shows great promise to detect OP compounds in complicated biological matrix and broadens the mind for high sensitivity detection of OP pesticide residues in agricultural products. Copyright © 2011 Elsevier B.V. All rights reserved.

Inhibition of acetylcholinesterase activity by rivastigmine decreases lipopolysaccharide-induced IL-1β expression in the hypothalamus of ewes.

PubMed

Herman, A P; Krawczyńska, A; Bochenek, J; Haziak, K; Antushevitch, H; Herman, A; Tomaszewska-Zaremba, D

2013-04-01

The present study was designed to determine the effect of subcutaneous rivastigmine treatment on IL-1β expression and IL-1 type I receptor (IL-1R1) gene expression in the hypothalamic structures (preoptic area [POA], anterior hypothalamus [AHA], and medial basal hypothalamus [MBH]) of ewes after lipopolysaccharide (LPS) treatment. Endotoxin treatment increased (P ≤ 0.01) both IL-1β and IL-1R1 gene expression in the POA, AHA, and MBH compared with the control group, whereas concomitant rivastigmine and LPS injection abolished this stimulatory effect. It was also found that LPS elevated (P ≤ 0.01) IL-1β concentration in the hypothalamus (71.0 ± 2.3 pg/mg) compared with controls (16.1 ± 3.6 pg/mg). The simultaneous injection of LPS and rivastigmine did not increase IL-1β concentration in the hypothalamus (24.6 ± 13.0 pg/mg). This central change in IL-1β synthesis seems to be an effect of acetylcholinesterase (AChE) inhibition by rivastigmine, which decreases (P ≤ 0.01) the activity of this enzyme from 78.5 ± 15.0 μmol · min(-1) · g(-1) of total protein in the control and 68.8 ± 9.8 μmol · min(-1) · g(-1) of total protein in LPS-treated animals to 45.2 ± 5.6 μmol · min(-1) · g(-1) of total protein in the rivastigmine and LPS-treated group. Our study showed that rivastigmine could effectively reverse the stimulatory effect of immune stress induced by LPS injection on IL-1β synthesis through a decrease in AChE activity in the hypothalamic area of sheep. Our results also proved that the cholinergic anti-inflammatory pathway could directly modulate the central response to endotoxin. Copyright © 2013 Elsevier Inc. All rights reserved.

Decursin inhibits UVB-induced MMP expression in human dermal fibroblasts via regulation of nuclear factor-κB.

PubMed

Hwang, Bo-Mi; Noh, Eun-Mi; Kim, Jong-Suk; Kim, Jeong-Mi; Hwang, Jin-Ki; Kim, Hye-Kyung; Kang, Jae-Seon; Kim, Do-Sung; Chae, Han-Jung; You, Yong-Ouk; Kwon, Kang-Beom; Lee, Young-Rae

2013-02-01

Decursin, a coumarin compound, was originally isolated from the roots of Angelica gigas almost four decades ago, and it was found to exhibit cytotoxicity against various types of human cancer cells and anti-amnesic activity in vivo through the inhibition of AChE activity. However, the anti-skin photoaging effects of decursin have not been reported to date. In the present study, we investigated the inhibitory effects of decursin on the expression of matrix metalloproteinase (MMP)-1 and MMP-3 in human dermal fibroblast (HDF) cells. Western blot analysis and real-time PCR revealed that decursin inhibited the ultraviolet (UV)B-induced expression of MMP-1 and MMP-3 in a dose-dependent manner. Decursin significantly blocked the UVB-induced activation of nuclear factor-κB (NF-κB). However, decursin showed no effect on MAPK or AP-1 activity. In this study, decursin prevented the UVB-induced expression of MMPs via the inhibition of NF-κB activation. In conclusion, decursin may be a potential agent for the prevention and treatment of skin photoaging.

Physiologically-Based Pharmacokinetic and Pharmacodynamic Modeling for the Inhibition of Acetylcholinesterase by Acotiamide, A Novel Gastroprokinetic Agent for the Treatment of Functional Dyspepsia, in Rat Stomach.

PubMed

Yoshii, Kazuyoshi; Iikura, Minami; Hirayama, Masamichi; Toda, Ryoko; Kawabata, Yoshihiro

2016-02-01

Acotiamide, a gastroprokinetic agent used to treat functional dyspepsia, is transported to at least two compartments in rat stomach. However, the role of these stomach compartments in pharmacokinetics and pharmacodynamics of acotiamide remains unclear. Thus, the purpose of this study was to elucidate the relationship of the blood and stomach concentration of acotiamide with its inhibitory effect on acetylcholinesterase (AChE). Concentration profiles of acotiamide and acetylcholine (ACh) were determined after intravenous administration to rats and analyzed by physiologically-based pharmacokinetic and pharmacodynamic (PBPK/PD) model containing vascular space, precursor pool and deep pool of stomach. Acotiamide was eliminated from the blood and stomach in a biexponential manner. Our PBPK/PD model estimated that acotiamide concentration in the precursor pool exceeded 2 μM at approximately 2 h after administration. Acotiamide inhibited AChE activity in vitro with a 50% inhibitory concentration of 1.79 μM. ACh reached the maximum concentration at 2 h after administration. Our PBPK model well described the profile of acotiamide and ACh concentration in the stomach in the assumption that acotiamide was distributed by carrier mediated process and inhibited AChE in the precursor pool of stomach. Thus, Acotiamide in the precursor pool plays an important role for producing the pharmacological action.

Effect of carbaryl (carbamate insecticide) on acetylcholinesterase activity of two strains of Daphnia magna (Crustacea, Cladocera).

PubMed

Toumi, Hela; Bejaoui, Mustapha; Touaylia, Samir; Burga Perez, Karen F; Ferard, Jean François

2016-11-01

The present study was designed to investigate the effect of carbaryl (carbamate insecticide) on the acetylcholinesterase activity in two strains (same clone A) of the crustacean cladoceran Daphnia magna. Four carbaryl concentrations (0.4, 0.9, 1.8 and 3.7 µg L(-1)) were compared against control AChE activity. Our results showed that after 48 h of carbaryl exposure, all treatments induced a significant decrease of AChE activities whatever the two considered strains. However, different responses were registered in terms of lowest observed effect concentrations (LOEC: 0.4 µg L(-1) for strain 1 and 0.9 µg L(-1) for strains 2) revealing differences in sensitivity among the two tested strains of D. magna. These results suggest that after carbaryl exposure, the AChE activity responses can be also used as a biomarker of susceptibility. Moreover, our results show that strain1 is less sensitive than strain 2 in terms of IC50-48 h of AChE activity. Comparing the EC50-48 h of standard ecotoxicity test and IC50-48 h of AChE inhibition, there is the same order of sensitivity with both strains.

Novel Method of Preparation and Activity Research on Arctigenin from Fructus Arctii.

PubMed

Cai, Enbo; Han, Jiahong; Yang, Limin; Zhang, Weiyuan; Zhao, Yan; Chen, Qiulian; Guo, Meng; He, Xinhong

2018-01-01

Arctigenin has many pharmacological activities with clinical significance and is derived from Arctium lappa L. However, the present extraction method is inefficient and does not have meaningful industrial production. A new method to directly prepare arctigenin was established by combining enzyme-assisted extraction and central composite design. Arctigenin's further pharmacological activity was also surveyed in vitro . β-D-Glucosidase, a food-grade enzyme, was added directly to the fruits of A. lappa L. to hydrolyze the arctiin to arctigenin, and the obtained samples were subsequently subjected to ethanol (30%, v/v) extraction. The pharmacological activity of the extraction and arctigenin was determined by inhibiting acetylcholinesterase (AChE) and scavenging nitrite. The factors investigated include the enzyme concentration (0.5%-2.5%), ultrasound time (10 min -3 0 min), and extraction temperature (30°C-50°C). From the analysis of the results by Design-Expert (V8.0.6), the optimal extraction conditions were obtained: enzyme concentration (1.4%), ultrasound time (25 min), and extraction temperature (45°C). The highest yield of arctigenin, obtained under the optimal conditions was 6.39%, representing an increase of 28.15% compared to the reference extraction without enzyme processing. The IC 50 values of the extraction and arctigenin, respectively, for inhibiting AChE were 0.572 mg/ml and 0.462 mg/ml, and those for nitrite-scavenging were 34.571 mg/ml and 17.49 mg/ml. The results demonstrate that using an enzyme directly in the production is an effective means for extracting arctigenin from Fructus arctii. The extraction has the activities of inhibiting AChE and scavenging nitrite, probably because there has arctigenin in it. It is implied that the extraction and arctigenin could contribute to human health in clinical applications. The new method of adding enzyme directly to the preparation of arctigenin was carried out instead of preparing arctigenin by two

Novel Method of Preparation and Activity Research on Arctigenin from Fructus Arctii

PubMed Central

Cai, Enbo; Han, Jiahong; Yang, Limin; Zhang, Weiyuan; Zhao, Yan; Chen, Qiulian; Guo, Meng; He, Xinhong

2018-01-01

Background: Arctigenin has many pharmacological activities with clinical significance and is derived from Arctium lappa L. However, the present extraction method is inefficient and does not have meaningful industrial production. Objective: A new method to directly prepare arctigenin was established by combining enzyme-assisted extraction and central composite design. Arctigenin's further pharmacological activity was also surveyed in vitro. Materials and Methods: β-D-Glucosidase, a food-grade enzyme, was added directly to the fruits of A. lappa L. to hydrolyze the arctiin to arctigenin, and the obtained samples were subsequently subjected to ethanol (30%, v/v) extraction. The pharmacological activity of the extraction and arctigenin was determined by inhibiting acetylcholinesterase (AChE) and scavenging nitrite. Results: The factors investigated include the enzyme concentration (0.5%–2.5%), ultrasound time (10 min−3 0 min), and extraction temperature (30°C–50°C). From the analysis of the results by Design-Expert (V8.0.6), the optimal extraction conditions were obtained: enzyme concentration (1.4%), ultrasound time (25 min), and extraction temperature (45°C). The highest yield of arctigenin, obtained under the optimal conditions was 6.39%, representing an increase of 28.15% compared to the reference extraction without enzyme processing. The IC50 values of the extraction and arctigenin, respectively, for inhibiting AChE were 0.572 mg/ml and 0.462 mg/ml, and those for nitrite-scavenging were 34.571 mg/ml and 17.49 mg/ml. Conclusions: The results demonstrate that using an enzyme directly in the production is an effective means for extracting arctigenin from Fructus arctii. The extraction has the activities of inhibiting AChE and scavenging nitrite, probably because there has arctigenin in it. It is implied that the extraction and arctigenin could contribute to human health in clinical applications. SUMMARY The new method of adding enzyme directly to the

Acetylcholinesterase and butyrylcholinesterase inhibitory activity of Pinus species essential oils and their constituents.

PubMed

Bonesi, Marco; Menichini, Federica; Tundis, Rosa; Loizzo, Monica R; Conforti, Filomena; Passalacqua, Nicodemo G; Statti, Giancarlo A; Menichini, Francesco

2010-10-01

This study aimed to investigate the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity of the essential oils from Pinus nigra subsp. nigra, P. nigra var. calabrica, and P. heldreichii subsp. leucodermis. This activity is relevant to the treatment of Alzheimer's disease (AD), since cholinesterase drugs are currently the only drugs available to treat AD. P. heldreichii subsp. leucodermis exhibited the most promising activity, with IC(50) values of 51.1 and 80.6 microg/mL against AChE and BChE, respectively. An interesting activity against AChE was also observed with P. nigra subsp. nigra essential oil, with an IC(50) value of 94.4 microg/mL. Essential oils were analyzed by GC and GC-MS with the purpose of investigating their relationships with the observed activities. Among the identified constituents, terpinolene, beta-phellandrene, linalyl acetate, trans-caryophyllene, and terpinen-4-ol were tested. trans-Caryophyllene and terpinen-4-ol inhibited BChE with IC(50) values of 78.6 and 107.6 microg/mL, respectively. beta-Phellandrene was selective against AChE (IC(50) value of 120.2 microg/mL).

Acetylcholinesterase, butyrylcholinesterase and paraoxonase 1 activities in rats treated with cannabis, tramadol or both.

PubMed

Abdel-Salam, Omar M E; Youness, Eman R; Khadrawy, Yasser A; Sleem, Amany A

2016-11-01

To investigate the effect of Cannabis sativa resin and/or tramadol, two commonly drugs of abuse on acetylcholinesterase and butyrylcholinesterase activities as a possible cholinergic biomarkers of neurotoxicity induced by these agents. Rats were treated with cannabis resin (5, 10 or 20 mg/kg) (equivalent to the active constituent Δ 9 -tetrahydrocannabinol), tramadol (5, 10 and 20 mg/kg) or tramadol (10 mg/kg) combined with cannabis resin (5, 10 and 20 mg/kg) subcutaneously daily for 6 weeks. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in brain and serum. We also measured the activity of paraoxonase-1 (PON1) in serum of rats treated with these agents. (i) AChE activity in brain increased after 10-20 mg/kg cannabis resin (by 16.3-36.5%). AChE activity in brain did not change after treatment with 5-20 mg/kg tramadol. The administration of both cannabis resin (5, 10 or 20 mg/kg) and tramadol (10 mg/kg) resulted in decreased brain AChE activity by 14.1%, 12.9% and 13.6%, respectively; (ii) BChE activity in serum was markedly and dose-dependently inhibited by cannabis resin (by 60.9-76.9%). BChE activity also decreased by 17.6-36.5% by 10-20 mg/kg tramadol and by 57.2-63.9% by the cannabis resin/tramadol combined treatment; (iii) Cannabis resin at doses of 20 mg/kg increased serum PON1 activity by 25.7%. In contrast, tramadol given at 5, 10 and 20 mg/kg resulted in a dose-dependent decrease in serum PON1 activity by 19%, 36.7%, and 46.1%, respectively. Meanwhile, treatment with cannabis resin plus tramadol resulted in 40.2%, 35.8%, 30.7% inhibition of PON1 activity compared to the saline group. These data suggest that cannabis resin exerts different effects on AChE and BChE activities which could contribute to the memory problems and the decline in cognitive function in chronic users. Copyright © 2016 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.

Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry.

PubMed

Li, He; Schopfer, Lawrence M; Nachon, Florian; Froment, Marie-Thérèse; Masson, Patrick; Lockridge, Oksana

2007-11-01

Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser-O-P(O)-(Y)-(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X-R moiety is dealkylated by breaking either the P-X or the X-R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of (18)O, if cleavage was at the P-X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25 degrees C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O-C bond cleavage, i.e., the classical X-R scission pathway. In contrast, isomalathion aged through both X-R and P-X pathways; the main aged product resulted from P-S bond cleavage and a minor product resulted from O-C and/or S-C bond cleavage.

Competitive inhibition of the nondepolarizing muscle relaxant rocuronium on nicotinic acetylcholine receptor channels in the rat superior cervical ganglia.

PubMed

Zhang, Chengmi; Wang, Zhenmeng; Zhang, Jinmin; Qiu, Haibo; Sun, Yuming; Yang, Liqun; Wu, Feixiang; Zheng, Jijian; Yu, Weifeng

2014-05-01

A number of case reports now indicate that rocuronium can induce a number of serious side effects. We hypothesized that these side effects might be mediated by the inhibition of nicotinic acetylcholine receptors (nAChRs) at superior cervical ganglion (SCG) neurons. Conventional patch clamp recordings were used to study the effects of rocuronium on nAChR currents from enzymatically dissociated rat SCG neurons. We found that ACh induced a peak transient inward current in rat SCG neurons. Additionally, rocuronium suppressed the peak ACh-evoked currents in rat SCG neurons in a concentration-dependent and competitive manner, and it increased the extent of desensitization of nAChRs. The inhibitory rate of rocuronium on nAChR currents did not change significantly at membrane potentials between -70 and -20 mV, suggesting that this inhibition was voltage independent. Lastly, rocuronium preapplication enhanced its inhibitory effect, indicating that this drug might prefer to act on the closed state of nAChR channels. In conclusion, rocuronium, at clinically relevant concentrations, directly inhibits nAChRs at the SCG by interacting with both opened and closed states. This inhibition is competitive, dose dependent, and voltage independent. Blockade of synaptic transmission in the sympathetic ganglia by rocuronium might have potentially inhibitory effects on the cardiovascular system.

D2 dopamine receptor activation inhibits basal and forskolin-evoked acetylcholine release from dissociated striatal cholinergic interneurons.

PubMed

Login, I S

1997-02-21

We tested whether D2 ligands inhibit basal and forskolin-stimulated [3H]ACh release from dissociated striata, as opposed to striatal slices. Quinpirole inhibited both basal (40% maximal inhibition; IC50 approximately 50 nM) and 10 microM forskolin-stimulated release (80% inhibition; IC50 approximately 25 nM quinpirole) and both actions were blocked by a D2 antagonist. Vesamicol prevented the quinpirole and forskolin actions. The ability of D2 agonists to inhibit basal and cyclase-stimulated acetylcholine release emanating from vesamicol-sensitive vesicles appears to be tonically suppressed by inhibitory elements within striatal circuitry.

In vitro effects of acetylcholinesterase reactivators on monoamine oxidase activity.

