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Sample records for ach receptor nachr

  1. 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. PMID:27385587

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

    PubMed

    Hamouda, Ayman K; Deba, Farah; Wang, Ze-Jun; Cohen, Jonathan B

    2016-05-01

    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 theα4 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 [(3)H]CMPI upon photolysis at 312 nm to identify its binding sites inTorpedonAChRs. Recording fromXenopusoocytes, we found that CMPI potentiated maximally the responses of (α4)3(β2)2nAChR to 10μM ACh (EC10) by 400% and with anEC50of ∼1µM. CMPI produced a left shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (∼35% at 10µM) ACh responses of (α4)2(β2)3nAChRs and fully inhibited human muscle andTorpedonAChRs with IC50values of ∼0.5µM. Upon irradiation at 312 nm, [(3)H]CMPI photoincorporated into eachTorpedo[(α1)2β1γδ] nAChR subunit. Sequencing of peptide fragments isolated from [(3)H]CMPI-photolabeled nAChR subunits established photolabeling of amino acids contributing to the ACh binding sites (αTyr(190),αTyr(198),γTrp(55),γTyr(111),γTyr(117),δTrp(57)) that was fully inhibitable by agonist and lower-efficiency, state-dependent [(3)H]CMPI photolabeling within the ion channel. Our results establish that CMPI is a potent potentiator of nAChRs containing anα4:α4 subunit interface, and that its intrinsic photoreactivy makes it of potential use to identify its binding sites in the (α4)3(β2)2nAChR. PMID:26976945

  3. Nicotinic acetylcholine receptors: a comparison of the nAChRs of Caenorhabditis elegans and parasitic nematodes.

    PubMed

    Holden-Dye, Lindy; Joyner, Michelle; O'Connor, Vincent; Walker, Robert J

    2013-12-01

    Nicotinic acetylcholine receptors (nAChRs) play a key role in the normal physiology of nematodes and provide an established target site for anthelmintics. The free-living nematode, Caenorhabditis elegans, has a large number of nAChR subunit genes in its genome and so provides an experimental model for testing novel anthelmintics which act at these sites. However, many parasitic nematodes lack specific genes present in C. elegans, and so care is required in extrapolating from studies using C. elegans to the situation in other nematodes. In this review the properties of C. elegans nAChRs are reviewed and compared to those of parasitic nematodes. This forms the basis for a discussion of the possible subunit composition of nAChRs from different species of parasitic nematodes. Currently our knowledge on this is largely based on studies using heterologous expression and pharmacological analysis of receptor subunits in Xenopus laevis oocytes. It is concluded that more information is required regarding the subunit composition and pharmacology of endogenous nAChRs in parasitic nematodes. PMID:23500392

  4. 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

  5. Synthesis and Biological Evaluation of Novel Carbon-11 Labeled Pyridyl Ethers: Candidate Ligands for In Vivo Imaging of α4β2 Nicotinic Acetylcholine Receptors (α4β2-nAChRs) in the brain with Positron Emission Tomography

    PubMed Central

    Gao, Yongjun; Ravert, Hayden T.; Kuwabara, Hiroto; Xiao, Yingxian; Endres, Christopher J.; Hilton, John; Holt, Daniel P.; Kumar, Anil; Alexander, Mohab; Wong, Dean F.; Dannals, Robert F.; Horti, Andrew G.

    2009-01-01

    The most abundant subtype of cerebral nicotinic acetylcholine receptors (nAChR), α4β2, plays a critical role in various brain functions and pathological states. Imaging agents suitable for visualization and quantification of α4β2 nAChRs by positron emission tomography (PET) would present unique opportunities to define the function and pharmacology of the nAChRs in the living human brain. In this study, we report the synthesis, nAChR binding affinity, and pharmacological properties of several novel 3-pyridyl ether compounds. Most of these derivatives displayed a high affinity to the nAChR and a high subtype selectivity for α4β2-nAChR. Three of these novel nAChR ligands were radiolabeled with the positron-emitting isotope 11C and evaluated in animal studies as potential PET radiotracers for imaging of cerebral nAChRs with improved brain kinetics. PMID:19481945

  6. Nicotinic acetylcholine receptors (nAChRs) at zebrafish red and white muscle show different properties during development.