PubMed

Fišar, Zdeněk; Hroudová, Jana; Korábečný, Jan; Musílek, Kamil; Kuča, Kamil

2011-03-05

Administration of acetylcholinesterase (AChE) reactivators (oximes) is usually used in order to counteract the poisoning effects of nerve agents. The possibility was suggested that oximes may show some therapeutic and/or adverse effects through their action in central nervous system. There are no sufficient data about interaction of oximes with monoaminergic neurotransmitter's systems in the brain. Oxime-type AChE reactivators pralidoxime, obidoxime, trimedoxime, methoxime and HI-6 were tested for their potential to affect the activity of monoamine oxidase of type A (MAO-A) and type B (MAO-B) in crude mitochondrial fraction of pig brains. The compounds were found to inhibit fully MAO-A with half maximal inhibitory concentration (IC(50)) of 0.375 mmol/l (pralidoxime), 1.53 mmol/l (HI-6), 2.31 mmol/l (methoxime), 2.42 mmol/l (obidoxime) and 4.98 mmol/l (trimedoxime). Activity of MAO-B was fully inhibited by HI-6 and pralidoxime only with IC(50) 4.81 mmol/l and 11.01 mmol/l, respectively. Methoxime, obidoxime and trimedoxime displayed non-monotonic concentration dependent effect on MAO-B activity. Because oximes concentrations effective for MAO inhibition could not be achieved in vivo at the cerebral level, we suppose that oximes investigated do not interfere with brain MAO at therapeutically relevant concentrations. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Evaluation of Antioxidant, Antidiabetic and Anticholinesterase Activities of Smallanthus sonchifolius Landraces and Correlation with Their Phytochemical Profiles

PubMed Central

Russo, Daniela; Valentão, Patrícia; Andrade, Paula B.; Fernandez, Eloy C.; Milella, Luigi

2015-01-01

The present study aimed to investigate the phytochemical profile of leaf methanol extracts of fourteen Smallanthus sonchifolius (yacon) landraces and their antioxidant, anticholinesterase and antidiabetic activities that could lead to the finding of more effective agents for the treatment and management of Alzheimer’s disease and diabetes. For this purpose, antioxidant activity was assessed using different tests: ferric reducing ability power (FRAP), 2,2-diphenyl-1-picryl hydrazyl (DPPH), nitric oxide (˙NO) and superoxide (O2˙−) scavenging and lipid peroxidation inhibition assays. Anticholinesterase activity was investigated by quantifying the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities, whereas antidiabetic activity was investigated by α-amylase and α-glucosidase inhibition tests. To understand the contribution of metabolites, phytochemical screening was also performed by high performance liquid chromatography-diode array detector (HPLC-DAD) system. Among all, methanol extract of PER09, PER04 and ECU44 landraces exhibited the highest relative antioxidant capacity index (RACI). ECU44 was found to be rich in 4,5-di-O-caffeoylquinic acid (CQA) and 3,5-di-O-CQA and displayed a good α-amylase and α-glucosidase inhibition, showing the lowest IC50 values. Flavonoids, instead, seem to be involved in the AChE and BChE inhibition. The results of this study revealed that the bioactive compound content differences could be determinant for the medicinal properties of this plant especially for antioxidant and antidiabetic activities. PMID:26263984

Evaluation of Antioxidant, Antidiabetic and Anticholinesterase Activities of Smallanthus sonchifolius Landraces and Correlation with Their Phytochemical Profiles.

PubMed

Russo, Daniela; Valentão, Patrícia; Andrade, Paula B; Fernandez, Eloy C; Milella, Luigi

2015-07-31

The present study aimed to investigate the phytochemical profile of leaf methanol extracts of fourteen Smallanthus sonchifolius (yacon) landraces and their antioxidant, anticholinesterase and antidiabetic activities that could lead to the finding of more effective agents for the treatment and management of Alzheimer's disease and diabetes. For this purpose, antioxidant activity was assessed using different tests: ferric reducing ability power (FRAP), 2,2-diphenyl-1-picryl hydrazyl (DPPH), nitric oxide (˙NO) and superoxide (O2˙-) scavenging and lipid peroxidation inhibition assays. Anticholinesterase activity was investigated by quantifying the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities, whereas antidiabetic activity was investigated by α-amylase and α-glucosidase inhibition tests. To understand the contribution of metabolites, phytochemical screening was also performed by high performance liquid chromatography-diode array detector (HPLC-DAD) system. Among all, methanol extract of PER09, PER04 and ECU44 landraces exhibited the highest relative antioxidant capacity index (RACI). ECU44 was found to be rich in 4,5-di-O-caffeoylquinic acid (CQA) and 3,5-di-O-CQA and displayed a good α-amylase and α-glucosidase inhibition, showing the lowest IC50 values. Flavonoids, instead, seem to be involved in the AChE and BChE inhibition. The results of this study revealed that the bioactive compound content differences could be determinant for the medicinal properties of this plant especially for antioxidant and antidiabetic activities.

Nicotinic ACh Receptors as Therapeutic Targets in CNS Disorders

PubMed Central

Dineley, Kelly T.; Pandya, Anshul A.; Yakel, Jerrel L.

2015-01-01

The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor channels (nAChRs). These receptors are widely distributed throughout the central nervous system, being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in the mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimer’s disease), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain. PMID:25639674

Nicotinic ACh receptors as therapeutic targets in CNS disorders.

PubMed

Dineley, Kelly T; Pandya, Anshul A; Yakel, Jerrel L

2015-02-01

The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor (nAChR) channels. These receptors are widely distributed throughout the central nervous system (CNS), being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however, the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimer's disease, AD), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain. Published by Elsevier Ltd.

New multipotent tetracyclic tacrines with neuroprotective activity.

PubMed

Marco-Contelles, José; León, Rafael; de los Ríos, Cristóbal; García, Antonio G; López, Manuela G; Villarroya, Mercedes

2006-12-15

The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues (5-13) are described. Compounds 7, 8 and 11 showed a significant neuroprotective effect on neuroblastoma cells subjected to Ca(2+) overload or free radical induced toxicity. These compounds are modest AChE inhibitors [the best inhibitor (11) is 50-fold less potent than tacrine], but proved to be very selective, as for most of them no BuChE inhibition was observed. In addition, the propidium displacement experiments showed that these compounds bind AChE to the peripheral anionic site (PAS) of AChE and, consequently, are potential agents that can prevent the aggregation of beta-amyloid. Overall, compound 8 is a modest and selective AChE inhibitor, but an efficient neuroprotective agent against 70mM K(+) and 60microM H(2)O(2). Based on these results, some of these molecules can be considered as lead candidates for the further development of anti-Alzheimer drugs.

Oxidation at C-16 enhances butyrylcholinesterase inhibition in lupane triterpenoids.

PubMed

Castro, María Julia; Richmond, Victoria; Faraoni, María Belén; Murray, Ana Paula

2018-05-17

A set of triterpenoids with different grades of oxidation in the lupane skeleton were prepared and evaluated as cholinesterase inhibitors. Allylic oxidation with selenium oxide and Jones's oxidation were employed to obtain mono-, di- and tri-oxolupanes, starting from calenduladiol (1) and lupeol (3). All the derivatives showed a selective inhibition of butyrylcholinesterase over acetylcholinesterase (BChE vs. AChE). A kinetic study proved that compounds 2 and 9, the more potent inhibitors of the series, act as competitive inhibitors. Molecular modeling was used to understand their interaction with BChE, the role of carbonyl at C-16 and the selectivity towards this enzyme over AChE. These results indicate that oxidation at C-16 of the lupane skeleton is a key transformation in order to improve the cholinesterase inhibition of these compounds. Copyright © 2018 Elsevier Inc. All rights reserved.

Phenolic composition, antioxidant and anti-acetylcholinesterase activities of the Tunisian Scabiosa arenaria.

PubMed

Besbes Hlila, Malek; Omri, Amel; Ben Jannet, Hichem; Lamari, Ali; Aouni, Mahjoub; Selmi, Boulbaba

2013-05-01

There is a need for the discovery of novel natural antioxidants and acetylcholinesterase inhibitors (AChEIs) that are safe and effective at a global level. This is the first study on antioxidant and anti-acethylcholinesterase activity of Scabiosa arenaria Forssk (Dipsacaceae). The antioxidant potential and anti-acetylcholinesterase (AChE) activity of S. arenaria were investigated. The crude, ethyl acetate (EtOAc), butanol (n-BuOH) and water extracts prepared from flowers, fruits and stems and leaves of S. arenaria were tested to determine their total polyphenol content (TPC), total flavonoid content (TFC), total condensed tannin content (CTC) and their antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), reducing power and β-carotene bleaching inhibition activity. Anti-AChE activity was also determined. EtOAc and n-BuOH fractions of fruits had both the highest (TPC) (269.09 mg gallic acid equivalents/g dry weight). The crude extract of stems and leaves had the highest TFC (10.9 mg quercetin equivalent/g dry weight). The n-BuOH fraction of stems and leaves had the highest CTC (489.75 mg catechin equivalents/g dry weight). The EtOAc fraction of flowers exhibit a higher activity in each antioxidant system with a special attention for DPPH assay (IC50 = 0.017 mg/mL) and reducing power (EC50 = 0.02 mg/mL). The EtOAc and n-BuOH fractions of stems and leaves showed strong inhibition of AChE (IC50 = 0.016 and 0.029 mg/mL, respectively). These results suggest the potential of S. arenaria as a possible source of novel compounds and as an alternative antioxidant and AChEIs.

Molecular docking and ex vivo and in vitro anticholinesterase activity studies of Salvia sp. and highlighted rosmarinic acid.

PubMed

Demirezer, Lütfiye Ömür; Gürbüz, Perihan; Kelicen Uğur, Emine Pelin; Bodur, Mine; Özenver, Nadire; Uz, Ayse; Güvenalp, Zühal

2015-01-01

To evaluate acetylcholinesterase (AChE) inhibitory activity and antioxidant capacity of the major molecule from Salvia sp., rosmarinic acid, as a drug candidate molecule for treatment of Alzheimer disease (AD). The AChE inhibitory activity of different extracts from Salvia trichoclada, Salvia verticillata, and Salvia fruticosa was determined by the Ellman and isolated guinea pig ileum methods, and the antioxidant capacity was determined with DPPH. The AChE inhibitory activity of the major molecule rosmarinic acid was determined by in silico docking and isolated guinea pig ileum methods. The methanol extract of Salvia trichoclada showed the highest inhibition on AChE. The same extract and rosmarinic acid showed significant contraction responses on isolated guinea pig ileum. All the extracts and rosmarinic acid showed high radical scavenging capacities. Docking results of rosmarinic acid showed high affinity to the selected target, AChE. In this study in vitro and ex vivo studies and in silico docking research of rosmarinic acid were used simultaneously for the first time. Rosmarinic acid showed promising results in all the methods tested.

Binary mixtures of azinphos-methyl oxon and chlorpyrifos oxon produce in vitro synergistic cholinesterase inhibition in Planorbarius corneus.

PubMed

Cacciatore, Luis Claudio; Kristoff, Gisela; Verrengia Guerrero, Noemí R; Cochón, Adriana C

2012-07-01

In this study, the cholinesterase (ChE) and carboxylesterase (CES) activities present in whole organism homogenates from Planorbarius corneus and their in vitro sensitivity to organophosphorous (OP) pesticides were studied. Firstly, a characterization of ChE and CES activities using different substrates and selective inhibitors was performed. Secondly, the effects of azinphos-methyl oxon (AZM-oxon) and chlorpyrifos oxon (CPF-oxon), the active oxygen analogs of the OP insecticides AZM and CPF, on ChE and CES activities were evaluated. Finally, it was analyzed whether binary mixtures of the pesticide oxons cause additive, antagonistic or synergistic ChE inhibition in P. corneus homogenates. The results showed that the extracts of P. corneus preferentially hydrolyzed acetylthiocholine (AcSCh) over propionylthiocholine (PrSCh) and butyrylthiocholine (BuSCh). Besides, AcSCh hydrolyzing activity was inhibited by low concentrations of BW284c51, a selective inhibitor of AChE activity, and also by high concentrations of substrate. These facts suggest the presence of a typical AChE activity in this species. However, the different dose-response curves observed with BW284c51 when using PrSCh or BuSCh instead of AcSCh suggest the presence of at least another ChE activity. This would probably correspond to an atypical BuChE. Regarding CES activity, the highest specific activity was obtained when using 2-naphthyl acetate (2-NA), followed by 1-naphthyl acetate (1-NA); p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). The comparison of the IC(50) values revealed that, regardless of the substrate used, CES activity was approximately one order of magnitude more sensitive to AZM-oxon than ChE activity. Although ChE activity was very sensitive to CPF-oxon, CES activity measured with 1-NA, 2-NA, and p-NPA was poorly inhibited by this pesticide. In contrast, CES activity measured with p-NPB was equally sensitive to CPF-oxon than ChE activity. Several specific binary

Acetylcholinesterase inhibitory activity of Thai traditional nootropic remedy and its herbal ingredients.

PubMed

Tappayuthpijarn, Pimolvan; Itharat, Arunporn; Makchuchit, Sunita

2011-12-01

The incidence of Alzheimer disease (AD) is increasing every year in accordance with the increasing of elderly population and could pose significant health problems in the future. The use of medicinal plants as an alternative prevention or even for a possible treatment of the AD is, therefore, becoming an interesting research issue. Acetylcholinesterase (AChE) inhibitors are well-known drugs commonly used in the treatment of AD. The aim of the present study was to screen for AChE inhibitory activity of the Thai traditional nootropic recipe and its herbal ingredients. The results showed that ethanolic extracts of four out of twenty-five herbs i.e. Stephania pierrei Diels. Kaempfera parviflora Wall. ex Baker, Stephania venosa (Blume) Spreng, Piper nigrum L at 0.1 mg/mL showed % AChE inhibition of 89, 64, 59, 50; the IC50 were 6, 21, 29, 30 microg/mL respectively. The other herbs as well as combination of the whole recipe had no synergistic inhibitory effect on AChE activity. However some plants revealed antioxidant activity. More research should have be performed on this local wisdom remedy to verify the uses in scientific term.

Immune responses to HTLV-I(ACH) during acute infection of pig-tailed macaques.

PubMed

McGinn, Therese M; Wei, Qing; Stallworth, Jackie; Fultz, Patricia N

2004-04-01

Human T cell lymphotropic virus type 1 (HTLV-I) is causally linked to adult T cell leukemia/lymphoma (ATL) and a chronic progressive neurological disease, HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A nonhuman primate model that reproduces disease symptoms seen in HTLV-I-infected humans might facilitate identification of initial immune responses to the virus and an understanding of pathogenic mechanisms in HTLV-I-related disease. Previously, we showed that infection of pig-tailed macaques with HTLV-I(ACH) is associated with multiple signs of disease characteristic of both HAM/TSP and ATL. We report here that within the first few weeks after HTLV-I(ACH) infection of pig-tailed macaques, serum concentrations of interferon (IFN)-alpha increased and interleukin-12 decreased transiently, levels of nitric oxide were elevated, and activation of CD4(+) and CD8(+) lymphocytes and CD16(+) natural killer cells in peripheral blood were observed. HTLV-I(ACH) infection elicited virus-specific antibodies in all four animals within 4 to 6 weeks; however, Tax-specific lymphoproliferative responses were not detected until 25-29 weeks after infection in all four macaques. IFN-gamma production by peripheral blood cells stimulated with a Tax or Gag peptide was detected to varying degrees in all four animals by ELISPOT assay. Peripheral blood lymphocytes from one animal that developed only a marginal antigen-specific cellular response were unresponsive to mitogen stimulation during the last few weeks preceding its death from a rapidly progressive disease syndrome associated with HTLV-I(ACH) infection of pig-tailed macaques. The results show that during the first few months after HTLV-I(ACH) infection, activation of both innate and adaptive immunity, limited virus-specific cellular responses, sustained immune system activation, and, in some cases, immunodeficiency were evident. Thus, this animal model might be valuable for understanding early stages of infection

Biocontrol of blue mold on apple fruits by Aureobasidium pullulans (strain Ach 1-1): in vitro and in situ evidence for the possible involvement of competition for nutrients.

PubMed

Bencheqroun, S Krimi; Bajji, M; Massart, S; Bentata, F; Labhilili, M; Achbani, H; El Jaafari, S; Jijakli, M H

2006-01-01

Aureobasidium pullulans strain Ach1-1 was recently isolated for its biocontrol effectiveness against Penicillium expansum, the causal agent of blue mold on harvested apples. In the present study, strain Ach1-1 was found to be very effective in controlling P. expansum on apple wounds. For in vitro tests, strain Ach1-1 and P. expansum were cocultured in the presence of apple juice (0 - 5%) using a system preventing direct contact between both agents. The presence of the antagonist greatly reduced germination of conidia at low (0.1, 0.5 and 1%) but not at high (5%) juice concentrations. Germination of previously inhibited conidia at 0.5% apple juice was partially restored in the presence of the antagonist when fresh juice was added at a final concentration of 5%, and completely recovered at both 0.5 and 5% juice concentrations in the absence of the antagonist. These data show that P. expansum conidia are able to germinate when cocultered with strain Ach1-1 in conditions of sufficient rather than limited nutrient availability and that the antagonist does not affect the viability of these conidia, indicating that the inhibitory effect of strain Ach1-1 on conidia germination may be due to a competition for nutrients. Such observation was confirmed in situ since the application of high amounts of exogenous amino acids, vitamins or sugars on apple wounds significantly reduced the protective level of strain Ach1-1 against P. expansum, the most important effect was obtained with amino acids followed by vitamins and then by sugars. The present work provides both in vitro and in situ evidence that the biocontrol activity of strain Ach1-1 against P. expansum essentially relies on competition for apple fruit nutrients, especially amino acids.

Effect of allyl isothiocyanate on ultra-structure and the activities of four enzymes in adult Sitophilus zeamais.

PubMed

Wu, Hua; Liu, Xue-ru; Yu, Dong-dong; Zhang, Xing; Feng, Jun-tao

2014-02-01

Rarefaction and vacuolization of the mitochondrial matrix of AITC-treated (allyl isothiocyanate-treated) adult Sitophilus zeamais were evident according to the ultra-structural by TEM. Four important enzymes in adult S. zeamais were further studied after fumigation treatment with allyl isothiocyanate (AITC) extracted from Armoracia rusticana roots and shoots. The enzymes were glutathione S-transferase (GST), catalase (CAT), cytochrome c oxidase, and acetylcholinesterase (AChE). The results indicated that the activities of the four enzymes were strongly time and dose depended. With prolonged exposure time, treatment with 0.74μg/mL AITC inhibited the activities of cytochrome c oxidase, AChE, and CAT, but induced the activity of GST. The activities of cytochrome c oxidase, AChE, and CAT were remarkably induced at a low AITC dosage (0.25μg/mL), but were restrained with increased AITC dosage. The activity of GST was inhibited at a low AITC dosage (0.5μg/mL), but was induced at a high AITC dosage (1.5μg/mL). According to the results of TEM, toxic symptoms and enzymes activities, it suggested that mitochondrial maybe the one site of action of AITC against the adult S. zeamais and it also suggested that cytochrome c oxidase maybe one target protein of AITC against the adult S. zeamais, which need to further confirmed by protein function tested. Copyright © 2014 Elsevier Inc. All rights reserved.

(-)-Phenserine inhibits neuronal apoptosis following ischemia/reperfusion injury.

PubMed

Chang, Cheng-Fu; Lai, Jing-Huei; Wu, John Chung-Che; Greig, Nigel H; Becker, Robert E; Luo, Yu; Chen, Yen-Hua; Kang, Shuo-Jhen; Chiang, Yung-Hsiao; Chen, Kai-Yun

2017-12-15

Stroke commonly leads to adult disability and death worldwide. Its major symptoms are spastic hemiplegia and discordant motion, consequent to neuronal cell death induced by brain vessel occlusion. Acetylcholinesterase (AChE) is upregulated and allied with inflammation and apoptosis after stroke. Recent studies suggest that AChE inhibition ameliorates ischemia-reperfusion injury and has neuroprotective properties. (-)-Phenserine, a reversible AChE inhibitor, has a broad range of actions independent of its AChE properties, including neuroprotective ones. However, its protective effects and detailed mechanism of action in the rat middle cerebral artery occlusion model (MCAO) remain to be elucidated. This study investigated the therapeutic effects of (-)-phenserine for stroke in the rat focal cerebral ischemia model and oxygen-glucose deprivation/reperfusion (OGD/RP) damage model in SH-SY5Y neuronal cultures. (-)-Phenserine mitigated OGD/PR-induced SH-SY5Y cell death, providing an inverted U-shaped dose-response relationship between concentration and survival. In MCAO challenged rats, (-)-phenserine reduced infarction volume, cell death and improved body asymmetry, a behavioral measure of stoke impact. In both cellular and animal studies, (-)-phenserine elevated brain-derived neurotrophic factor (BDNF) and B-cell lymphoma 2 (Bcl-2) levels, and decreased activated-caspase 3, amyloid precursor protein (APP) and glial fibrillary acidic protein (GFAP) expression, potentially mediated through the ERK-1/2 signaling pathway. These actions mitigated neuronal apoptosis in the stroke penumbra, and decreased matrix metallopeptidase-9 (MMP-9) expression. In synopsis, (-)-phenserine significantly reduced neuronal damage induced by ischemia/reperfusion injury in a rat model of MCAO and cellular model of OGD/RP, demonstrating that its anti-apoptotic/neuroprotective/neurotrophic cholinergic and non-cholinergic properties warrant further evaluation in conditions of brain injury

AChR-specific immunosuppressive therapy of myasthenia gravis.

PubMed

Luo, Jie; Lindstrom, Jon

2015-10-15

Myasthenia gravis (MG) is an organ-specific autoimmune disease characterized by muscle fatigability. In most cases, it is mediated by autoantibodies targeting muscle nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction. Experimental autoimmune myasthenia gravis (EAMG) is an animal model for MG, which is usually induced by immunization with AChR purified from fish electric organ. Pathological autoantibodies to AChRs are directed at the extracellular surface, especially the main immunogenic region (MIR). Current treatments for MG can help many but not all patients. Antigen-specific immunosuppressive therapy for MG that specifically suppresses the autoimmune response without affecting the entire immune system and avoids side effects of general immunosuppression is currently unavailable. Early attempts at antigen-specific immunosuppression for EAMG using AChR extracellular domain sequences that form epitopes for pathological autoantibodies risked provoking autoimmunity rather than suppressing it. We discovered a novel approach to specific immunosuppression of EAMG with a therapeutic vaccine consisting of bacterially-expressed human AChR cytoplasmic domains, which has the potential to specifically suppress MG without danger of causing exacerbation. This approach prevents development of chronic EAMG when initiated immediately after the acute phase of EAMG, and rapidly reverses established chronic EAMG when started during the chronic phase of EAMG. Successfully treated rats exhibited long-term resistance to re-induction of EAMG. In this review we also discuss the current understanding of the mechanisms by which the therapy works. Vaccination with AChR cytoplasmic domains in adjuvant is promising as a safe, antigen-specific, potent, effective, rapidly acting, and long lasting approach to therapy of MG. Copyright © 2015 Elsevier Inc. All rights reserved.

Mode of action of triflumezopyrim: A novel mesoionic insecticide which inhibits the nicotinic acetylcholine receptor.

PubMed

Cordova, Daniel; Benner, Eric A; Schroeder, Mark E; Holyoke, Caleb W; Zhang, Wenming; Pahutski, Thomas F; Leighty, Robert M; Vincent, Daniel R; Hamm, Jason C

2016-07-01

Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace (3)H-imidacloprid with a Ki value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC50 of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 μM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick β2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism. Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein-mimicking nanowire-inspired electro-catalytic biosensor for probing acetylcholinesterase activity and its inhibitors.

PubMed

Zhang, Qingqing; Hu, Yufang; Wu, Di; Ma, Shaohua; Wang, Jiao; Rao, Jiajia; Xu, Lihua; Xu, Huan; Shao, Huili; Guo, Zhiyong; Wang, Sui

2018-06-01

A highly sensitive electrochemical biosensor based on the synthetized L-Cysteine-Ag(I) coordination polymer (L-Cys-Ag(I) CP), which looks like a protein-mimicking nanowire, was constructed to detect acetylcholinesterase (AChE) activity and screen its inhibitors. This sensing strategy involves the reaction of acetylcholine chloride (ACh) with acetylcholinesterase (AChE) to form choline that is in turn catalytically oxidized by choline oxidase (ChOx) to produce hydrogen peroxide (H 2 O 2 ), thus L-Cys-Ag(I) CP possesses the electro-catalytic property to H 2 O 2 reduction. Herein, the protein-mimicking nanowire-based platform was capable of investigating successive of H 2 O 2 effectively by amperometric i-t (current-time) response, and was further applied for the turn-on electrochemical detection of AChE activity. The proposed sensor is highly sensitive (limit of detection is 0.0006 U/L) and is feasible for screening inhibitors of AChE. The model for AChE inhibition was further established and two traditional AChE inhibitors (donepezil and tacrine) were employed to verify the feasibility of the system. The IC 5 0 of donepezil and tacrine were estimated to be 1.4 nM and 3.5 nM, respectively. The developed protocol provides a new and promising platform for probing AChE activity and screening its inhibitors with low cost, high sensitivity and selectivity. Copyright © 2018 Elsevier B.V. All rights reserved.

Design, synthesis, acetylcholinesterase inhibition and larvicidal activity of girgensohnine analogs on Aedes aegypti, vector of dengue fever.

PubMed

Carreño Otero, Aurora L; Vargas Méndez, Leonor Y; Duque L, Jonny E; Kouznetsov, Vladimir V

2014-05-06

Girgensohnine alkaloid was used as a natural model in the design and generation of new alkaloid-like α-aminonitrile series that was completed by the use of SSA-catalyzed Strecker reaction between commercial and inexpensive substituted benzaldehydes, piperidine (pyrrolidine, morpholine and N-methylpiperazine) and acetone cyanohydrin. Calculated ADMETox parameters of the designed analogs revealed their good pharmacokinetic profiles indicating lipophilic characteristics. In vitro AChE enzyme test showed that obtained α-aminonitriles could be considered as AChEIs with micromolar IC50 values ranging from 42.0 to 478.0 μM (10.3-124.0 μg/mL). Among this series, the best AChE inhibitor was the pyrrolidine α-aminonitrile 3 (IC50 = 42 μM), followed by the piperidine α-aminonitriles 2 and 6 (IC50 = 45 μM and IC50 = 51 μM, respectively), and the compound 7 (IC50 = 51 μM). In vivo insecticidal activity of more active AChEIs against Aedes aegypti larvae was also performed showing a good larvicidal activity at concentrations less than 140 ppm, highlighting products 2 and 7 that could serve as lead compounds to develop new potent and selective insecticides. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Mechanisms of flow and ACh-induced dilation in rat soleus arterioles are altered by hindlimb unweighting

NASA Technical Reports Server (NTRS)

Schrage, William G.; Woodman, Christopher R.; Laughlin, M. Harold

2002-01-01

The purpose of this study was to test the hypothesis that endothelium-dependent dilation (flow-induced dilation and ACh-induced dilation) in rat soleus muscle arterioles is impaired by hindlimb unweighting (HLU). Male Sprague-Dawley rats (approximately 300 g) were exposed to HLU or weight-bearing control (Con) conditions for 14 days. Soleus first-order (1A) and second-order (2A) arterioles were isolated, cannulated, and exposed to step increases in luminal flow at constant pressure. Flow-induced dilation was not impaired by HLU in 1A or 2A arterioles. The cyclooxygenase inhibitor indomethacin (Indo; 50 microM) did not alter flow-induced dilation in 1As or 2As. Inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-L-arginine (L-NNA; 300 microM) reduced flow-induced dilation by 65-70% in Con and HLU 1As. In contrast, L-NNA abolished flow-induced dilation in 2As from Con rats but had no effect in HLU 2As. Combined treatment with L-NNA + Indo reduced tone in 1As and 2As from Con rats, but flow-induced dilation in the presence of L-NNA + Indo was not different from responses without inhibitors in either Con or HLU 1As or 2As. HLU also did not impair ACh-induced dilation (10(-9)-10(-4) M) in soleus 2As. L-NNA reduced ACh-induced dilation by approximately 40% in Con 2As but abolished dilation in HLU 2As. Indo did not alter ACh-induced dilation in Con or HLU 2As, whereas combined treatment with L-NNA + Indo abolished ACh-induced dilation in 2As from both groups. We conclude that flow-induced dilation (1As and 2As) was preserved after 2 wk HLU, but HLU decreased the contribution of NOS in mediating flow-induced dilation and increased the contribution of a NOS- and cyclooxygenase-independent mechanism (possibly endothelium-derived hyperpolarizing factor). In soleus 2As, ACh-induced dilation was preserved after 2-wk HLU but the contribution of NOS in mediating ACh-induced dilation was increased.

Nicotine/cigarette smoke promotes metastasis of pancreatic cancer through α7nAChR-mediated MUC4 upregulation.

PubMed

Momi, N; Ponnusamy, M P; Kaur, S; Rachagani, S; Kunigal, S S; Chellappan, S; Ouellette, M M; Batra, S K

2013-03-14

Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared with the normal pancreas, and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette smoke extract and nicotine, which is the major component of CS, significantly upregulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via the α7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the α7nAChR antagonists, α-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. In addition, we demonstrated that nicotine-mediated MUC4 upregulation promotes the PC cell migration through the activation of the downstream effectors, such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in vivo studies showed a marked increase in the mean pancreatic tumor weight (low dose (100 mg/m(3) total suspended particulate (TSP)), P=0.014; high dose (247 mg/m(3) TSP), P=0.02) and significant tumor metastasis to various distant organs in the CS-exposed mice, orthotopically implanted with luciferase-transfected PC cells, as compared with the sham controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine (low dose, 155.88±35.96 ng/ml; high dose, 216.25±29.95 ng/ml) and increased MUC4, α7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings

Behavioral changes in young and adult rats: Indications of cholinesterase inhibition

EPA Science Inventory

Inhibition of acetylcholinesterase (AChE) has long been accepted as the basis for neurotoxicity produced by organophosphorus (OP) and N-methyl carbamate chemicals. Functional or behavioral alterations result from acute exposure to these chemicals. We have conducted behavioral eva...

Anticholinesterase activity of potential therapeutic 5-(1,3,3-trimethylindolinyl) carbamates. (Reannouncement with new availability information)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lieske, C.N.; Gepp, R.T.; Clark, J.H.

1991-12-31

Six N-alkyl and N-aryl 5-(1,3,3-trimethylindolinyl) carbamates were synthesized and studied for their structure-activity relationships in inhibiting eel acetylcholinesterase (AChE). The carbamates were 5-(1,3,3,trimethylindolinyl) N,N-dimethylcarbamate (Cui Xing Ning) (I), 5-(1,3,3-trimethylindolinyl) N-methylcarbamate (II), 5-(1,3,3-trimethylindolinyl) N-ethylcarbamate (III), 5-(1,3,3-trimethylindolinyl) N,Ndiethylcarbamate (IV), 5-(1,3,3-trimethylindolinyl) N-heptylcarbamate (V), and 5-(1,3,3-trimethylindolinyl) N-(3-cholorophenyl)carbamate (VI). The inhibition studies were carried out at 25.0 deg C at pH 7.60. The rank order of the ki values for eel AChE inhibition is 11 > V > I > Ill > Vi > IV. Compound 11 has a greater affinity for the enzyme than any irreversible inhibitor cited in the literature (Kd = 7.14more » x 10(-8)M). Our findings should aid in the application of these carbamates (1) for counteracting the cholinergic problems associated with various diseases,and (2) for developing potential pretreatment compounds for organophosphate poisoning. Acetylcholinesterase, carbamates, inhibition« less

Acetylcholine from the mesopontine tegmental nuclei differentially affects methamphetamine induced locomotor activity and neurotransmitter levels in the mesolimbic pathway

PubMed Central

Dobbs, Lauren K.; Mark, Gregory P.

2012-01-01

Methamphetamine (MA) increases dopamine (DA) levels within the mesolimbic pathway and acetylcholine (ACh), a neurotransmitter known to increase DA cell firing and release and mediate reinforcement, within the ventral tegmental area (VTA). The laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei provide cholinergic input to the VTA; however, the contribution of LDT- and PPT-derived ACh to MA-induced DA and ACh levels and locomotor activation remains unknown. The first experiment examined the role of LDT-derived ACh in MA locomotor activation by reversibly inhibiting these neurons with bilateral intra-LDT microinjections of the M2 receptor agonist oxotremorine (OXO). Male C57BL/6 J mice were given a bilateral 0.1 µl OXO (0, 1, or 10 nM/side) microinjection immediately prior to IP saline or MA (2 mg/kg). The highest OXO concentration significantly inhibited both saline-and MA-primed locomotor activity. In a second set of experiments we characterized the individual contributions of ACh originating in the LDT or pedunculopontine tegmental nucleus (PPT) to MA-induced levels of ACh and DA by administering intra-LDT or PPT OXO and performing in vivo microdialysis in the VTA and NAc. Intra-LDT OXO dose-dependently attenuated the MA-induced increase in ACh within the VTA but had no effect on DA in NAc. Intra-PPT OXO had no effect on ACh or DA levels within the VTA or NAc, respectively. We conclude that LDT, but not PPT, ACh is important in locomotor behavior and the cholinergic, but not dopaminergic, response to systemic MA. PMID:21945297

Cholinesterase inhibitory activities of Apai-sa-le recipe and its ingredients.

PubMed

Senavong, Pimolvan; Sattaponpan, Chitsanucha; Silavat Suk-um; Itharat, Arunporn

2014-08-01

Acetylcholinesterase and butyrylcholoinesterase inhibitors are well-known drugs commonly used in the treatment ofAlzheimer's disease (AD) to improve cognitive function. These enzyme inhibitors were reported to be found in manyplants. Apai-sa-le recipe was a Thai tradition used as nootropic recipe and formerly claimed to improve memory. Therefore, it is interesting to investigate cholinesterase inhibitory activity ofthe recipe and its ingredients. To determine the whole recipe ofApai-sa-le and its ingredients for inhibitory effect on acetylcholinesterase (AChE) and human butyrylcholinesterase (BuChE) activities. Thirty grams of each plant and 181 grams of the whole recipe were separately extracted by 95% ethanol, after filtered the filtrate were evaporated and vacuum-dried at 45°C. By Elman method, the inhibitory activities of both enzymes were assessed. The volatile constituents ofeach extract were determined by GCMS. The constituents in the non- volatile extract were examined by TLC and the antioxidant activity was determined. Four plants exhibited specific BuChE inhibitor were Lepidium sativum Linn. (Ls), Piper nigrum L. (Pn), Angelica dahurica Benth (Ad) andAtractylodes lancea DC. (Al), which shown the lC50 of 5.59, 24.52, 73.23, 96.25 μg/ml, respectively whereas galantamine and the whole recipe showed IC50 of 0.59 and 236 μg/ml. Only Pn extract inhibited AChE at lCso of 25.46 μg/ml. By GCMS and TLC fingerprints revealed the main constituents in LS, Ad, Al andPn as apiol, cumialdehyde, furanodiene and piperine. Moreover nine plant extracts and the whole recipe showed antioxidant activity. Lepidium sativum Linn. (Ls) extract showed the most potency on BuChE inhibitory effect. Three ingredients and the whole recipe exhibited mild activity. Only Piper nigrum L demonstrated inhibition effect on both AChE and BuChE.

Synthesis and acetylcholinesterase/butyrylcholinesterase inhibition activity of 4-amino-2, 3-diaryl-5, 6, 7, 8-tetrahydrofuro(and thieno)[2, 3-b]-quinolines, and 4-amino-5, 6, 7, 8, 9-pentahydro-2, 3-diphenylcyclohepta[e]furo(and thieno)-[2, 3-b]pyridines.