    PubMed

    Ahmed, Kazi T; Ali, Declan W

    2016-08-01

    Nicotinic acetylcholine receptors (nAChRs) are highly expressed at the vertebrate neuromuscular junction (NMJ) where they are required for muscle activation. Understanding the factors that underlie NMJ development is critical for a full understanding of muscle function. In this study we performed whole cell and outside-out patch clamp recordings, and single-cell RT-qPCR from zebrafish red and white muscle to examine the properties of nAChRs during the first 5 days of development. In red fibers miniature endplate currents (mEPCs) exhibit single exponential time courses at 1.5 days postfertilization (dpf) and double exponential time courses from 2 dpf onwards. In white fibers, mEPCs decay relatively slowly, with a single exponential component at 1.5 dpf. By 2 and 3 dpf, mEPC kinetics speed up, and decay with a double exponential component, and by 4 dpf the exponential decay reverts back to a single component. Single channel recordings confirm the presence of two main conductance classes of nAChRs (∼45 pS and ∼65 pS) in red fibers with multiple time courses. Two main conductance classes are also present in white fibers (∼55 pS and ∼73 pS), but they exhibit shorter mean open times by 5 dpf compared with red muscle. RT-qPCR of mRNA for nicotinic receptor subunits supports a switch from γ to ε subunits in white fibers but not in red. Our findings provide a developmental profile of mEPC properties from red and white fibers in embryonic and larval zebrafish, and reveal previously unknown differences between the NMJs of these muscle fibers.© 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 916-936, 2016. PMID:26585318

  7. Biochemical and functional properties of distinct nicotinic acetylcholine receptors in the superior cervical ganglion of mice with targeted deletions of nAChR subunit genes.

    PubMed

    David, Reinhard; Ciuraszkiewicz, Anna; Simeone, Xenia; Orr-Urtreger, Avi; Papke, Roger L; McIntosh, J M; Huck, Sigismund; Scholze, Petra

    2010-03-01

    Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Here, we determined the subunit composition of hetero-pentameric nAChRs in the mouse superior cervical ganglion (SCG), the function of distinct receptors (obtained by deletions of nAChR subunit genes) and mechanisms at the level of nAChRs that might compensate for the loss of subunits. As shown by immunoprecipitation and Western blots, wild-type (WT) mice expressed: alpha 3 beta 4 (55%), alpha 3 beta 4 alpha 5 (24%) and alpha 3 beta 4 beta 2 (21%) nAChRs. nAChRs in beta 4 knockout (KO) mice were reduced to < 15% of controls and no longer contained the alpha 5 subunit. Compound action potentials, recorded from the postganglionic (internal carotid) nerve and induced by preganglionic nerve stimulation, did not differ between alpha 5 beta 4 KO and WT mice, suggesting that the reduced number of receptors in the KO mice did not impair transganglionic transmission. Deletions of alpha 5 or beta2 did not affect the overall number of receptors and we found no evidence that the two subunits substitute for each other. In addition, dual KOs allowed us to study the functional properties of distinct alpha 3 beta4 and alpha 3 beta 2 receptors that have previously only been investigated in heterologous expression systems. The two receptors strikingly differed in the decay of macroscopic currents, the efficacy of cytisine, and their responses to the alpha-conotoxins AuIB and MII. Our data, based on biochemical and functional experiments and several mouse KO models, clarify and significantly extend previous observations on the function of nAChRs in heterologous systems and the SCG. PMID:20377613

  8. [18F]ASEM, a radiolabeled antagonist for imaging the α7-nicotinic acetylcholine receptor (α7-nAChR) with positron emission tomography (PET)

    PubMed Central

    Horti, Andrew G.; Gao, Yongjun; Kuwabara, Hiroto; Wang, Yuchuan; Abazyan, Sofya; Yasuda, Robert P.; Tran, Thao; Xiao, Yingxian; Sahibzada, Niaz; Holt, Daniel P.; Kellar, Kenneth J.; Pletnikov, Mikhail V.; Pomper, Martin G.; Wong, Dean F.; Dannals, Robert F.

    2014-01-01

    The α7-nicotinic cholinergic receptor (α7-nAChR) is a key mediator of brain communication and has been implicated in a wide variety of central nervous system disorders. None of the currently available PET radioligands for α7-nAChR are suitable for quantitative PET imaging, mostly due to insufficient specific binding. The goal of this study was to evaluate the potential of [18F]ASEM ([18F]JHU82132) as an α7-nAChR radioligand for PET. Methods Inhibition binding assay and receptor functional properties of ASEM were assessed in vitro. The brain regional distribution of [18F]ASEM in baseline and blockade were evaluated in DISC1 mice (dissection) and baboons (PET). Results ASEM is an antagonist for the α7-nAChR with high binding affinity (Ki = 0.3 nM). [18F]ASEM readily entered the baboon brain and specifically labeled α7-nAChR. The in vivo specific binding of [18F]ASEM in the brain regions enriched with α7-nAChRs was 80–90%. SSR180711, an α7-nAChR selective partial agonist, blocked [18F]ASEM binding in the baboon brain in a dose-dependent manner, suggesting that the binding of [18F]ASEM was mediated by α7-nAChRs and the radioligand was suitable for drug evaluation studies. In the baboon baseline studies, the brain regional volume of distribution (VT) values for [18F]ASEM were 23 (thalamus), 22 (insula), 18 (hippocampus) and 14 (cerebellum), whereas in the binding selectivity (blockade) scan, all regional VT values were reduced to less than 4. The range of regional binding potential (BPND) values in the baboon brain was from 3.9 to 6.6. In vivo cerebral binding of [18F]ASEM and α7-nAChR expression in mutant DISC1 mice, a rodent model of schizophrenia, was significantly lower than in control animals, which is in agreement with previous post-mortem human data. Conclusion [18F]ASEM holds promise as a radiotracer with suitable imaging properties for quantification of α7-nAChR in the human brain. PMID:24556591

  9. Targeting the Nicotinic Acetylcholine Receptors (nAChRs) in Astrocytes as a Potential Therapeutic Target in Parkinson's Disease.