PubMed

Marco, José L; De Los Ríos, Cristóbal; Carreiras, María C; Baños, Josep E; Badia, Albert; Vivas, Nuria M

2002-07-01

The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition activities of a series of 4-amino-2, 3-diaryl-5, 6, 7, 8-tetrahydrofuro[2, 3-b]quinolines (10-12)/4-amino-5, 6, 7, 8-tetrahydro-2, 3-diphenylthieno[2, 3-b]quinoline (14) and 4-amino-5, 6, 7, 8, 9-pentahydro-2, 3-diphenylcyclohepta[e]furo[2, 3-b]pyridine (13)/4-amino-5, 6, 7, 8, 9-pentahydro-2, 3-phenylcyclohepta[e]thieno[2, 3-b]pyridine (15) are described. These compounds are tacrine (THA) analogues which have been prepared either from readily available 2-amino-3-cyano-4, 5-diarylfurans (16-18) or from 2-amino-3-cyano-4, 5-diphenylthiophene (19), via Friedländer condensation with cyclohexanone or cycloheptanone. These compounds are competitive inhibitors for acetylcholinesterase, the more potent being compound (13) which is three-fold less active than tacrine. The butyrylcholinesterase inhibition activity is significant only in compounds 10 and133, which are ten-fold less active than tacrine. It is found that the products 11 and 12 strongly inhibit acetylcholinesterase, and show excellent selectivity regarding butyrylcholinesterase.

Rescue of Amyloid-Beta-Induced Inhibition of Nicotinic Acetylcholine Receptors by a Peptide Homologous to the Nicotine Binding Domain of the Alpha 7 Subtype

PubMed Central

Trujillo, Cleber A.; Sathler, Luciana B.; Juliano, Maria A.; Juliano, Luiz; Ulrich, Henning; Ferreira, Sergio T.

2013-01-01

Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD. PMID:23894286

RgIA4 Potently Blocks Mouse α9α10 nAChRs and Provides Long Lasting Protection against Oxaliplatin-Induced Cold Allodynia.

PubMed

Christensen, Sean B; Hone, Arik J; Roux, Isabelle; Kniazeff, Julie; Pin, Jean-Philippe; Upert, Grégory; Servent, Denis; Glowatzki, Elisabeth; McIntosh, J Michael

2017-01-01

Transcripts for α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are found in diverse tissues. The function of α9α10 nAChRs is best known in mechanosensory cochlear hair cells, but elsewhere their roles are less well-understood. α9α10 nAChRs have been implicated as analgesic targets and α-conotoxins that block α9α10 nAChRs produce analgesia. However, some of these peptides show large potency differences between species. Additionally several studies have indicated that these conotoxins may also activate GABA B receptors (GABA B Rs). To further address these issues, we cloned the cDNAs of mouse α9 and α10 nAChR subunits. When heterologously expressed in Xenopus oocytes, the resulting α9α10 nAChRs had the expected pharmacology of being activated by acetylcholine and choline but not by nicotine. A conotoxin analog, RgIA4, potently, and selectively blocked mouse α9α10 nAChRs with low nanomolar affinity indicating that RgIA4 may be effectively used to study murine α9α10 nAChR function. Previous reports indicated that RgIA4 attenuates chemotherapy-induced cold allodynia. Here we demonstrate that RgIA4 analgesic effects following oxaliplatin treatment are sustained for 21 days after last RgIA4 administration indicating that RgIA4 may provide enduring protection against nerve damage. RgIA4 lacks activity at GABA B receptors; a bioluminescence resonance energy transfer assay was used to demonstrate that two other analgesic α-conotoxins, Vc1.1 and AuIB, also do not activate GABA B Rs expressed in HEK cells. Together these findings further support the targeting of α9α10 nAChRs in the treatment of pain.

Comparative study on the inhibitory effect of caffeic and chlorogenic acids on key enzymes linked to Alzheimer's disease and some pro-oxidant induced oxidative stress in rats' brain-in vitro.

PubMed

Oboh, Ganiyu; Agunloye, Odunayo M; Akinyemi, Ayodele J; Ademiluyi, Adedayo O; Adefegha, Stephen A

2013-02-01

This study sought to investigate and compare the interaction of caffeic acid and chlorogenic acid on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and some pro-oxidants (FeSO(4), sodium nitroprusside and quinolinic acid) induced oxidative stress in rat brain in vitro. The result revealed that caffeic acid and chlorogenic acid inhibited AChE and BChE activities in dose-dependent manner; however, caffeic acid had a higher inhibitory effect on AChE and BChE activities than chlorogenic acid. Combination of the phenolic acids inhibited AChE and BChE activities antagonistically. Furthermore, pro-oxidants such as, FeSO(4), sodium nitroprusside and quinolinic acid caused increase in the malondialdehyde (MDA) contents of the brain which was significantly decreased dose-dependently by the phenolic acids. Inhibition of AChE and BChE activities slows down acetylcholine and butyrylcholine breakdown in the brain. Therefore, one possible mechanism through which the phenolic acids exert their neuroprotective properties is by inhibiting AChE and BChE activities as well as preventing oxidative stress-induced neurodegeneration. However, esterification of caffeic acid with quinic acid producing chlorogenic acid affects these neuroprotective properties.

Recent advances in acetylcholinesterase Inhibitors and Reactivators: an update on the patent literature (2012-2015).

PubMed

McHardy, Stanton F; Wang, Hua-Yu Leo; McCowen, Shelby V; Valdez, Matthew C

2017-04-01

Acetylcholinesterase (AChE) is the major enzyme that hydrolyzes acetylcholine, a key neurotransmitter for synaptic transmission, into acetic acid and choline. Mild inhibition of AChE has been shown to have therapeutic relevance in Alzheimer's disease (AD), myasthenia gravis, and glaucoma among others. In contrast, strong inhibition of AChE can lead to cholinergic poisoning. To combat this, AChE reactivators have to be developed to remove the offending AChE inhibitor, restoring acetylcholine levels to normal. Areas covered: This article covers recent advances in the development of acetylcholinesterase modulators, including both inhibitors of acetylcholinesterase for the efforts in development of new chemical entities for treatment of AD, as well as re-activators for resurrection of organophosphate bound acetylcholinesterase. Expert opinion: Over the past three years, research efforts have continued to identify novel small molecules as AChE inhibitors for both CNS and peripheral diseases. The more recent patent activity has focused on three AChE ligand design areas: derivatives of known AChE ligands, natural product based scaffolds and multifunctional ligands, all of which have produced some unique chemical matter with AChE inhibition activities in the mid picomolar to low micromolar ranges. New AChE inhibitors with polypharmacology or dual inhibitory activity have also emerged as highlighted by new AChE inhibitors with dual activity at L-type calcium channels, GSK-3, BACE1 and H3, although most only show low micromolar activity, thus further research is warranted. New small molecule reactivators of organophosphate-inhibited AChE have also been disclosed, which focused on the design of neutral ligands with improved pharmaceutical properties and blood-brain barrier (BBB) penetration. Gratifyingly, some research in this area is moving away from the traditional quaternary pyridinium oximes AChE reactivators, while still employing the necessary reactivation group (oximes

Are linear AChR epitopes the real culprit in ocular myasthenia gravis?

PubMed

Wu, Xiaorong; Tüzün, Erdem

2017-02-01

Extraocular muscle weakness occurs in most of the myasthenia gravis (MG) patients and it is often the initial complaint. Approximately 10-20% of MG patients with extraocular muscle weakness display only ocular symptoms and rest of the patients subsequently develop generalized muscle weakness. It is not entirely clear why some MG patients develop only ocular symptoms and why extraocular muscle weakness almost always precedes generalized muscle weakness. These facts are often explained by increased susceptibility of extraocular muscles due to their reduced endplate safety factor and lower complement inhibitor expression. Findings of a recently developed animal model of ocular MG suggest that additional factors might be in play. While immunization of HLA transgenic and wild-type (WT) mice with the native acetylcholine receptor (AChR) pentamer carrying conformational epitopes generates severe generalized muscle weakness, immunization of the same mouse strains with recombinant unfolded AChR subunits containing linear epitopes induces ptosis with or without mild generalized muscle weakness. Notably, immunization of mice with deficient T helper cell-mediated antigen presentation with recombinant AChR subunits or whole native AChR pentamer also induces ocular symptoms, AChR-reactive B cells and AChR antibodies. Based on these findings, we hypothesize that ocular symptoms observed in the earlier stages of MG might be triggered by linear and non-conformational AChR epitopes expressed by thymic cells or invading microorganisms. This initial AChR autoimmunity might be managed by T cell-independent and B cell mediated mechanisms yielding low affinity AChR antibodies. These antibodies are putatively capable of inducing muscle weakness only in extraocular muscles which have increased vulnerability due to their inherent biological properties. After this initial attack, as AChR bearing immune complexes form and the immune system gains access to the native AChR expressed by muscle

Design, synthesis and biological activity of novel donepezil derivatives bearing N-benzyl pyridinium moiety as potent and dual binding site acetylcholinesterase inhibitors.

PubMed

Lan, Jin-Shuai; Zhang, Tong; Liu, Yun; Yang, Jing; Xie, Sai-Sai; Liu, Jing; Miao, Ze-Yang; Ding, Yue

2017-06-16

A series of new donepezil derivatives were designed synthesized and evaluated as multifunctional cholinesterase inhibitors against Alzheimer's disease (AD). In vitro studies showed that most of them exhibited significant potency to inhibit acetylcholinesterase and self-induced β-amyloid (Aβ) aggregation, and moderate antioxidant activity. Especially, compound 5b presented the greatest ability to inhibit cholinesterase (IC 50 , 1.9 nM for eeAChE and 0.8 nM for hAChE), good inhibition of Aβ aggregation (53.7% at 20 μM) and good antioxidant activity (0.54 trolox equivalents). Kinetic and molecular modeling studies indicated that compound 5b was a mixed-type inhibitor, binding simultaneously to the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, compound 5b could reduce PC12 cells death induced by oxidative stress and Aβ (1-42). Moreover, in vivo experiments showed that compound 5b was nontoxic and tolerated at doses up to 2000 mg/kg. These results suggested that compound 5b might be an excellent multifunctional agent for AD treatment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Nicotine/Cigarette-smoke Promotes Metastasis of Pancreatic Cancer Through α7nAChR-mediated MUC4 Up-regulation

PubMed Central

Momi, Navneet; Ponnusamy, Moorthy P.; Kaur, Sukhwinder; Rachagani, Satyanarayana; Kunigal, Sateesh S; Chellappan, Srikumar; Ouellette, Michel M; Batra, Surinder K

2012-01-01

Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well-established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared to the normal pancreas and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette-smoke-extract (CSE) and nicotine, which is the major component of CS, significantly up-regulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via α7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the α7nAChR antagonists, α-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. Additionally, we demonstrated that nicotine-mediated MUC4 up-regulation promotes the PC cell migration through the activation of the downstream effectors such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in-vivo studies demonstrated a dramatic increase in the mean pancreatic tumor weight [low-dose (100 mg/m3 TSP), p=0.014; high-dose (247 mg/m3 TSP), p=0.02] and significant tumor metastasis to various distant organs in the CS-exposed-mice, orthotopically implanted with luciferase-transfected PC cells, as compared to the sham-controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine [low-dose, 155.88±35.96 ng/ml; high-dose, 216.25±29.95 ng/ml] and increased MUC4, α7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings revealed for the first time that CS up

β-Cryptoxanthin Reduced Lung Tumor Multiplicity and Inhibited Lung Cancer Cell Motility by Downregulating Nicotinic Acetylcholine Receptor α7 Signaling.

PubMed

Iskandar, Anita R; Miao, Benchun; Li, Xinli; Hu, Kang-Quan; Liu, Chun; Wang, Xiang-Dong

2016-11-01

Despite the consistent association between a higher intake of the provitamin A carotenoid β-cryptoxanthin (BCX) and a lower risk of lung cancer among smokers, potential mechanisms supporting BCX as a chemopreventive agent are needed. We first examined the effects of BCX on 4-[methyl nitrosamino]-1-[3-pyridyl]-1-butanone (NNK)-induced lung tumorigenesis in A/J mice. BCX supplementation was given daily to the mice starting 2 weeks prior to the injection of NNK and continued 16 weeks after NNK injection. BCX supplementation resulted in a dose-dependent increase of BCX concentration in both serum and lungs of the mice without a significant alteration of vitamin A (retinol and retinyl palmitate) concentration. BCX significantly reduced the multiplicity of the NNK-induced lung tumor by 52% to 63% compared with the NNK-treated mice without BCX supplementation. The protective effect of BCX in the lungs was associated with reductions of both mRNA and protein of the homopentameric neuronal nicotinic acetylcholine receptor α7 (α7-nAChR), which has been implicated in lung tumorigenesis. We then conducted an in vitro cell culture study and found that BCX treatment suppressed α7-nAChR expression and inhibited the migration and invasion of α7-nAChR-positive lung cancer cells but not in cells lacking α7-nAChR. The activities of BCX were significantly attenuated by activators of α7-nAChR/PI3K signaling or by overexpression of constitutively active PI3K. Collectively, the results suggest that BCX inhibits lung tumorigenesis and cancer cell motility through the downregulation of α7-nAChR/PI3K signaling, independent of its provitamin A activity. Therefore, BCX can be used as a chemopreventive agent or a chemotherapeutic compound against lung cancer. Cancer Prev Res; 9(11); 875-86. ©2016 AACR. ©2016 American Association for Cancer Research.

Rosuvastatin ameliorates cognitive impairment in rats fed with high-salt and cholesterol diet via inhibiting acetylcholinesterase activity and amyloid beta peptide aggregation.

PubMed

Husain, I; Akhtar, M; Abdin, M Zainul; Islamuddin, M; Shaharyar, M; Najmi, A K

2018-04-01

Amyloid beta (Aβ) peptide aggregation and cholinergic neurodegeneration are involved in the development of cognitive impairment. Therefore, in this article, we examined rosuvastatin (RSV), an oral hypolipidemic drug, to determine its potential as a dual inhibitor of acetylcholinesterase (AChE) and Aβ peptide aggregation for the treatment of cognitive impairment. Molecular docking study was done to examine the affinity of RSV with Aβ 1-42 and AChE in silico. We also employed neurobehavioral activity tests, biochemical estimation, and histopathology to study the anti-Aβ 1-42 aggregation capability of RSV in vivo. Molecular docking study provided evidence that RSV has the best binding conformer at its receptor site or active site of an enzyme. The cognitive impairment in female Wistar rats was induced by high-salt and cholesterol diet (HSCD) ad libitum for 8 weeks. RSV ameliorated serum cholesterol level, AChE activity, and Aβ 1-42 peptide aggregations in HSCD induced cognitive impairment. In addition, RSV-treated rats showed greater scores in the open field (locomotor activity) test. Moreover, the histopathological studies in the hippocampus and cortex of rat brain also supported that RSV markedly reduced the cognitive impairment and preserved the normal histoarchitectural pattern of the hippocampus and cortex. Taken together, these data indicate that RSV may act as a dual inhibitor of AChE and Aβ 1-42 peptide aggregation, therefore suggesting a therapeutic strategy for cognitive impairment treatment.

Nicotine withdrawal-induced inattention is absent in alpha7 nAChR knockout mice

PubMed Central

Higa, K. K.; Grim, A.; Kamenski, M. E.; van Enkhuizen, J.; Zhou, X.; Li, K.; Naviaux, J. C.; Wang, L.; Naviaux, R. K.; Geyer, M. A.; Markou, A.; Young, J. W.

2017-01-01

Rationale Smoking is the leading cause of preventable death in the U.S., but quit attempts result in withdrawal-induced cognitive dysfunction and predicts relapse. Greater understanding of the neural mechanism(s) underlying these cognitive deficits is required to develop targeted treatments to aid quit attempts. Objectives We examined nicotine withdrawal-induced inattention in mice lacking the α7 nicotinic acetylcholine receptor (nAChR) using the 5-choice continuous performance test (5C-CPT). Methods Mice were trained in the 5C-CPT prior to osmotic minipump implantation containing saline or nicotine. Experiment 1 used 40 mg/kg/day nicotine treatment and tested C57BL/6 mice 4, 28, and 52 h after pump removal. Experiment 2 used 14 and 40 mg/kg/day nicotine treatment in α7 nAChR knockout (KO) and wildtype (WT) littermates tested 4 h after pump removal. Subsets of WT mice were sacrificed before and after pump removal to assess changes in receptor expression associated with nicotine administration and withdrawal. Results Nicotine withdrawal impaired attention in the 5C-CPT, driven by response inhibition and target detection deficits. The overall attentional deficit was absent in α7 nAChR KO mice despite response disinhibition in these mice. Synaptosomal glutamate mGluR5 and dopamine D4 receptor expression were reduced during chronic nicotine but increased during withdrawal, potentially contributing to cognitive deficits. Conclusions The α7 nAChR may underlie nicotine withdrawal-induced deficits in target detection but is not required for response disinhibition deficits. Alterations to the glutamatergic and dopaminergic pathways may also contribute to withdrawal-induced attentional deficits, providing novel targets to alleviate the cognitive symptoms of withdrawal during quit attempts. PMID:28243714

Silibinin inhibits acetylcholinesterase activity and amyloid β peptide aggregation: a dual-target drug for the treatment of Alzheimer's disease.