    PubMed

    Jurado-Coronel, Juan Camilo; Avila-Rodriguez, Marco; Capani, Francisco; Gonzalez, Janneth; Moran, Valentina Echeverria; Barreto, George E

    2016-01-01

    Parkinson's disease (PD) is a relatively common disorder of the Central Nervous System (CNS), whose etiology is characterized by a selective and progressive degeneration of dopaminergic neurons, and the presence of Lewy bodies in the pars compacta of the substantia nigra, and gaping dopamine depletion in the striatum. Patients with this disease suffer from tremors, slowness of movements, gait instability, and rigidity. These patients may also present functional disability, reduced quality of life, and rapid cognitive decline. It has been shown that nicotine exerts beneficial effects in patients with PD and in in-vitro and in-vivo models of this disease. Astrocytes are an important component in the immune response associated with PD, and that nicotine might be able to inhibit the inflammation-related apoptosis of these cells, being this a potential strategy for PD treatment. This action of nicotine could be due mainly to activation of α7 nicotinic acetylcholine receptors (α7-nAChRs) expressed in glial cells. However, nicotine administration can protect dopaminergic neurons against degeneration by inhibiting astrocytes activation in the substantia nigra pars compacta (SNpc) and therefore reduce inflammation. Owing to the toxicity and capacity of nicotine to induce addiction, analogues of this substance have been designed and tested in various experimental paradigms, and targeting α7-nAChRs expressed in glial cells may be a novel therapeutic strategy for PD treatment. PMID:26972289

  10. Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

    SciTech Connect

    Machaalani, R.; Ghazavi, E.; Hinton, T.; Waters, K.A.; Hennessy, A.

    2014-05-01

    Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.

  11. 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

    2016-03-01

    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. PMID:26961950

  12. Synthesis and biological activities of indolizine derivatives as alpha-7 nAChR agonists.

    PubMed

    Xue, Yu; Tang, Jingshu; Ma, Xiaozhuo; Li, Qing; Xie, Bingxue; Hao, Yuchen; Jin, Hongwei; Wang, Kewei; Zhang, Guisen; Zhang, Liangren; Zhang, Lihe

    2016-06-10

    Human α7 nicotinic acetylcholine receptor (nAChR) is a promising therapeutic target for the treatment of schizophrenia accompanied with cognitive impairment. Herein, we report the synthesis and agonistic activities of a series of indolizine derivatives targeting to α7 nAChR. The results show that all synthesized compounds have affinity to α7 nAChR and some give strong agonistic activity, particularly most active agonists show higher potency than control EVP-6124. The docking and structure-activity relationship studies provide insights to develop more potent novel α7 nAChR agonists. PMID:26994846

  13. Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist {alpha}-conotoxin OmIA that discriminates {alpha}3 vs. {alpha}6 nAChR subtypes

    SciTech Connect

    Chi, Seung-Wook; Kim, Do-Hyoung; Olivera, Baldomero M.; McIntosh, J. Michael; Han, Kyou-Hoon . E-mail: khhan600@kribb.re.kr

    2006-06-23

    {alpha}-Conotoxin OmIA from Conus omaria is the only {alpha}-conotoxin that shows a {approx}20-fold higher affinity to the {alpha}3{beta}2 over the {alpha}6{beta}2 subtype of nicotinic acetylcholine receptor. We have determined a three-dimensional structure of {alpha}-conotoxin OmIA by nuclear magnetic resonance spectroscopy. {alpha}-Conotoxin OmIA has an '{omega}-shaped' overall topology with His{sup 5}-Asn{sup 12} forming an {alpha}-helix. Structural features of {alpha}-conotoxin OmIA responsible for its selectivity are suggested by comparing its surface characteristics with other functionally related {alpha}4/7 subfamily conotoxins. Reduced size of the hydrophilic area in {alpha}-conotoxin OmIA seems to be associated with the reduced affinity towards the {alpha}6{beta}2 nAChR subtype.

  14. Roles for N-terminal Extracellular Domains of Nicotinic Acetylcholine Receptor (nAChR) β3 Subunits in Enhanced Functional Expression of Mouse α6β2β3- and α6β4β3-nAChRs*

    PubMed Central

    Dash, Bhagirathi; Li, Ming D.; Lukas, Ronald J.

    2014-01-01

    Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where “*” indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5–8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′S subunits were substituted for mβ3V9′S subunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops “C” (Glu221 and Phe223), “E” (Ser144 and Ser148), and “β2-β3” (Gln94 and Glu101) increased function of mα6mβ2*- (∼2–3-fold) or mα6mβ4* (∼2–4-fold)-nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs. PMID:25028511

  15. Beyond acetylcholinesterase inhibitors for treating Alzheimer's disease: α7-nAChR agonists in human clinical trials.

    PubMed

    Russo, Patrizia; Del Bufalo, Alessandra; Frustaci, Alessandra; Fini, Massimo; Cesario, Alfredo

    2014-01-01

    The neuronal nicotinic alpha7-acetylcholine receptor (α7-nAChR) is a promising and attractive drug target for improving cognitive deficits in neuropsychiatric and neurological disorders such as Alzheimer's disease (AD). α7-nAChR belongs to the family of ligand gated ion channels. α7-nAChR is expressed in key brain regions (e.g. pre- and frontal cortex, hippocampus). It is involved in essential cognitive functions such as memory, thinking, comprehension, learning capacity, calculation, orientation, language, and judgment. α7-nAChR binds to amyloid peptide (Aβ) inducing either receptor activation or inhibition in an Aβ concentration-dependent mode. Aβ oligomers induce τ phosphorylation via α7-nAChR activation. α7-nAChR agonists and/or α7-nAChR positive allosteric modulators may be useful in AD therapy. The current review enlightens: (i) α7-nAChR neurobiology, (ii) α7-nAChR role in cognition and (iii) in AD, and (iv) the clinical status of the most promising molecules for the treatment of cognitive dysfunction in AD. PMID:24641224

  16. Docking studies of benzylidene anabaseine interactions with α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding proteins (AChBPs): Application to the design of related α7 selective ligands

    PubMed Central

    Kombo, David C.; Mazurov, Anatoly; Tallapragada, Kartik; Hammond, Philip S.; Chewning, Joseph; Hauser, Terry A.; Vasquez-Valdivieso, Montserrat; Yohannes, Daniel; Talley, Todd T.; Taylor, Palmer; Caldwell, William S.

    2016-01-01

    AChBPs isolated from Lymnaea stagnalis (Ls), Aplysia californica (Ac) and Bulinus truncatus (Bt) have been extensively used as structural prototypes to understand the molecular mechanisms that underlie ligand-interactions with nAChRs [1]. Here, we describe docking studies on interactions of benzylidene anabaseine analogs with AChBPs and α7 nAChR. Results reveal that docking of these compounds using Glide software accurately reproduces experimentally-observed binding modes of DMXBA and of its active metabolite, in the binding pocket of Ac. In addition to the well-known nicotinic pharmacophore (positive charge, hydrogen-bond acceptor, and hydrophobic aromatic groups), a hydrogen-bond donor feature contributes to binding of these compounds to Ac, Bt, and the α7 nAChR. This is consistent with benzylidene anabaseine analogs with OH and NH2 functional groups showing the highest binding affinity of these congeners, and the position of the ligand shown in previous X-ray crystallographic studies of ligand-Ac complexes. In the predicted ligand-Ls complex, by contrast, the ligand OH group acts as hydrogen-bond acceptor. We have applied our structural findings to optimizing the design of novel spirodiazepine and spiroimidazoline quinuclidine series. Binding and functional studies revealed that these hydrogen-bond donor containing compounds exhibit improved affinity and selectivity for the α7 nAChR subtype and demonstrate partial agonism. The gain in affinity is also due to conformational restriction, tighter hydrophobic enclosures, and stronger cation-π interactions. The use of AChBPs structure as a surrogate to predict binding affinity to α7 nAChR has also been investigated. On the whole, we found that molecular docking into Ls binding site generally scores better than when a α7 homology model, Bt or Ac crystal structure is used. PMID:21986237

  17. Tribendimidine: Mode of Action and nAChR Subtype Selectivity in Ascaris and Oesophagostomum

    PubMed Central

    Robertson, Alan P.; Puttachary, Sreekanth; Buxton, Samuel K.; Martin, Richard J.

    2015-01-01

    The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like bephenium. The different nAChR subtype

  18. Kinetic evidence that desensitized nAChR may promote transitions of active nAChR to desensitized states during sustained exposure to agonists in skeletal muscle.