PubMed

Duan, Songwei; Guan, Xiaoyin; Lin, Runxuan; Liu, Xincheng; Yan, Ying; Lin, Ruibang; Zhang, Tianqi; Chen, Xueman; Huang, Jiaqi; Sun, Xicui; Li, Qingqing; Fang, Shaoliang; Xu, Jun; Yao, Zhibin; Gu, Huaiyu

2015-05-01

Alzheimer's disease (AD) is characterized by amyloid β (Aβ) peptide aggregation and cholinergic neurodegeneration. Therefore, in this paper, we examined silibinin, a flavonoid extracted from Silybum marianum, to determine its potential as a dual inhibitor of acetylcholinesterase (AChE) and Aβ peptide aggregation for AD treatment. To achieve this, we used molecular docking and molecular dynamics simulations to examine the affinity of silibinin with Aβ and AChE in silico. Next, we used circular dichroism and transmission electron microscopy to study the anti-Aβ aggregation capability of silibinin in vitro. Moreover, a Morris Water Maze test, enzyme-linked immunosorbent assay, immunohistochemistry, 5-bromo-2-deoxyuridine double labeling, and a gene gun experiment were performed on silibinin-treated APP/PS1 transgenic mice. In molecular dynamics simulations, silibinin interacted with Aβ and AChE to form different stable complexes. After the administration of silibinin, AChE activity and Aβ aggregations were down-regulated, and the quantity of AChE also decreased. In addition, silibinin-treated APP/PS1 transgenic mice had greater scores in the Morris Water Maze. Moreover, silibinin could increase the number of newly generated microglia, astrocytes, neurons, and neuronal precursor cells. Taken together, these data suggest that silibinin could act as a dual inhibitor of AChE and Aβ peptide aggregation, therefore suggesting a therapeutic strategy for AD treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

Anticholinesterase activities of cold and hot aqueous extracts of F. racemosa stem bark.

PubMed

Ahmed, Faiyaz; Urooj, Asna

2010-04-01

The present study evaluated the anticholinesterase activity of cold and hot aqueous extracts of Ficus racemosa stem bark against rat brain acetylcholinesterase in vitro. Both the cold aqueous extract (FRC) and the hot aqueous extract (FRH) exhibited a dose dependent inhibition of rat brain acetylcholinesterase. FRH showed significantly higher (P activity compared to FRC; however, both the extracts did not show 50% inhibition of AChE at the doses tested (200-1000 mug ml(-1)). The IC(50) values of 1813 and 1331 mug ml(-1) were deduced for FRC and FRH, respectively (calculated by extrapolation using Boltzmann's dose response analysis).

Acetylcholinesterase inhibition and gill lesions in Rasbora caverii, an indigenous fish inhabiting rice field associated waterbodies in Sri Lanka.

PubMed

Wijeyaratne, W M D N; Pathiratne, Asoka

2006-10-01

The present study was aimed at applying condition factor (CF), brain acetylcholinesterase (AChE) and gill histology as biomarkers for detecting possible exposure/effect induced by pesticides in fish residing rice field associated waterbodies in Sri Lanka. Biomarkers of an indigenous fish, Rasbora caverii collected from five sampling sites including canals near rice fields, a river and a reservoir (the reference site) were evaluated at four sampling stages covering pesticide application periods during rice cultivation season in 2004. Results indicated that CF of the fish did not show significant alterations regardless of the sampling sites or sampling stages. Site specific differences in AChE activities of the fish were not evident either prior to application of pesticides or at 7 days after Paraquat application to the rice fields. Two days after the application of a mixture of Fenthion and Phenthoate to the rice fields, AChE activity of the fish collected from canals near rice fields was significantly depressed (65-75%) compared to the fish in the reference site. The activities remain depressed to 50-56% even at 65 days after the insecticides application. Laboratory studies showed that prior exposure of R. caverii to Paraquat (2 microg l(-1), 7 days) enhanced the extent of inhibition of brain AChE activity induced by Fenthion (3 microg l(-1)) or a mixture of Fenthion (3 microg l(-1)) and Phenthoate (5 microg l(-1)). Gills of fish collected from canals near rice fields exhibited abnormal multiple divisions at the tips of some secondary lamellae in addition to hyperplasia, hypertrophy and club shaped deformities. Results indicate that application of pesticides in rice culture could manifest a threat to native fish populations residing rice field associated waterbodies. The response of brain AChE and histological changes in the gills of R. caverii allowed differentiating sampling sites after insecticide applications to the rice fields. Hence, R. caverii may be

Antioxidant and acetylcholinesterase inhibitory activities of ginger root (Zingiber officinale Roscoe) extract.

PubMed

Tung, Bui Thanh; Thu, Dang Kim; Thu, Nguyen Thi Kim; Hai, Nguyen Thanh

2017-05-04

Background Zingiber officinale Roscoe has been used in traditional medicine for the treatment of neurological disorder. This study aimed to investigate the phenolic contents, antioxidant, acetylcholinesterase enzyme (AChE) inhibitory activities of different fraction of Z. officinale root grown in Vietnam. Methods The roots of Z. officinale are extracted with ethanol 96 % and fractionated with n-hexane, ethyl acetate (EtOAc) and butanol (BuOH) solvents. These fractions evaluated the antioxidant activity by 1,1-Diphenyl -2-picrylhydrazyl (DPPH) assay and AChE inhibitory activity by Ellman's colorimetric method. Results Our data showed that the total phenolic content of EtOAc fraction was highest equivalents to 35.2±1.4 mg quercetin/g of fraction. Our data also demonstrated that EtOAc fraction had the strongest antioxidant activity with IC50 was 8.89±1.37 µg/mL and AChE inhibitory activity with an IC50 value of 22.85±2.37 μg/mL in a dose-dependent manner, followed by BuOH fraction and the n-hexane fraction is the weakest. Detailed kinetic analysis indicated that EtOAc fraction was mixed inhibition type with Ki (representing the affinity of the enzyme and inhibitor) was 30.61±1.43 µg/mL. Conclusions Our results suggest that the EtOAc fraction of Z. officinale may be a promising source of AChE inhibitors for Alzheimer's disease.

Design, synthesis and evaluation of novel 7-aminoalkyl-substituted flavonoid derivatives with improved cholinesterase inhibitory activities.

PubMed

Luo, Wen; Chen, Ying; Wang, Ting; Hong, Chen; Chang, Li-Ping; Chang, Cong-Cong; Yang, Ya-Cheng; Xie, Song-Qiang; Wang, Chao-Jie

2016-02-15

A novel series of 7-aminoalkyl-substituted flavonoid derivatives 5a-5r were designed, synthesized and evaluated as potential cholinesterase inhibitors. The results showed that most of the synthesized compounds exhibited potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities at the micromolar range. Compound 2-(naphthalen-1-yl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5q) showed the best inhibitory activity (IC50, 0.64μM for AChE and 0.42μM for BChE) which were better than our previously reported compounds and the commercially available cholinergic agent Rivastigmine. The results from a Lineweaver-Burk plot indicated a mixed-type inhibition for compound 5q with AChE and BChE. Furthermore, molecular modeling study showed that 5q targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, these compounds (5a-5r) did not affect PC12 and HepG2 cell viability at the concentration of 10μM. Consequently, these flavonoid derivatives should be further investigated as multipotent agents for the treatment of Alzheimer's disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation.

PubMed

Kaplan, Mehmet Mahsum; Sultana, Nasreen; Benedetti, Ariane; Obermair, Gerald J; Linde, Nina F; Papadopoulos, Symeon; Dayal, Anamika; Grabner, Manfred; Flucher, Bernhard E

2018-06-26

Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (Ca V 1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Inhibition of acetylcholinesterase by (1S,3S)-isomalathion proceeds with loss of thiomethyl: kinetic and mass spectral evidence for an unexpected primary leaving group.

PubMed

Doorn, J A; Gage, D A; Schall, M; Talley, T T; Thompson, C M; Richardson, R J

2000-12-01

Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S, 3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k(3)) were measured. Oxime-mediated k(3) values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)-isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k(3) values of 1.88 x 10(5) and 4.18 x 10(5) min(-)(1), respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k(3) values of 0 and 6.05 x 10(3) min(-)(1), respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct that renders EEAChE stable toward reactivation after inhibition with (1S,3S)-isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate adduct was found in the mass spectra of (1S, 3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were

The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: Intracellular calcium increase, calpain/caspase 3 activation, and functional upregulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Rates, Sara; Camarasa, Jordi; Sanchez-Garcia, Ana I.

2010-05-01

Previous work by our group demonstrated that homomeric alpha7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca{sup 2+} increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibitedmore » the response induced by ACh, nicotine, and the specific alpha7 agonist PNU 282987 with IC{sub 50} values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human alpha7 but not with alpha4beta2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and alpha-bungarotoxin but not by dihydro-beta-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on alpha7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca{sup 2+} release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca{sup 2+} levels and induced an increase in alpha-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and alpha7 nAChR. Sustained calcium entry and calpain activation could favor

Comparative functional expression of nAChR subtypes in rodent DRG neurons.

PubMed

Smith, Nathan J; Hone, Arik J; Memon, Tosifa; Bossi, Simon; Smith, Thomas E; McIntosh, J Michael; Olivera, Baldomero M; Teichert, Russell W

2013-01-01

We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly α3β4 and α6β4 nAChRs; (2) neurons that express predominantly α7 nAChRs; (3) neurons that express a combination of α3β4/α6β4 and α7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70-80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15-30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca(2) (+) transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.

Inhibitors of histone demethylation and histone deacetylation cooperate in regulating gene expression and inhibiting growth in human breast cancer cells

PubMed Central

Vasilatos, Shauna N.; Boric, Lamia; Shaw, Patrick G.; Davidson, Nancy E.

2013-01-01

Abnormal activities of histone lysine demethylases (KDMs) and lysine deacetylases (HDACs) are associated with aberrant gene expression in breast cancer development. However, the precise molecular mechanisms underlying the crosstalk between KDMs and HDACs in chromatin remodeling and regulation of gene transcription are still elusive. In this study, we showed that treatment of human breast cancer cells with inhibitors targeting the zinc cofactor dependent class I/II HDAC, but not NAD+ dependent class III HDAC, led to significant increase of H3K4me2 which is a specific substrate of histone lysine-specific demethylase 1 (LSD1) and a key chromatin mark promoting transcriptional activation. We also demonstrated that inhibition of LSD1 activity by a pharmacological inhibitor, pargyline, or siRNA resulted in increased acetylation of H3K9 (AcH3K9). However, siRNA knockdown of LSD2, a homolog of LSD1, failed to alter the level of AcH3K9, suggesting that LSD2 activity may not be functionally connected with HDAC activity. Combined treatment with LSD1 and HDAC inhibitors resulted in enhanced levels of H3K4me2 and AcH3K9, and exhibited synergistic growth inhibition of breast cancer cells. Finally, microarray screening identified a unique subset of genes whose expression was significantly changed by combination treatment with inhibitors of LSD1 and HDAC. Our study suggests that LSD1 intimately interacts with histone deacetylases in human breast cancer cells. Inhibition of histone demethylation and deacetylation exhibits cooperation and synergy in regulating gene expression and growth inhibition, and may represent a promising and novel approach for epigenetic therapy of breast cancer. PMID:21452019

Anti-inflammatory and acetylcholinesterase activity of extract, fractions and five compounds isolated from the leaves and twigs of Artemisia annua growing in Cameroon.

PubMed

Chougouo, Rosine D K; Nguekeu, Yves M M; Dzoyem, Jean P; Awouafack, Maurice D; Kouamouo, Jonas; Tane, Pierre; McGaw, Lyndy J; Eloff, Jacobus N

2016-01-01

Natural products, including those derived from higher plants have, over the years, contributed greatly to the development of modern therapeutic drugs. Due to the medicinal importance in traditional practice and the diversified biology and chemistry of the constituents from Artemisia spp., the genus has been receiving growing attention. The aim of this study was to investigate the ability of the ethanol extract, four fractions (F1-F4) and five compounds namely artemisinin (1), scopoletin (2), chrysosplenetin (3), eupatin (4) and 3-O-β-d-glucopyranoside of sitosterol (5) isolated from A. annua to modulate the activity of anticholinesterase (AchE) and the production of nitric oxide (NO) in LPS-activated RAW 264.7 macrophages. At the lowest concentration tested (6.25 µg/mL), the crude extract and fraction F2 had the highest NO inhibitory activity (72.39 and 71.00 % inhibition respectively) without significant toxicity on the viability of macrophage cells (93.86 and 79.87 % of cell viability respectively). The crude extract inhibited AchE activity by 71.83 % (at 1 mg/mL) with an IC50 value of 87.43 µg/mL while F2 and F4 were the most active fractions (IC50 values of 36.75 and 28.82 µg/mL). Artemisinin (1) and chrysosplenetin (3) had the highest AChE activity with 71.67 and 80.00 % inhibition (at 0.1 mg/mL) and IC50 values of 29.34 and 27.14 µg/mL, respectively. Our results validate the traditional use of A. annua and could help to support the usefulness of this plant in the treatment of inflammatory and neurological disorders especially where nitric oxide and a cholinesterase are involved.

Phytochemical compositions of extract from peel of hawthorn fruit, and its antioxidant capacity, cell growth inhibition, and acetylcholinesterase inhibitory activity.

PubMed

Wu, Panpan; Li, Fajie; Zhang, Jianyong; Yang, Bin; Ji, Zhaojie; Chen, Weidong

2017-03-11

Hawthorn fruit (HF) is a well-known traditional medicine in China with the effects of improving digestion and regulating qi-flowing for removing blood stasis. Modern pharmacological experiments showed that HF extract has various pharmaceutical properties and flavonoids are considered as the main bioactive compounds. In this paper, Diaion HP-20 adsorption chromatography was used to enrich flavonoids in PHF, and the phytochemical composition of EPHF was analyzed by high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS). In addition, EPHF's antioxidant capacity, acetylcholinesterase (AChE) inhibitory activity and cytotoxic activity were evaluated. EPHF was obtained by Diaion HP-20 adsorption chromatography. Phytochemical composition of EPHF was analyzed qualitatively and quantitatively using HPLC and LC-MS. Radical scavenging capacity of EPHF was estimated using 2,2-diphenyl-1-picryhydrazyl (DPPH) assay and oxygen radical absorbance capacity (ORAC) assay. The AChE inhibitory activity of EPHF was evaluated by Ellman method. Cytotoxic activity of EPHF was assessed by means of MTT assay. Eight kinds of components were identified, in which ideain with the value of 179.4 mg/g was identified to be present in the highest level in EPHF, followed by (-)-epicatechin, chlorogenic acid, cyanidin 3-arabinoside, hyperoside and isoquercitrin at the concentrations of 40.9, 10.0, 1.4, 0.4 and 0.2 mg/g, respectively. The contents of these compounds in EPHF were much higher than those in PHF and HF. In addition, EPHF exhibited strong antioxidant and AChE inhibitory activity (ORAC value: 11.65 ± 2.37 μM Trolox equivalents (TE)/mg, DPPH IC 50 value: 6.72 μg/mL, anti-AChE activity IC 50 value: 11.72 μg/mL) compared with PHF and HF. Moreover, EPHF exhibited high levels of cytotoxicity on MCF-7 and SKOV-3 human tumour cell lines in a dose-dependent manner with the IC 50 of 2.76 and 80.11 μg/mL, respectively. Macroporous resin

Congo red modulates ACh-induced Ca2+ oscillations in single pancreatic acinar cells of mice

PubMed Central

Huang, Ze-bing; Wang, Hai-yan; Sun, Na-na; Wang, Jing-ke; Zhao, Meng-qin; Shen, Jian-xin; Gao, Ming; Hammer, Ronald P; Fan, Xue-gong; Wu, Jie

2014-01-01

Aim: Congo red, a secondary diazo dye, is usually used as an indicator for the presence of amyloid fibrils. Recent studies show that congo red exerts neuroprotective effects in a variety of models of neurodegenerative diseases. However, its pharmacological profile remains unknown. In this study, we investigated the effects of congo red on ACh-induced Ca2+ oscillations in mouse pancreatic acinar cells in vitro. Methods: Acutely dissociated pancreatic acinar cells of mice were prepared. A U-tube drug application system was used to deliver drugs into the bath. Intracellular Ca2+ oscillations were monitored by whole-cell recording of Ca2+-activated Cl− currents and by using confocal Ca2+ imaging. For intracellular drug application, the drug was added in pipette solution and diffused into cell after the whole-cell configuration was established. Results: Bath application of ACh (10 nmol/L) induced typical Ca2+ oscillations in dissociated pancreatic acinar cells. Addition of congo red (1, 10, 100 μmol/L) dose-dependently enhanced Ach-induced Ca2+ oscillations, but congo red alone did not induce any detectable response. Furthermore, this enhancement depended on the concentrations of ACh: congo red markedly enhanced the Ca2+ oscillations induced by ACh (10–30 nmol/L), but did not alter the Ca2+ oscillations induced by ACh (100–10000 nmol/L). Congo red also enhanced the Ca2+ oscillations induced by bath application of IP3 (30 μmol/L). Intracellular application of congo red failed to alter ACh-induced Ca2+ oscillations. Conclusion: Congo red significantly modulates intracellular Ca2+ signaling in pancreatic acinar cells, and this pharmacological effect should be fully considered when developing congo red as a novel therapeutic drug. PMID:25345744

Cholinesterase inhibitory activity of isoquinoline alkaloids from three Cryptocarya species (Lauraceae).

PubMed

Wan Othman, Wan Nurul Nazneem; Liew, Sook Yee; Khaw, Kooi Yeong; Murugaiyah, Vikneswaran; Litaudon, Marc; Awang, Khalijah

2016-09-15

Alzheimer's disease is the most common form of dementia among older adults. Acetylcholinesterase and butyrylcholinesterase are two enzymes involved in the breaking down of the neurotransmitter acetylcholine. Inhibitors for these enzymes have potential to prolong the availability of acetylcholine. Hence, the search for such inhibitors especially from natural products is needed in developing potential drugs for Alzheimer's disease. The present study investigates the cholinesterase inhibitory activity of compounds isolated from three Cryptocarya species towards acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Nine alkaloids were isolated; (+)-nornantenine 1, (-)-desmethylsecoantofine 2, (+)-oridine 3, (+)-laurotetanine 4 from the leaves of Cryptocarya densiflora BI., atherosperminine 5, (+)-N-methylisococlaurine 6, (+)-N-methyllaurotetanine 7 from the bark of Cryptocarya infectoria Miq., 2-methoxyatherosperminine 8 and (+)-reticuline 9 from the bark of Cryptocarya griffithiana Wight. In general, most of the alkaloids showed higher inhibition towards BChE as compared to AChE. The phenanthrene type alkaloid; 2-methoxyatherosperminine 8, exhibited the most potent inhibition against BChE with IC50 value of 3.95μM. Analysis of the Lineweaver-Burk (LB) plot of BChE activity over a range of substrate concentration suggested that 2-methoxyatherosperminine 8 exhibited mixed-mode inhibition with an inhibition constant (Ki) of 6.72μM. Molecular docking studies revealed that 2-methoxyatherosperminine 8 docked well at the choline binding site and catalytic triad of hBChE (butyrylcholinesterase from Homo sapiens); hydrogen bonding with Tyr 128 and His 438 residues respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

Neurotoxic responses in brain tissues of rainbow trout exposed to imidacloprid pesticide: Assessment of 8-hydroxy-2-deoxyguanosine activity, oxidative stress and acetylcholinesterase activity.