    PubMed

    Manthey, Arthur A

    2006-06-01

    During prolonged exposure of postjunctional nicotinic acetylcholine receptors (nAChR) of skeletal muscle to acetylcholine (ACh), agonist-activated nAChR (nAChRa) gradually fall into a refractory "desensitized" state (nAChRd), which no longer supports the high-conductance channel openings characteristic of the initially active nAChRa. In the present study, the possibility was examined that nAChRd, rather than simply constituting a passive "trap" for nAChRa, may actively promote further conversions of nAChRa to nAChRd in a formally autocatalytic manner. Single-ion whole-cell voltage-clamp currents (Na+ and Li+ in separate trials) were measured using two KCl-filled capillary electrodes (5-10 MOmega) implanted at the postjunctional locus of single frog skeletal muscle fibers (Rana pipiens) equilibrated in 30 mM K+ bath media to eliminate mechanical responses. Various nAChR agonists (carbamylcholine, acetylcholine, suberyldicholine) at different concentrations were delivered focally by positive pressure microjet. It was found that the decline of postmaximal agonist-induced currents under these different conditions (driven by the growth of the subpool of nAChRd) consistently followed an autocatalytic logistic rule modified for population growth of fixed units in a planar array: [Formula: see text] (where y represents the remaining agonist-induced current at time t, A=initial maximum current, and n is a constant). Some further experimental features that might result from a self-promoting growth of nAChRd were also tested, namely, (1) the effect of increased nAChRa and (2) the effect of increased nAChRd. Increase in agonist concentration of the superfusate, by increasing the planar density of active nAChRa at the outset, should enhance the probability of autocatalytic interactions with emerging nAChRd, hence, the rate of decline of agonist-induced current, and this was a consistent finding under all conditions tested. Raising the initial level of desensitized nAChRd by

  19. Functional expression and axonal transport of α7 nAChRs by peptidergic nociceptors of rat dorsal root ganglion.

    PubMed

    Shelukhina, Irina; Paddenberg, Renate; Kummer, Wolfgang; Tsetlin, Victor

    2015-07-01

    In recent pain studies on animal models, α7 nicotinic acetylcholine receptor (nAChR) agonists demonstrated analgesic, anti-hyperalgesic and anti-inflammatory effects, apparently acting through some peripheral receptors. Assuming possible involvement of α7 nAChRs on nociceptive sensory neurons, we investigated the morphological and neurochemical features of the α7 nAChR-expressing subpopulation of dorsal root ganglion (DRG) neurons and their ability to transport α7 nAChR axonally. In addition, α7 receptor activity and its putative role in pain signal neurotransmitter release were studied. Medium-sized α7 nAChR-expressing neurons prevailed, although the range covered all cell sizes. These cells accounted for one-fifth of total medium and large DRG neurons and <5% of small ones. 83.2% of α7 nAChR-expressing DRG neurons were peptidergic nociceptors (CGRP-immunopositive), one half of which had non-myelinated C-fibers and the other half had myelinated Aδ- and likely Aα/β-fibers, whereas 15.2% were non-peptidergic C-fiber nociceptors binding isolectin B4. All non-peptidergic and a third of peptidergic α7 nAChR-bearing nociceptors expressed TRPV1, a capsaicin-sensitive noxious stimulus transducer. Nerve crush experiments demonstrated that CGRPergic DRG nociceptors axonally transported α7 nAChRs both to the spinal cord and periphery. α7 nAChRs in DRG neurons were functional as their specific agonist PNU282987 evoked calcium rise enhanced by α7-selective positive allosteric modulator PNU120596. However, α7 nAChRs do not modulate neurotransmitter CGRP and glutamate release from DRG neurons since nicotinic ligands affected neither their basal nor provoked levels, showing the necessity of further studies to elucidate the true role of α7 nAChRs in those neurons. PMID:24706047

  20. Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and {beta}-adrenergic receptor signaling pathways

    SciTech Connect

    Shin, Vivian Yvonne; Jin, H.C.; Ng, Enders K.O.; Yu Jun; Leung, W.K.; Cho, C.H.; Sung, J.J.Y.

    2008-12-01

    Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor ({alpha}7 nAChR) and {beta}-adrenergic receptors. Treatment of cells with {alpha}-bungarotoxin ({alpha}-BTX, {alpha}7nAChR antagonist) or propranolol ({beta}-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE{sub 2} and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE{sub 2} induction can only be suppressed by propranolol, but not {alpha}-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.

  1. Anti-inflammatory role of microglial alpha7 nAChRs and its role in neuroprotection.

    PubMed

    Egea, Javier; Buendia, Izaskun; Parada, Esther; Navarro, Elisa; León, Rafael; Lopez, Manuela G

    2015-10-15

    Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous system, being expressed in neurons and non-neuronal cells, where they participate in a variety of physiological responses like memory, learning, locomotion, attention, among others. We will focus on the α7 nAChR subtype, which has been implicated in neuroprotection, synaptic plasticity and neuronal survival, and is considered as a potential therapeutic target for several neurological diseases. Oxidative stress and neuroinflammation are currently considered as two of the most important pathological mechanisms common in neurodegenerative diseases such as Alzheimer, Parkinson or Huntington diseases. In this review, we will first analysed the distribution and expression of nAChR in mammalian brain. Then, we focused on the function of the α7 nAChR subtype in neuronal and non-neuronal cells and its role in immune responses (cholinergic anti-inflammatory pathway). Finally, we will revise the anti-inflammatory pathway promoted via α7 nAChR activation that is related to recruitment and activation of Jak2/STAT3 pathway, which on the one hand inhibits NF-κB nuclear translocation, and on the other hand, activates the master regulator of oxidative stress Nrf2/HO-1. This review provides a profound insight into the role of the α7 nAChR subtype in microglia and point out to microglial α7/HO-1 pathway as an anti-inflammatory therapeutic target. PMID:26232730

  2. Cross-talk between α7 nAchR and NMDAR revealed by protein profiling.

    PubMed

    Zhang, Hailong; Li, Tao; Li, Shupeng; Liu, Fang

    2016-01-10

    Functional regulation of NMDA receptor (NMDAR) by the activation of α7 nicotinic acetylcholine receptor (α7nAChR) has been reported, although the molecular signaling pathway underlying this process remains largely unknown. We employed a label-free quantitative proteomics approach to identify potential intracellular molecules and pathways that might be involved in the functional cross-talk between NMDAR and α7nAChR. 43 proteins showed significantly expression changes after choline treatment in which 35 out of 43 proteins was significantly altered by co-treatment with NMDA. Western blot analysis verified proteins expression determined by LC-MS. Furthermore, protein expression in vivo in neurons from fetal rats were visualized and quantified by Confocal microscopy,which showed consistency of relative changes of AHA-1 expressionmeasured by LC-MS and Western blot. Biological network analysis of differently expressed proteins found 21 kind of biological pathways involved. Of those pathways, 6 pathways were directly involved in regulation of neurotransmitters. Lastly, the levels of neurotransmitters (dopamine, glutamate, GABA and 5-HT) were measured by HPLC-ECD. Co-treatment choline/NMDA significantly enhances the release of dopamine, glutamate and GABA and dramatically decrease 5-HT to only 65% of control level, which is consist with this protein interaction network analysis, providing an additional evidence for the cross-talk between NMDAR and α7nAChR. PMID:26498070

  3. 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. PMID:26979166

  4. The Role of nAChR and Calcium Signaling in Pancreatic Cancer Initiation and Progression

    PubMed Central

    Schaal, Courtney; Padmanabhan, Jaya; Chellappan, Srikumar

    2015-01-01

    Pancreatic cancer shows a strong correlation with smoking and the current therapeutic strategies have been relatively ineffective in improving the survival of patients. Efforts have been made over the past many years to understand the molecular events that drive the initiation and progression of pancreatic cancer, especially in the context of smoking. It has become clear that components of tobacco smoke not only initiate these cancers, especially pancreatic ductal adenocarcinomas (PDACs) through their mutagenic properties, but can also promote the growth and metastasis of these tumors by stimulating cell proliferation, angiogenesis, invasion and epithelial-mesenchymal transition. Studies in cell culture systems, animal models and human samples have shown that nicotinic acetylcholine receptor (nAChR) activation enhances these tumor-promoting events by channeling signaling through multiple pathways. In this context, signaling through calcium channels appear to facilitate pancreatic cancer growth by itself or downstream of nAChRs. This review article highlights the role of nAChR downstream signaling events and calcium signaling in the growth, metastasis as well as drug resistance of pancreatic cancer. PMID:26264026

  5. Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer.

    PubMed

    Kouvatsos, Nikolaos; Giastas, Petros; Chroni-Tzartou, Dafni; Poulopoulou, Cornelia; Tzartos, Socrates J

    2016-08-23

    In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 Å. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(-) binding site on the heteromeric low sensitivity α2β2 nAChR and validated the functional importance of specific residues in α2 and β2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases. PMID:27493220

  6. Evidence for a role for α6(∗) nAChRs in l-dopa-induced dyskinesias using Parkinsonian α6(∗) nAChR gain-of-function mice.

    PubMed

    Bordia, T; McGregor, M; McIntosh, J M; Drenan, R M; Quik, M

    2015-06-01

    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, 3μg/ml) into the medial forebrain bundle. Three to 4wk later, they were administered l-dopa (3mg/kg) plus benserazide (15mg/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 300μg/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 20μg/ml. However, the nAChR antagonist mecamylamine (1mg/kg ip 30min 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

  7. Effect of calcium on nicotine-induced current expressed by an atypical alpha-bungarotoxin-insensitive nAChR2.

    PubMed

    Thany, Steeve H; Courjaret, Raphael; Lapied, Bruno

    2008-06-27

    Two distinct native alpha-bungarotoxin (alpha-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 microM alpha-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2. PMID:18485593

  8. From crystal structure of α-conotoxin GIC in complex with Ac-AChBP to molecular determinants of its high selectivity for α3β2 nAChR

    PubMed Central

    Lin, Bo; Xu, Manyu; Zhu, Xiaopeng; Wu, Yong; Liu, Xi; Zhangsun, Dongting; Hu, Yuanyan; Xiang, Shi-Hua; Kasheverov, Igor E.; Tsetlin, Victor I.; Wang, Xinquan; Luo, Sulan

    2016-01-01

    Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking data, we suggest the key residues of GIC in determining its high affinity and selectivity for human α3β2 vs α3β4 nAChRs. These suggestions were checked by radioligand and electrophysiology experiments, which confirmed the functional role of detected contacts for GIC interactions with Ac-AChBP and α3β2 nAChR subtypes. While GIC elements responsible for its high affinity binding with Ac-AChBP and α3β2 nAChR were identified, our study also showed the limitations of computer modelling in extending the data from the X-ray structures of the AChBP complexes to all nAChR subtypes. PMID:26925840

  9. From crystal structure of α-conotoxin GIC in complex with Ac-AChBP to molecular determinants of its high selectivity for α3β2 nAChR.