PubMed

Topal, Ahmet; Alak, Gonca; Ozkaraca, Mustafa; Yeltekin, Aslı Cilingir; Comaklı, Selim; Acıl, Gurdal; Kokturk, Mine; Atamanalp, Muhammed

2017-05-01

The extensive use of imidacloprid, a neonicotinoid insecticide, causes undesirable toxicity in non-targeted organisms including fish in aquatic environments. We investigated neurotoxic responses by observing 8-hydroxy-2-deoxyguanosine (8-OHdG) activity, oxidative stress and acetylcholinesterase (AChE) activity in rainbow trout brain tissue after 21 days of imidacloprid exposure at levels of (5 mg/L, 10 mg/L, 20 mg/L). The obtained results indicated that 8-OHdG activity did not change in fish exposed to 5 mg/L of imidacloprid, but 10 mg/L and 20 mg/L of imidacloprid significantly increased 8-OHdG activity compared to the control (p < 0.05). An immunopositiv reaction to 8-OHdG was detected in brain tissues. The brain tissues indicated a significant increase in antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) compared to the control and there was a significant increase in malondialdehyde (MDA) levels (p < 0.05). High concentrations of imidacloprid caused a significant decrease in AChE enzyme activity (p < 0.05). These results suggested that imidacloprid can be neurotoxic to fish by promoting AChE inhibition, an increase in 8-OHdG activity and changes in oxidative stress parameters. Therefore, these data may reflect one of the molecular pathways that play a role in imidacloprid toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Restitution of defective glucose-stimulated insulin secretion in diabetic GK rat by acetylcholine uncovers paradoxical stimulatory effect of beta-cell muscarinic receptor activation on cAMP production.

PubMed

Dolz, Manuel; Bailbé, Danielle; Giroix, Marie-Hélène; Calderari, Sophie; Gangnerau, Marie-Noelle; Serradas, Patricia; Rickenbach, Katharina; Irminger, Jean-Claude; Portha, Bernard

2005-11-01

Because acetylcholine (ACh) is a recognized potentiator of glucose-stimulated insulin release in the normal beta-cell, we have studied ACh's effect on islets of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. We first verified that ACh was able to restore the insulin secretory glucose competence of the GK beta-cell. Then, we demonstrated that in GK islets 1) ACh elicited a first-phase insulin release at low glucose, whereas it had no effect in Wistar; 2) total phospholipase C activity, ACh-induced inositol phosphate production, and intracellular free calcium concentration ([Ca2+]i) elevation were normal; 3) ACh triggered insulin release, even in the presence of thapsigargin, which induced a reduction of the ACh-induced [Ca2+]i response (suggesting that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on GK exocytosis); 4) inhibition of protein kinase C did not affect [Ca2+]i nor the insulin release responses to ACh; and 5) inhibition of cAMP-dependent protein kinases (PKAs), adenylyl cyclases, or cAMP generation, while not affecting the [Ca2+]i response, significantly lowered the insulinotropic response to ACh (at low and high glucose). In conclusion, ACh acts mainly through activation of the cAMP/PKA pathway to potently enhance Ca2+-stimulated insulin release in the GK beta-cell and, in doing so, normalizes its defective glucose responsiveness.

In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae

PubMed Central

Formagio, A.S.N.; Vieira, M.C.; Volobuff, C.R.F.; Silva, M.S.; Matos, A.I.; Cardoso, C.A.L.; Foglio, M.A.; Carvalho, J.E.

2015-01-01

The aim of this research was to investigate the antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species in vitro. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for acetylcholinesterase (AchE) inhibitors using Ellman's reagent. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ADR, and K-562 cell lines with growth inhibition (GI50) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 µg/mL, respectively. In addition, the Annona crassiflora and A. coriacea seed extracts were the most active among the tested extracts and the most effective against the tumor cell lines, with GI50 values below 8.90 µg/mL. The A. cacans extract displayed the lowest activity. Based on the microplate assay, the percent AchE inhibition of the extracts ranged from 12 to 52%, and the A. coriacea seed extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the A. crassiflora seed samples. The A. coriacea seeds contained ferulic and sinapic acid. Overall, the results indicated that A. crassiflora and A. coriacea extracts have antiproliferative and anticholinesterase properties, which opens up new possibilities for alternative pharmacotherapy drugs. PMID:25714885

In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae.

PubMed

Formagio, A S N; Vieira, M C; Volobuff, C R F; Silva, M S; Matos, A I; Cardoso, C A L; Foglio, M A; Carvalho, J E

2015-04-01

The aim of this research was to investigate the antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species in vitro. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for acetylcholinesterase (AchE) inhibitors using Ellman's reagent. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ADR, and K-562 cell lines with growth inhibition (GI50) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 µg/mL, respectively. In addition, the Annona crassiflora and A. coriacea seed extracts were the most active among the tested extracts and the most effective against the tumor cell lines, with GI50 values below 8.90 µg/mL. The A. cacans extract displayed the lowest activity. Based on the microplate assay, the percent AchE inhibition of the extracts ranged from 12 to 52%, and the A. coriacea seed extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the A. crassiflora seed samples. The A. coriacea seeds contained ferulic and sinapic acid. Overall, the results indicated that A. crassiflora and A. coriacea extracts have antiproliferative and anticholinesterase properties, which opens up new possibilities for alternative pharmacotherapy drugs.

Probing the reactivation process of sarin-inhibited acetylcholinesterase with α-nucleophiles: hydroxylamine anion is predicted to be a better antidote with DFT calculations.

PubMed

Khan, Md Abdul Shafeeuulla; Lo, Rabindranath; Bandyopadhyay, Tusar; Ganguly, Bishwajit

2011-08-01

Inactivation of acetylcholinesterase (AChE) due to inhibition by organophosphorus (OP) compounds is a major threat to human since AChE is a key enzyme in neurotransmission process. Oximes are used as potential reactivators of OP-inhibited AChE due to their α-effect nucleophilic reactivity. In search of more effective reactivating agents, model studies have shown that α-effect is not so important for dephosphylation reactions. We report the importance of α-effect of nucleophilic reactivity towards the reactivation of OP-inhibited AChE with hydroxylamine anion. We have demonstrated with DFT [B3LYP/6-311G(d,p)] calculations that the reactivation process of sarin-serine adduct 2 with hydroxylamine anion is more efficient than the other nucleophiles reported. The superiority of hydroxylamine anion to reactivate the sarin-inhibited AChE with sarin-serine adducts 3 and 4 compared to formoximate anion was observed in the presence and absence of hydrogen bonding interactions of Gly121 and Gly122. The calculated results show that the rates of reactivation process of adduct 4 with hydroxylamine anion are 261 and 223 times faster than the formoximate anion in the absence and presence of such hydrogen bonding interactions. The DFT calculated results shed light on the importance of the adjacent carbonyl group of Glu202 for the reactivation of sarin-serine adduct, in particular with formoximate anion. The reverse reactivation reaction between hydroxylamine anion and sarin-serine adduct was found to be higher in energy compared to the other nucleophiles, which suggests that this α-nucleophile can be a good antidote agent for the reactivation process. Copyright © 2011 Elsevier Inc. All rights reserved.

Generation of Recombinant Human AChE OP-Scavengers with Extended Circulatory Longevity

DTIC Science & Technology

2006-11-01

PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Avigdor Shafferman Ph.D. 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail: avigdor...glycans of rHuAChE, and in particular the distal termini of these glycan 8 projections , constitute a major factor in determining the circulatory...experiments funded by sources other than the USAMRMC contract, since they may have a major impact on the present project . Section III focuses on the

The impact of a parkinsonian lesion on dynamic striatal dopamine transmission depends on nicotinic receptor activation

PubMed Central

Jennings, Katie A.; Platt, Nicola J.; Cragg, Stephanie J.

2015-01-01

Dopamine function is disturbed in Parkinson's disease (PD), but whether and how release of dopamine from surviving neurons is altered has long been debated. Nicotinic acetylcholine receptors (nAChRs) on dopamine axons powerfully govern dopamine release and could be critical contributing factors. We revisited whether fundamental properties of dopamine transmission are changed in a parkinsonian brain and tested the potentially profound masking effects of nAChRs. Using real-time detection of dopamine in mouse striatum after a partial 6-hydroxydopamine lesion and under nAChR inhibition, we reveal that dopamine signals show diminished sensitivity to presynaptic activity. This effect manifested as diminished contrast between DA release evoked by the lowest versus highest frequencies. This reduced activity-dependence was underpinned by loss of short-term facilitation of dopamine release, consistent with an increase in release probability (Pr). With nAChRs active, the reduced activity-dependence of dopamine release after a parkinsonian lesion was masked. Consequently, moment-by-moment variation in activity of nAChRs may lead to dynamic co-variation in dopamine signal impairments in PD. PMID:26117304

Environmental monitoring using acetylcholinesterase inhibition in vitro. A case study in two Mexican lagoons.

PubMed

Rodríguez-Fuentes, G; Gold-Bouchot, G

2000-01-01

Cholinesterase inhibition is considered a specific biomarker of exposure and effect for organophosphorous pesticides. Its use for monitoring has been hindered, particularly in tropical countries where organophosphates are widely used for malaria and dengue control, because of the frequent lack of suitable controls. An in vitro technique is proposed as a biochemical method for monitoring pollutant mixtures in sediment toxicity tests. Brain homogenate from the fish Oreochromis niloticus is used as the enzyme source. Optimum incubation time, extraction solvent and effect of crude oil on acetylcholinesterase (AChE) are reported. The method described was used in sediments from two Mexican lagoons, located in an oil extraction area where pesticides are used in agriculture and vector control campaigns. AChE inhibitions from 3 to 21% were found in these lagoons, even in the presence of high concentrations of petroleum.

PON1 status does not influence cholinesterase activity in Egyptian agricultural workers exposed to chlorpyrifos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ellison, Corie A., E-mail: cellison@buffalo.edu; Crane, Alice L., E-mail: alcrane@buffalo.edu; Bonner, Matthew R., E-mail: mrbonner@buffalo.edu

Animal studies have shown that paraoxonase 1 (PON1) genotype can influence susceptibility to the organophosphorus pesticide chlorpyrifos (CPF). However, Monte Carlo analysis suggests that PON1 genotype may not affect CPF-related toxicity at low exposure conditions in humans. The current study sought to determine the influence of PON1 genotype on the activity of blood cholinesterase as well as the effect of CPF exposure on serum PON1 in workers occupationally exposed to CPF. Saliva, blood and urine were collected from agricultural workers (n = 120) from Egypt's Menoufia Governorate to determine PON1 genotype, blood cholinesterase activity, serum PON1 activity towards chlorpyrifos-oxon (CPOase)more » and paraoxon (POase), and urinary levels of the CPF metabolite 3,5,6-trichloro-2-pyridinol (TCPy). The PON1 55 (P ≤ 0.05) but not the PON1 192 genotype had a significant effect on CPOase activity. However, both the PON1 55 (P ≤ 0.05) and PON1 192 (P ≤ 0.001) genotypes had a significant effect on POase activity. Workers had significantly inhibited AChE and BuChE after CPF application; however, neither CPOase activity nor POase activity was associated with ChE depression when adjusted for CPF exposure (as determined by urinary TCPy levels) and stratified by PON1 genotype. CPOase and POase activity were also generally unaffected by CPF exposure although there were alterations in activity within specific genotype groups. Together, these results suggest that workers retained the capacity to detoxify chlorpyrifos-oxon under the exposure conditions experienced by this study population regardless of PON1 genotype and activity and that effects of CPF exposure on PON1 activity are minimal. -- Highlights: ► CPF exposure resulted in an increase in TCPy and decreases in BuChE and AChE. ► CPOase activity decreased in subjects with the PON1 55LM and PON1 55 MM genotypes. ► Neither PON1 genotype nor CPOase activity had an effect on BuChE or AChE inhibition.« less

Strychnine, but not PMBA, inhibits neuronal nicotinic acetylcholine receptors expressed by rabbit retinal ganglion cells.

PubMed

Renna, J M; Strang, C E; Amthor, F R; Keyser, K T

2007-01-01

Strychnine is considered a selective competitive antagonist of glycine gated Cl- channels (Saitoh et al., 1994) and studies have used strychnine at low micromolar concentrations to study the role of glycine in rabbit retina (Linn, 1998; Protti et al., 2005). However, other studies have shown that strychnine, in the concentrations commonly used, is also a potent competitive antagonist of alpha7 nicotinic acetylcholine receptors (nAChRs; Matsubayashi et al., 1998). We tested the effects of low micromolar concentrations of strychnine and 3-[2'-phosphonomethyl[1,1'-biphenyl]-3-yl] alanine (PMBA), a specific glycine receptor blocker (Saitoh et al., 1994; Hosie et al., 1999) on the activation of both alpha7 nAChRs on retinal ganglion cells and on ganglion cell responses to a light flash. Extracellular recordings were obtained from ganglion cells in an isolated retina/choroid preparation and 500 microM choline was used as an alpha7 agonist (Alkondon et al., 1997). We recorded from brisk sustained and brisk transient OFF cells, many of which have been previously shown to have alpha7 receptors (Strang et al., 2005). Further, we tested the effect of strychnine, PMBA and alpha-bungarotoxin on the binding of tetramethylrhodamine alpha-bungarotoxin in the inner plexiform layer. Our data indicates that strychnine, at doses as low as 1.0 microM, can inhibit the alpha7 nAChR-mediated response to choline, but PMBA at concentrations as high as 0.4 microM does not. Binding studies show strychnine and alpha-bungarotoxin inhibit binding of labeled alpha-bungarotoxin in the IPL. Thus, the effects of strychnine application may be to inhibit glycine receptors expressed by ganglion cell or to inhibit amacrine cell alpha7 nAChRs, both of which would result in an increase in the ganglion cell responses. Further research will be required to disentangle the effects of strychnine previously believed to be caused by a single mechanism of glycine receptor inhibition.

The role of the laterodorsal tegmental nucleus in methamphetamine conditioned place preference and locomotor activity.

PubMed

Dobbs, Lauren K; Cunningham, Christopher L

2014-05-15

Methamphetamine (METH) indirectly stimulates the laterodorsal tegmental nucleus (LDT) acetylcholine (ACh) neurons to increase ACh within the ventral tegmental area (VTA). LDT ACh inhibition attenuates METH and saline locomotor activity. The aim of these experiments was to determine whether LDT ACh contributes to METH conditioned place preference (CPP). C57BL/6J mice received a bilateral electrolytic or sham lesion of the LDT. After recovery, mice received alternating pairings of METH (0.5 mg/kg) and saline with distinct tactile floor cues over 8 days. During preference tests, mice were given access to both floor types and time spent on each was recorded. Mice were tested again after exposure to both extinction and reconditioning trials. Brains were then processed for choline acetyltransferase immunohistochemistry to label LDT ACh neurons. Lesioned mice had significantly fewer LDT ACh neurons and showed increased saline and METH locomotor activity during the first conditioning trial compared to sham mice. Locomotor activity (saline and METH) was negatively correlated with the number of LDT ACh neurons. Lesioned and sham mice showed similar METH CPP following conditioning, extinction and reconditioning trials. LDT ACh neurons are not necessary for METH reward as indexed by CPP, but may be important for basal and METH-induced locomotor activity. Copyright © 2014 Elsevier B.V. All rights reserved.

Design, synthesis and evaluation of rivastigmine and curcumin hybrids as site-activated multitarget-directed ligands for Alzheimer's disease therapy.