    PubMed

    Lin, Bo; Xu, Manyu; Zhu, Xiaopeng; Wu, Yong; Liu, Xi; Zhangsun, Dongting; Hu, Yuanyan; Xiang, Shi-Hua; Kasheverov, Igor E; Tsetlin, Victor I; Wang, Xinquan; Luo, Sulan

    2016-01-01

    Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking data, we suggest the key residues of GIC in determining its high affinity and selectivity for human α3β2 vs α3β4 nAChRs. These suggestions were checked by radioligand and electrophysiology experiments, which confirmed the functional role of detected contacts for GIC interactions with Ac-AChBP and α3β2 nAChR subtypes. While GIC elements responsible for its high affinity binding with Ac-AChBP and α3β2 nAChR were identified, our study also showed the limitations of computer modelling in extending the data from the X-ray structures of the AChBP complexes to all nAChR subtypes. PMID:26925840

  10. Enantiopure Cyclopropane-Bearing Pyridyldiazabicyclo[3.3.0]octanes as Selective α4β2-nAChR Ligands.

    PubMed

    Onajole, Oluseye K; Eaton, J Brek; Lukas, Ronald J; Brunner, Dani; Thiede, Lucinda; Caldarone, Barbara J; Kozikowski, Alan P

    2014-11-13

    We report the synthesis and characterization of a series of enantiopure 5-cyclopropane-bearing pyridyldiazabicyclo[3.3.0]octanes that display low nanomolar binding affinities and act as functional agonists at α4β2-nicotinic acetylcholine receptor (nAChR) subtype. Structure-activity relationship studies revealed that incorporation of a cyclopropane-containing side chain at the 5-position of the pyridine ring provides ligands with improved subtype selectivity for nAChR β2 subunit-containing nAChR subtypes (β2*-nAChRs) over β4*-nAChRs compared to the parent compound 4. Compound 15 exhibited subnanomolar binding affinity for α4β2- and α4β2*-nAChRs with negligible interaction. Functional assays confirm selectivity for α4β2-nAChRs. Furthermore, using the SmartCube assay system, this ligand showed antidepressant, anxiolytic, and antipsychotic features, while mouse forced-swim assay further confirm the antidepressant-like property of 15. PMID:25408831

  11. Functionality and stability data of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

    PubMed

    Padilla-Morales, Luis F; Colón-Sáez, José O; González-Nieves, Joel E; Quesada-González, Orestes; Lasalde-Dominicci, José A

    2016-03-01

    The presented data provides additional information about the assessment of affinity purified nicotinic acetylcholine receptor (nAChR) rich membrane solubilized with long chain (16 saturated carbons) lysophospholipid with glycerol headgroup (LFG-16). The assessment of stability and functionality of solubilized membrane protein is a critical step prior to further crystallization trails. One of the key factors for this task is the appropriate choice of a detergent that can support nAChR activity and stability comparable to the crude membranes. The stability of the nAChR-LFG-16 complex incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of fluorescence recovery after photobleaching (FRAP) and the functionality was evaluated after its incorporation into Xenopus oocyte by means of the two electrode voltage clamp technique. PMID:26870753

  12. Inactivity–Induced Increase in nAChRs Up–Regulates Shal K+ Channels to Stabilize Synaptic Potentials

    PubMed Central

    Ping, Yong; Tsunoda, Susan

    2011-01-01

    Long–term synaptic changes, which are essential for learning and memory, are dependent on homeostatic mechanisms that stabilize neural activity. Homeostatic responses have also been implicated in pathological conditions, including nicotine addiction. Although multiple homeostatic pathways have been described, little is known about how compensatory responses are tuned to prevent them from overshooting their optimal range of activity. We show that prolonged inhibition of nicotinic acetylcholine receptors (nAChRs), the major excitatory receptor in the Drosophila CNS, results in a homeostatic increase in the Dα7 nAChR. This response then induces an increase in the transient A–type K+ current carried by Shal/Kv4 channels. While increasing Dα7 boosts mEPSCs, the ensuing increase in Shal channels serves to stabilize postsynaptic potentials. This identifies a novel mechanism to fine–tune the homeostatic response. PMID:22081160

  13. Enhanced synthesis and release of dopamine in transgenic mice with gain-of-function α6* nAChRs

    PubMed Central

    Wang, Yuexiang; Lee, Jang-Won; Oh, Gyeon; Grady, Sharon R.; McIntosh, J. Michael; Brunzell, Darlene H.; Cannon, Jason R.; Drenan, Ryan M.