PubMed

Li, Yujie; Peng, Peng; Tang, Li; Hu, Yunzhen; Hu, Yongzhou; Sheng, Rong

2014-09-01

A series of novel 2-methoxy-phenyl dimethyl-carbamate derivatives were designed, synthesized and evaluated as site-activated MTDLs based on rivastigmine and curcumin. Most of them exhibited good to excellent AChE and BuChE inhibitory activities with sub-micromolar IC50 values. Among all the compounds, 6a demonstrated the most potent AChE inhibition with IC50 value of 0.097μM, which is about 20-fold than that of rivastigmine. In addition, the three selected compounds 5a, 6a and 6e demonstrated inhibitory activity against Aβ self-aggregation similar to cucurmin in TEM assay, which is obviously different from the weak activity of rivastigmine. Moreover, the hydrolysate of 6a (compound 7) also showed potent ABTS(+) scavenging and moderate copper ion chelating activity in vitro. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evidence for a role for α6* nAChRs in L-dopa-induced dyskinesias using parkinsonian α6* nAChR gain-of-function mice

PubMed Central

Bordia, Tanuja; McGregor, Matthew; McIntosh, J.M.; Drenan, Ryan M.; Quik, Maryka

2015-01-01

L-Dopa-induced dyskinesias (LIDs) are a serious side effect of dopamine replacement therapy for Parkinson's disease. The mechanisms that underlie LIDs are currently unclear. However, preclinical studies indicate that nicotinic acetylcholine receptors (nAChRs) play a role, suggesting that drugs targeting these receptors may be of therapeutic benefit. To further understand the involvement of α6β2* nAChRs in LIDs, we used gain-of-function α6* nAChR (α6L9S) mice that exhibit a 20-fold enhanced sensitivity to nAChR agonists. Wildtype (WT) and α6L9S mice were lesioned by unilateral injection of 6-hydroxydopamine (6-OHDA, 3 μg/ml) into the medial forebrain bundle. Three to 4 wk later, they were administered L-dopa (3 mg/kg) plus benserazide (15 mg/kg) until stably dyskinetic. L-dopa-induced abnormal involuntary movements (AIMs) were similar in α6L9S and WT mice. WT mice were then given nicotine in the drinking water in gradually increasing doses to a final 300 μg/ml, which resulted in a 40% decline AIMs. By contrast, there was no decrease in AIMs in α6L9S mice at a maximally tolerated nicotine dose of 20 μg/ml. However, the nAChR antagonist mecamylamine (1 mg/kg ip 30 min before L-dopa) reduced L-dopa-induced AIMs in both α6L9S and WT mice. Thus, both a nAChR agonist and antagonist decreased AIMs in WT mice, but only the antagonist was effective in α6L9S mice. Since nicotine appears to reduce LIDs via desensitization, hypersensitive α6β2* nAChRs may desensitize less readily. The present data show that α6β2* nAChRs are key regulators of LIDs, and may be useful therapeutic targets for their management in Parkinson's disease. PMID:25813704

Acetylcholinesterase inhibition reveals endogenous nicotinic modulation of glutamate inputs to CA1 stratum radiatum interneurons in hippocampal slices

PubMed Central

Alkondon, Manickavasagom; Albuquerque, Edson X.; Pereira, Edna F.R.

2013-01-01

The involvement of brain nicotinic acetylcholine receptors (nAChRs) in the neurotoxicological effects of soman, a potent acetylcholinesterase (AChE) inhibitor and a chemical warfare agent, is not clear. This is partly due to a poor understanding of the role of AChE in brain nAChR-mediated functions. To test the hypothesis that AChE inhibition builds sufficient acetylcholine (ACh) in the brain and facilitates nAChR-dependent glutamate transmission, we used whole-cell patch-clamp technique to record spontaneous glutamate excitatory postsynaptic currents (EPSCs) from CA1 stratum radiatum interneurons (SRI) in hippocampal slices. First, the frequency, amplitude and kinetics of EPSCs recorded from slices of control guinea pigs were compared to those recorded from slices of guinea pigs after a single injection of the irreversible AChE inhibitor soman (25.2 μg/kg, s.c.). Second, EPSCs were recorded from rat hippocampal slices before and after their superfusion with the reversible AChE inhibitor donepezil (100 nM). The frequency of EPSCs was significantly higher in slices taken from guinea pigs 24 h but not 7 days after the soman injection than in slices from control animals. In 52% of the rat hippocampal slices tested, bath application of donepezil increased the frequency of EPSCs. Further, exposure to donepezil increased both burst-like and large-amplitude EPSCs, and increased the proportion of short (20–100 ms) inter-event intervals. Donepezil’s effects were suppressed significantly in presence of 10 μM mecamylamine or 10 nM methyllycaconitine. These results support the concept that AChE inhibition is able to recruit nAChR-dependent glutamate transmission in the hippocampus and such a mechanism can contribute to the acute neurotoxicological actions of soman. PMID:23511125

Behavioral impact of neurotransmitter-activated GPCRs: Muscarinic and GABAB receptors regulate C. elegans locomotion

PubMed Central

Dittman, Jeremy S; Kaplan, Joshua M

2008-01-01

Neurotransmitter released from presynaptic terminals activates both ligand-gated ion channels (ionotropic receptors) and a variety of G protein-coupled receptors (GPCRs). These neurotransmitter receptors are expressed on both pre- and postsynaptic cells. Thus, each neurotransmitter acts on multiple receptor classes, generating a large repertoire of physiological responses. The impact of many ionotropic receptors on neuronal activity and behavior has been clearly elucidated; however, much less is known about how neurotransmitter-gated GPCRs regulate neurons and circuits. In C. elegans, both Acetylcholine (ACh) and GABA are released in the nerve cord and mediate fast neuromuscular excitation and inhibition during locomotion. Here we identify a muscarinic receptor (GAR-2) and the GABAB receptor dimer (GBB-1/2) that detect synaptically released ACh and GABA, respectively. Both GAR-2 and GBB-1/2 inhibited cholinergic motor neurons when ACh and GABA levels were enhanced. Loss of either GPCR resulted in movement defects, suggesting that these receptors are activated during locomotion. When the negative feedback provided by GAR-2 was replaced with positive feedback, animals became highly sensitive to ACh levels and locomotion was severely impaired. Thus, conserved GPCRs act in the nematode motor circuit to provide negative feedback and to regulate locomotory behaviors that underlie navigation. PMID:18614679

Functional link between muscarinic receptors and large-conductance Ca2+-activated K+ channels in freshly-isolated human detrusor smooth muscle cells

PubMed Central

Parajuli, Shankar P.; Hristov, Kiril L.; Cheng, Qiuping; Malysz, John; Rovner, Eric S.; Petkov, Georgi V.

2014-01-01

Activation of muscarinic acetylcholine receptors (mAChRs) constitutes the primary mechanism for enhancing excitability and contractility of human detrusor smooth muscle (DSM). Since the large conductance Ca2+-activated K+ (KCa1.1) channels are key regulators of human DSM function, we investigated whether mAChR activation increases human DSM excitability by inhibiting KCa1.1 channels. We used the mAChR agonist, carbachol, to determine the changes in KCa1.1 channel activity upon mAChR activation in freshly-isolated human DSM cells obtained from open bladder surgeries using the perforated whole cell and single KCa1.1 channel patch-clamp recordings. Human DSM cells were collected from 29 patients (23 males and 6 females, average age of 65.9±1.5 years). Carbachol inhibited the amplitude and frequency of KCa1.1 channel-mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, which are triggered by the release of Ca2+ from ryanodine receptors. Carbachol also caused membrane potential depolarization, which was not observed in the presence of iberiotoxin, a KCa1.1 channel inhibitor, indicating the critical role of the KCa1.1 channels. The potential direct carbachol effects on KCa1.1channels were examined under conditions of removing the major cellular Ca2+ sources for KCa1.1 channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single KCa1.1 channel open probability and mean KCa1.1 channel conductance (cell-attached configuration) or depolarization-induced whole cell steady-state KCa1.1 currents. The data support the concept that mAChR activation triggers indirect functional KCa1.1 channel inhibition mediated by intracellular Ca2+, thus increasing the excitability in human DSM cells. PMID:24867682

α7-nAChR Knockout Mice Decreases Biliary Hyperplasia and Liver Fibrosis in Cholestatic Bile-Duct Ligated Mice.

PubMed

Ehrlich, Laurent; O'Brien, April; Hall, Chad; White, Tori; Chen, Lixian; Wu, Nan; Venter, Julie; Scrushy, Marinda; Mubarak, Muhammad; Meng, Fanyin; Dostal, David; Wu, Chaodong; Lairmore, Terry C; Alpini, Gianfranco; Glaser, Shannon

2018-03-26

α7-nAChR is a nicotinic acetylcholine receptor (specifically expressed on hepatic stellate cells, Kupffer cells, and cholangiocytes) that regulates inflammation and apoptosis in the liver. Thus, targeting α7-nAChR may be therapeutic in biliary diseases. Bile-duct ligation (BDL) was performed on wild-type (WT) and α7-nAChR-/- mice. We first evaluated the expression of α7-nAChR by immunohistochemistry (IHC) in liver sections. IHC was also performed to assess intrahepatic bile-duct mass (IBDM), and Sirius Red staining was performed to quantify the amount of collagen deposition. Immunofluorescence was performed to assess co-localization of α7-nAChR with bile ducts (co-stained with CK-19) and hepatic stellate cells (HSCs) (co-stained with desmin). The mRNA expression of α7-nAChR, Ki67/PCNA (proliferation), fibrosis genes (TGF-β1, Fibronectin-1, Col1α1, and α-SMA), and inflammatory markers (IL-6, IL-1β, and TNFα) was measured by real-time PCR. Biliary TGF-β1 and hepatic CD68 (Kupffer cell marker) expression was assessed using IHC. α7-nAChR immunoreactivity was observed in both bile ducts and HSCs and increased following BDL. α7-nAChR-/- BDL mice exhibited decreased: (i) bile duct mass, liver fibrosis, and inflammation; and (ii) immunoreactivity of TGF-1 as well as expression of fibrosis genes compared to WT BDL mice. α7-nAChR activation triggers biliary proliferation and liver fibrosis and may be a therapeutic target in managing extra-hepatic biliary obstruction.

Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery.

PubMed

González-Cestari, Tatiana F; Henderson, Brandon J; Pavlovicz, Ryan E; McKay, Susan B; El-Hajj, Raed A; Pulipaka, Aravinda B; Orac, Crina M; Reed, Damon D; Boyd, R Thomas; Zhu, Michael X; Li, Chenglong; Bergmeier, Stephen C; McKay, Dennis B

2009-02-01

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 microM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.

Effect of Novel Negative Allosteric Modulators of Neuronal Nicotinic Receptors on Cells Expressing Native and Recombinant Nicotinic Receptors: Implications for Drug Discovery

PubMed Central

González-Cestari, Tatiana F.; Henderson, Brandon J.; Pavlovicz, Ryan E.; McKay, Susan B.; El-Hajj, Raed A.; Pulipaka, Aravinda B.; Orac, Crina M.; Reed, Damon D.; Boyd, R. Thomas; Zhu, Michael X.; Li, Chenglong; Bergmeier, Stephen C.; McKay, Dennis B.

2009-01-01

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of α3β4* nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant α3β4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native α3β4* nAChR, with IC50 values ranging from 0.4 to 13.0 μM. Using cells expressing recombinant α3β4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC50 values ranging from 0.7 to 38.2 μM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 μM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery. PMID:18984653

The role of the oximes HI-6 and HS-6 inside human acetylcholinesterase inhibited with nerve agents: a computational study.

PubMed

Cuya, Teobaldo; Gonçalves, Arlan da Silva; da Silva, Jorge Alberto Valle; Ramalho, Teodorico C; Kuca, Kamil; C C França, Tanos

2017-10-27

The oximes 4-carbamoyl-1-[({2-[(E)-(hydroxyimino) methyl] pyridinium-1-yl} methoxy) methyl] pyridinium (known as HI-6) and 3-carbamoyl-1-[({2-[(E)-(hydroxyimino) methyl] pyridinium-1-yl} methoxy) methyl] pyridinium (known as HS-6) are isomers differing from each other only by the position of the carbamoyl group on the pyridine ring. However, this slight difference was verified to be responsible for big differences in the percentual of reactivation of acetylcholinesterase (AChE) inhibited by the nerve agents tabun, sarin, cyclosarin, and VX. In order to try to find out the reason for this, a computational study involving molecular docking, molecular dynamics, and binding energies calculations, was performed on the binding modes of HI-6 and HS-6 on human AChE (HssAChE) inhibited by those nerve agents.

Biological Activities and Composition of Ferulago carduchorum Essential Oil

PubMed Central

Golfakhrabadi, Fereshteh; Khanavi, Mahnaz; Ostad, Seyed Nasser; Saeidnia, Soodabeh; Vatandoost, Hassan; Abai, Mohammad Reza; Hafizi, Mitra; Yousefbeyk, Fatemeh; Rad, Yaghoob Razzaghi; Baghenegadian, Ameneh; Ardekani, Mohammad Reza Shams

2015-01-01

Background: Ferulago carduchorum Boiss and Hausskn belongs to the Apiaceae family. This plant grows in west part of Iran that local people added it to dairy and oil ghee to delay expiration date and give them a pleasant taste. The aim of this study was to investigate the antioxidant, antimicrobial, acetyl cholinesterase inhibition, cytotoxic, larvicidal activities and composition of essential oil of F. carduchorum. Methods: Acetyl cholinesterase (AChE) inhibitory, larvicidal activities and chemical composition of essential oil of F. carduchorum were investigated. Besides, antioxidant, antimicrobial and cytotoxic activities of essential oil were tested using DPPH, microdilution method and MTT assay, respectively. Results: The major components of essential oil were (z)-β-ocimene (43.3%), α-pinene (18.23%) and bornyl acetate (3.98%). Among 43 identified components, monoterpenes were the most compounds (84.63%). The essential oil had noticeable efficiency against Candida albicans (MIC= 2340 μg ml−1) and it was effective against Anopheles stephensi with LC50 and LC90 values of 12.78 and 47.43 ppm, respectively. The essential oil could inhibit AChE (IC50= 23.6 μl ml−1). The essential oil showed high cytotoxicity on T47D, HEP-G2 and HT-29 cell lines (IC50< 2 μg ml−1). Conclusion: The essential oil of F. carduchorum collected from west of Iran had anti-Candida, larvicidal and cytotoxicity effects and should be further investigated in others in vitro and in vivo experimental models. PMID:26114148

Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice

PubMed Central

Cole, R N; Ghazanfari, N; Ngo, S T; Gervásio, O L; Reddel, S W; Phillips, W D

2010-01-01

The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day−1i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51–73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR β-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering. PMID:20603331

Cholinesterase inhibition and acetylcholine accumulation following intracerebral administration of paraoxon in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ray, A.; Liu, J.; Karanth, S.

2009-05-01

We evaluated the inhibition of striatal cholinesterase activity following intracerebral administration of paraoxon assaying activity either in tissue homogenates ex vivo or by substrate hydrolysis in situ. Artificial cerebrospinal fluid (aCSF) or paraoxon in aCSF was infused unilaterally (0.5 {mu}l/min for 2 h) and ipsilateral and contralateral striata were harvested for ChE assay ex vivo. High paraoxon concentrations were needed to inhibit ipsilateral striatal cholinesterase activity (no inhibition at < 0.1 mM; 27% at 0.1 mM; 79% at 1 mM paraoxon). With 3 mM paraoxon infusion, substantial ChE inhibition was also noted in contralateral striatum. ChE histochemistry generally confirmed thesemore » concentration- and side-dependent effects. Microdialysates collected for up to 4 h after paraoxon infusion inhibited ChE activity when added to striatal homogenate, suggesting prolonged efflux of paraoxon. Since paraoxon efflux could complicate acetylcholine analysis, we evaluated the effects of paraoxon (0, 0.03, 0.1, 1, 10 or 100 {mu}M, 1.5 {mu}l/min for 45 min) administered by reverse dialysis through a microdialysis probe. ChE activity was then monitored in situ by perfusing the colorimetric substrate acetylthiocholine through the same probe and measuring product (thiocholine) in dialysates. Concentration-dependent inhibition was noted but reached a plateau of about 70% at 1 {mu}M and higher concentrations. Striatal acetylcholine was below the detection limit at all times with 0.1 {mu}M paraoxon but was transiently elevated (0.5-1.5 h) with 10 {mu}M paraoxon. In vivo paraoxon (0.4 mg/kg, sc) in adult rats elicited about 90% striatal ChE inhibition measured ex vivo, but only about 10% inhibition measured in situ. Histochemical analyses revealed intense AChE and glial fibrillary acidic protein staining near the cannula track, suggesting proliferation of inflammatory cells/glia. The findings suggest that ex vivo and in situ cholinesterase assays can provide very

Small molecular floribundiquinone B derived from medicinal plants inhibits acetylcholinesterase activity

PubMed Central

Jiang, Li; Qin, Rui; Su, Qiang; Chen, Fuxue; Du, Dongshu; Shu, Yilai; Chou, Kuo-Chen

2017-01-01

Being a neurodegenerative disorder, Alzheimer's disease (AD) is the one of the most terrible diseases. And acetylcholinesterase (AChE) is considered as an important target for treating AD. Acetylcholinesterase inhibitors (AChEI) are considered to be one of the effective drugs for the treatment of AD. The aim of this study is to find a novel potential AChEI as a drug for the treatment of AD. In this study, instead of using the synthetic compounds, we used those extracted from plants to investigate the interaction between floribundiquinone B (FB) and AChE by means of both the experimental approach such as fluorescence spectra, ultraviolet-visible (UV-vis) absorption spectrometry, circular dichroism (CD) and the theoretical approaches such as molecular docking. The findings reported here have provided many useful clues and hints for designing more effective and less toxic drugs against Alzheimer's disease. PMID:28915661

Transferable residues from dog fur and plasma cholinesterase inhibition in dogs treated with a flea control dip containing chlorpyrifos.

PubMed Central

Boone, J S; Tyler, J W; Chambers, J E

2001-01-01

We studied chlorpyrifos, an insecticide present in a commercial dip for treating ectoparasites in dogs, to estimate the amount of transferable residues that children could obtain from their treated pets. Although the chlorpyrifos dip is no longer supported by the manufacturer, the methodology described herein can help determine transferable residues from other flea control insecticide formulations. Twelve dogs of different breeds and weights were dipped using the recommended guidelines with a commercial, nonprescription chlorpyrifos flea dip for 4 consecutive treatments at 3-week intervals (nonshampoo protocol) and another 12 dogs were dipped with shampooing between dips (shampoo protocol). The samples collected at 4 hr and 7, 14, and 21 days after treatment in the nonshampoo protocol averaged 971, 157, 70, and 26 microg chlorpyrifos, respectively; in the shampoo protocol the samples averaged 459, 49, 15, and 10 microg, respectively. The highest single sample was about 7,000 microg collected at 4 hr. The pretreatment specific activities in the plasma of the dogs were about 75 nmol/min/mg protein for butyrylcholinesterase (BChE), and 9 nmol/min/mg protein for acetylcholinesterase (AChE). BChE was inhibited 50-75% throughout the study, and AChE was inhibited 11-18% in the nonshampoo protocol; inhibition was not as great in the shampoo protocol. There was no correlation (pinhibition was observed at 7 days than at 4 hr, probably reflecting the bioactivation of chlorpyrifos to chlorpyrifos-oxon. Plasma cholinesterase activity did not return to control levels during the 3-week period. The differences between the shampoo and nonshampoo protocols were explained by differences in the techniques of the dip

Antioxidant activity and cholinesterase inhibition studies of four flavouring herbs from Alentejo.

PubMed

Arantes, Sílvia; Piçarra, Andreia; Candeias, Fátima; Caldeira, A Teresa; Martins, M Rosário; Teixeira, Dora

2017-09-01

Essential oils (EOs) and aqueous extracts of aerial parts of four aromatic species, Calamintha nepeta, Foeniculum vulgare, Mentha spicata and Thymus mastichina, from southwest of Portugal were characterised chemically and analysed in order to evaluate their antioxidant potential and cholinesterase inhibitory activities. The main components of EOs were oxygenated monoterpenes, and aqueous extracts were rich in phenol and flavonoid compounds. EOs and aqueous extracts presented a high antioxidant potential, with ability to protect the lipid substrate, free radical scavenging and iron reducing power. Furthermore, EOs and extracts showed AChE and BChE inhibitory activities higher than rivastigmine, the standard drug. Results suggested the potential use of EOs and aqueous extracts of these flavouring herbs as nutraceutical or pharmaceutical preparations to minimise the oxidative stress and the progression of degenerative diseases.

Tumor necrosis factor-alpha inhibits insulin's stimulating effect on glucose uptake and endothelium-dependent vasodilation in humans.

PubMed

Rask-Madsen, Christian; Domínguez, Helena; Ihlemann, Nikolaj; Hermann, Thomas; Køber, Lars; Torp-Pedersen, Christian

2003-10-14

Inflammatory mechanisms could be involved in the pathogenesis of both insulin resistance and atherosclerosis. Therefore, we aimed at examining whether the proinflammatory cytokine tumor necrosis factor (TNF)-alpha inhibits insulin-stimulated glucose uptake and insulin-stimulated endothelial function in humans. Healthy, lean male volunteers were studied. On each study day, 3 acetylcholine (ACh) or sodium nitroprusside (SNP) dose-response studies were performed by infusion into the brachial artery. Before and during the last 2 dose-response studies, insulin and/or TNF-alpha were coinfused. During infusion of insulin alone for 20 minutes, forearm glucose uptake increased by 220+/-44%. This increase was completely inhibited during coinfusion of TNF-alpha (started 10 min before insulin) with a more pronounced inhibition of glucose extraction than of blood flow. Furthermore, TNF-alpha inhibited the ACh forearm blood flow response (P<0.001), and this inhibition was larger during insulin infusion (P=0.01) but not further increased by NG-monomethyl-L-arginine acetate (P=0.2). Insulin potentiated the SNP response less than the ACh response and the effect of TNF-alpha was smaller (P<0.001); TNF-alpha had no effect on the SNP response without insulin infusion. Thus, TNF-alpha inhibition of the combined response to insulin and ACh was likely mediated through inhibition of NO production. These results support the concept that TNF-alpha could play a role in the development of insulin resistance in humans, both in muscle and in vascular tissue.

Lanostanoids with acetylcholinesterase inhibitory activity from the mushroom Haddowia longipes.

PubMed

Zhang, Shuang-Shuang; Ma, Qing-Yun; Huang, Sheng-Zhuo; Dai, Hao-Fu; Guo, Zhi-Kai; Yu, Zhi-Fang; Zhao, You-Xing

2015-02-01

Nine lanostanoids, together with nine known ones, were isolated from the ethyl acetate extract of the fruiting bodies of the mushroom Haddowia longipes. Their structures were elucidated as 11-oxo-ganoderiol D, lanosta-8-en-7,11-dioxo-3β-acetyloxy-24,25,26-trihydroxy, lanosta-8-en-7-oxo-3β-acetyloxy-11β,24,25,26-tetrahydroxy, lanosta-7,9(11)-dien-3β-acetyloxy-24,25,26-trihydroxy, lanosta-7,9(11)-dien-3β-acetyloxy-24,26-dihydroxy-25-methoxy, 11-oxo-lucidadiol, 11β-hydroxy-lucidadiol, lucidone H and lanosta-7,9(11),24E-trien-3β-acetyloxy-26,27-dihydroxy by analysing their 1D/2D NMR and MS spectra. In addition, bioassays of inhibitory activity against acetylcholinesterase (AChE) of all compounds showed that thirteen compounds possessed inhibitory activity against AChE with the percentage inhibition ranging from 10.3% to 42.1% when tested at 100 μM. Copyright © 2015 Elsevier Ltd. All rights reserved.

PLGA-PEG Nanoparticles Coated with Anti-CD45RO and Loaded with HDAC Plus Protease Inhibitors Activate Latent HIV and Inhibit Viral Spread

NASA Astrophysics Data System (ADS)

Tang, Xiaolong; Liang, Yong; Liu, Xinkuang; Zhou, Shuping; Liu, Liang; Zhang, Fujina; Xie, Chunmei; Cai, Shuyu; Wei, Jia; Zhu, Yongqiang; Hou, Wei

2015-10-01

Activating HIV-1 proviruses in latent reservoirs combined with inhibiting viral spread might be an effective anti-HIV therapeutic strategy. Active specific delivery of therapeutic drugs into cells harboring latent HIV, without the use of viral vectors, is a critical challenge to this objective. In this study, nanoparticles of poly(lactic-co-glycolic acid)-polyethylene glycol diblock copolymers conjugated with anti-CD45RO antibody and loaded with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and/or protease inhibitor nelfinavir (Nel) were tested for activity against latent virus in vitro. Nanoparticles loaded with SAHA, Nel, and SAHA + Nel were characterized in terms of size, surface morphology, zeta potential, entrapment efficiency, drug release, and toxicity to ACH-2 cells. We show that SAHA- and SAHA + Nel-loaded nanoparticles can target latently infected CD4+ T-cells and stimulate virus production. Moreover, nanoparticles loaded with SAHA + NEL were capable of both activating latent virus and inhibiting viral spread. Taken together, these data demonstrate the potential of this novel reagent for targeting and eliminating latent HIV reservoirs.

Assessment of the expression and role of the α1-nAChR subunit in efferent cholinergic function during the development of the mammalian cochlea.

PubMed

Roux, Isabelle; Wu, Jingjing Sherry; McIntosh, J Michael; Glowatzki, Elisabeth

2016-08-01

Hair cell (HC) activity in the mammalian cochlea is modulated by cholinergic efferent inputs from the brainstem. These inhibitory inputs are mediated by calcium-permeable nicotinic acetylcholine receptors (nAChRs) containing α9- and α10-subunits and by subsequent activation of calcium-dependent potassium channels. Intriguingly, mRNAs of α1- and γ-nAChRs, subunits of the "muscle-type" nAChR have also been found in developing HCs (Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. J Neurosci 35: 5870-5883, 2015; Scheffer D, Sage C, Plazas PV, Huang M, Wedemeyer C, Zhang DS, Chen ZY, Elgoyhen AB, Corey DP, Pingault V. J Neurochem 103: 2651-2664, 2007; Sinkkonen ST, Chai R, Jan TA, Hartman BH, Laske RD, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller S. Sci Rep 1: 26, 2011) prompting proposals that another type of nAChR is present and may be critical during early synaptic development. Mouse genetics, histochemistry, pharmacology, and whole cell recording approaches were combined to test the role of α1-nAChR subunit in HC efferent synapse formation and cholinergic function. The onset of α1-mRNA expression in mouse HCs was found to coincide with the onset of the ACh response and efferent synaptic function. However, in mouse inner hair cells (IHCs) no response to the muscle-type nAChR agonists (±)-anatoxin A, (±)-epibatidine, (-)-nicotine, or 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) was detected, arguing against the presence of an independent functional α1-containing muscle-type nAChR in IHCs. In α1-deficient mice, no obvious change of IHC efferent innervation was detected at embryonic day 18, contrary to the hyperinnervation observed at the neuromuscular junction. Additionally, ACh response and efferent synaptic activity were detectable in α1-deficient IHCs, suggesting that α1 is not necessary for assembly and membrane targeting of nAChRs or for efferent synapse formation in IHCs.

Assessment of the expression and role of the α1-nAChR subunit in efferent cholinergic function during the development of the mammalian cochlea

PubMed Central

Wu (武靜靜), Jingjing Sherry; McIntosh, J. Michael; Glowatzki, Elisabeth

2016-01-01

Hair cell (HC) activity in the mammalian cochlea is modulated by cholinergic efferent inputs from the brainstem. These inhibitory inputs are mediated by calcium-permeable nicotinic acetylcholine receptors (nAChRs) containing α9- and α10-subunits and by subsequent activation of calcium-dependent potassium channels. Intriguingly, mRNAs of α1- and γ-nAChRs, subunits of the “muscle-type” nAChR have also been found in developing HCs (Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. J Neurosci 35: 5870–5883, 2015; Scheffer D, Sage C, Plazas PV, Huang M, Wedemeyer C, Zhang DS, Chen ZY, Elgoyhen AB, Corey DP, Pingault V. J Neurochem 103: 2651–2664, 2007; Sinkkonen ST, Chai R, Jan TA, Hartman BH, Laske RD, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller S. Sci Rep 1: 26, 2011) prompting proposals that another type of nAChR is present and may be critical during early synaptic development. Mouse genetics, histochemistry, pharmacology, and whole cell recording approaches were combined to test the role of α1-nAChR subunit in HC efferent synapse formation and cholinergic function. The onset of α1-mRNA expression in mouse HCs was found to coincide with the onset of the ACh response and efferent synaptic function. However, in mouse inner hair cells (IHCs) no response to the muscle-type nAChR agonists (±)-anatoxin A, (±)-epibatidine, (−)-nicotine, or 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) was detected, arguing against the presence of an independent functional α1-containing muscle-type nAChR in IHCs. In α1-deficient mice, no obvious change of IHC efferent innervation was detected at embryonic day 18, contrary to the hyperinnervation observed at the neuromuscular junction. Additionally, ACh response and efferent synaptic activity were detectable in α1-deficient IHCs, suggesting that α1 is not necessary for assembly and membrane targeting of nAChRs or for efferent synapse formation in IHCs. PMID:27098031

Chemical Composition and Acetylcholinesterase Inhibitory Activity of Essential Oils from Piper Species.

PubMed

Xiang, Cai-Peng; Han, Jia-Xin; Li, Xing-Cong; Li, Yun-Hui; Zhang, Yi; Chen, Lin; Qu, Yan; Hao, Chao-Yun; Li, Hai-Zhou; Yang, Chong-Ren; Zhao, San-Jun; Xu, Min

2017-05-10

The essential oils (EOs) derived from aromatic plants such as Piper species are considered to play a role in alleviating neuronal ailments that are associated with inhibition of acetylcholinesterase (AChE). The chemical compositions of 23 EOs prepared from 16 Piper spp. were analyzed by both gas chromatography with a flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 76 compounds were identified in the EOs from the leaves and stems of 19 samples, while 30 compounds were detected in the EOs from the fruits of four samples. Sesquiterpenes and phenylpropanoids were found to be rich in these EOs, of which asaricin, caryophyllene, caryophyllene oxide, isospathulenol, (+)-spathulenol, and β-bisabolene are the major constituents. The EOs from the leaves and stems of Piper austrosinense, P. puberulum, P. flaviflorum, P. betle, and P. hispidimervium showed strong AChE inhibitory activity with IC 50 values in the range of 1.51 to 13.9 mg/mL. A thin-layer chromatography (TLC) bioautography assay was employed to identify active compound(s) in the most active EO from P. hispidimervium. The active compound was isolated and identified as asaricin, which gave an IC 50 value of 0.44 ± 0.02 mg/mL against AChE, comparable to galantamine with an IC 50 0.15 ± 0.01 mg/mL.

In vitro screening for anti-cholinesterase and antioxidant activity of methanolic extracts of ayurvedic medicinal plants used for cognitive disorders.

PubMed

Mathew, Maya; Subramanian, Sarada

2014-01-01

Inhibition of Acetylcholinesterase (AChE) is still considered as the main therapeutic strategy against Alzheimer's disease (AD). Many plant derived phytochemicals have shown AChE inhibitory activity in addition to the currently approved drugs for AD. In the present study, methanolic extracts of 20 plants used in Indian Ayurvedic system of medicine for improving cognitive function were screened for acetylcholinesterase inhibitory activity by Ellman's microplate colorimetric method. Out of 20 extracts, Emblica officinalis, Nardostachys jatamansi, Nelumbo nucifera, Punica granatum and Raulfia Serpentina showed IC50 values <100 µg/ml for acetylcholinesterase inhibitory activity. Antioxidant activities of these plants were assessed by DPPH scavenging assay. Among the extracts used, antioxidant activity was highest for Terminalia chebula and Emblica officinalis with IC50 values <10 µg/ml. Considering the complex multifactorial etiology of AD, these plant extracts will be safer and better candidates for the future disease modifying therapies against this devastating disease.

Activation of the α7 nicotinic ACh receptor induces anxiogenic effects in rats which is blocked by a 5-HT1a receptor antagonist

PubMed Central

Pandya, Anshul A.; Yakel, Jerrel L.

2013-01-01

The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in different regions of the brain and is associated with cognitive function as well as anxiety. Agonists and positive allosteric modulators (PAMs) of the α7 subtype of nAChRs have been shown to improve cognition. Previously nicotine, which activates both α7 and non-α7 subtypes of nAChRs, has been shown to have an anxiogenic effect in behavioral tests. In this study, we compared the effects of the α7-selective agonist (PNU-282987) and PAM (PNU-120596) in a variety of behavioral tests in Sprague Dawley rats to look at their effects on learning and memory as well as anxiety. We found that neither PNU-282987 nor PNU-120596 improved spatial-learning or episodic memory by themselves. However when cognitive impairment was induced in the rats with scopolamine (1 mg/kg), both PNU-120596 and PNU-282987 were able to reverse this memory impairment and restore it back to normal levels. While PNU-120596 reversed the scopolamine-induced cognitive impairment, it did not have any adverse effect on anxiety. PNU-282987 on the other hand displayed an increase in anxiety-like behavior at a higher dose (10 mg/kg) that was significantly reduced by the serotonin 5-HT1a receptor antagonist WAY-100135. However the α7 receptor antagonist methyllycaconitine was unable to reverse these anxiety-like effects seen with PNU-282987. These results suggest that α7 nAChR PAMs are pharmacologically advantageous over agonists, and should be considered for further development as therapeutic drugs targeting the α7 receptors. PMID:23321689

Induction of plasma acetylcholinesterase activity in mice challenged with organophosphorus poisons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duysen, Ellen G.; Lockridge, Oksana, E-mail: olockrid@unmc.edu

2011-09-01

The restoration of plasma acetylcholinesterase activity in mice following inhibition by organophosphorus pesticides and nerve agents has been attributed to synthesis of new enzyme. It is generally assumed that activity levels return to normal, are stable and do not exceed the normal level. We have observed over the past 10 years that recovery of acetylcholinesterase activity levels in mice treated with organophosphorus agents (OP) exceeds pretreatment levels and remains elevated for up to 2 months. The most dramatic case was in mice treated with tri-cresyl phosphate and tri-ortho-cresyl phosphate, where plasma acetylcholinesterase activity rebounded to a level 250% higher thanmore » the pretreatment activity. The present report summarizes our observations on plasma acetylcholinesterase activity in mice treated with chlorpyrifos, chlorpyrifos oxon, diazinon, tri-ortho-cresyl phosphate, tri-cresyl phosphate, tabun thiocholine, parathion, dichlorvos, and diisopropylfluorophosphate. We have developed a hypothesis to explain the excess acetylcholinesterase activity, based on published observations. We hypothesize that acetylcholinesterase activity is induced when cells undergo apoptosis and that consequently there is a rise in the level of plasma acetylcholinesterase. - Highlights: > Acetylcholinesterase activity is induced by organophosphorus agents. > AChE induction is related to apoptosis. > Induction of AChE activity by OP is independent of BChE.« less

Pterostilbene-O-acetamidoalkylbenzylamines derivatives as novel dual inhibitors of cholinesterase with anti-β-amyloid aggregation and antioxidant properties for the treatment of Alzheimer's disease.

PubMed

Li, Yuxing; Qiang, Xiaoming; Li, Yan; Yang, Xia; Luo, Li; Xiao, Ganyuan; Cao, Zhongcheng; Tan, Zhenghuai; Deng, Yong

2016-04-15

A series of pterostilbene-O-acetamidoalkylbenzylamines were designed, synthesized and evaluated as dual inhibitors of AChE and BuChE. To further explore the multifunctional properties of the new derivatives, their antioxidant activities and inhibitory effects on self-induced Aβ1-42 aggregation and HuAChE-induced Aβ1-40 aggregation were also tested. The results showed that most of these compounds could effectively inhibit AChE and BuChE. Particularly, compound 21d exhibited the best AChE inhibitory activity (IC50=0.06 μM) and good inhibition of BuChE (IC50=28.04 μM). Both the inhibition kinetic analysis and molecular modeling study revealed that these compounds showed mixed-type inhibition, binding simultaneously to the CAS and PAS of AChE. In addition to cholinesterase inhibitory activities, these compounds showed different levels of antioxidant activity. However, the inhibitory activities against self-induced and HuAChE-induced Aβ aggregation of these new derivatives were unsatisfied. Taking into account the results of the biological evaluation, further modifications will be designed in order to increase the potency on the different targets. The results displayed in this Letter can be a new starting point for further development of multifunctional agents for Alzheimer's disease. Copyright © 2016 Elsevier Ltd. All rights reserved.