    2014-01-01

    α6β2* nAChRs in the ventral tegmental area (VTA) to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain-of-function α6β2* nAChRs (α6L9’S mice) that are hypersensitive to nicotine and endogenous acetylcholine (ACh). Evoked extracellular dopamine (DA) levels were enhanced in α6L9’S NAc slices compared to control, non-transgenic (nonTg) slices. Extracellular DA levels in both nonTg and α6L9’S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6β2* nAChR activation by ACh plays a role in enhancing DA levels, as α-conotoxin MII completely abolished evoked DA release in α6L9’S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9’S NAc tissue contained significantly more DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) compared to nonTg NAc tissue. Serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine (NE) were unchanged in α6L9’S compared to nonTg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9’S NAc. Overall, these results show that enhanced α6β2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels. PMID:24266758

  14. Vagus nerve stimulation mediates protection from kidney ischemia-reperfusion injury through α7nAChR+ splenocytes.

    PubMed

    Inoue, Tsuyoshi; Abe, Chikara; Sung, Sun-Sang J; Moscalu, Stefan; Jankowski, Jakub; Huang, Liping; Ye, Hong; Rosin, Diane L; Guyenet, Patrice G; Okusa, Mark D

    2016-05-01

    The nervous and immune systems interact in complex ways to maintain homeostasis and respond to stress or injury, and rapid nerve conduction can provide instantaneous input for modulating inflammation. The inflammatory reflex referred to as the cholinergic antiinflammatory pathway regulates innate and adaptive immunity, and modulation of this reflex by vagus nerve stimulation (VNS) is effective in various inflammatory disease models, such as rheumatoid arthritis and inflammatory bowel disease. Effectiveness of VNS in these models necessitates the integration of neural signals and α7 nicotinic acetylcholine receptors (α7nAChRs) on splenic macrophages. Here, we sought to determine whether electrical stimulation of the vagus nerve attenuates kidney ischemia-reperfusion injury (IRI), which promotes the release of proinflammatory molecules. Stimulation of vagal afferents or efferents in mice 24 hours before IRI markedly attenuated acute kidney injury (AKI) and decreased plasma TNF. Furthermore, this protection was abolished in animals in which splenectomy was performed 7 days before VNS and IRI. In mice lacking α7nAChR, prior VNS did not prevent IRI. Conversely, adoptive transfer of VNS-conditioned α7nAChR splenocytes conferred protection to recipient mice subjected to IRI. Together, these results demonstrate that VNS-mediated attenuation of AKI and systemic inflammation depends on α7nAChR-positive splenocytes. PMID:27088805

  15. Vagus nerve stimulation mediates protection from kidney ischemia-reperfusion injury through α7nAChR+ splenocytes

    PubMed Central

    Inoue, Tsuyoshi; Abe, Chikara; Sung, Sun-sang J.; Moscalu, Stefan; Jankowski, Jakub; Huang, Liping; Ye, Hong; Guyenet, Patrice G.

    2016-01-01

    The nervous and immune systems interact in complex ways to maintain homeostasis and respond to stress or injury, and rapid nerve conduction can provide instantaneous input for modulating inflammation. The inflammatory reflex referred to as the cholinergic antiinflammatory pathway regulates innate and adaptive immunity, and modulation of this reflex by vagus nerve stimulation (VNS) is effective in various inflammatory disease models, such as rheumatoid arthritis and inflammatory bowel disease. Effectiveness of VNS in these models necessitates the integration of neural signals and α7 nicotinic acetylcholine receptors (α7nAChRs) on splenic macrophages. Here, we sought to determine whether electrical stimulation of the vagus nerve attenuates kidney ischemia-reperfusion injury (IRI), which promotes the release of proinflammatory molecules. Stimulation of vagal afferents or efferents in mice 24 hours before IRI markedly attenuated acute kidney injury (AKI) and decreased plasma TNF. Furthermore, this protection was abolished in animals in which splenectomy was performed 7 days before VNS and IRI. In mice lacking α7nAChR, prior VNS did not prevent IRI. Conversely, adoptive transfer of VNS-conditioned α7nAChR splenocytes conferred protection to recipient mice subjected to IRI. Together, these results demonstrate that VNS-mediated attenuation of AKI and systemic inflammation depends on α7nAChR-positive splenocytes. PMID:27088805

  16. Enhanced synthesis and release of dopamine in transgenic mice with gain-of-function α6* nAChRs.

    PubMed

    Wang, Yuexiang; Lee, Jang-Won; Oh, Gyeon; Grady, Sharon R; McIntosh, J Michael; Brunzell, Darlene H; Cannon, Jason R; Drenan, Ryan M

    2014-04-01

    α6β2* nicotinic acetylcholine receptors (nAChRs)s in the ventral tegmental area to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain-of-function α6β2* nAChRs (α6L9'S mice) that are hypersensitive to nicotine and endogenous acetylcholine. Evoked extracellular dopamine (DA) levels were enhanced in α6L9'S NAc slices compared to control, non-transgenic (non-Tg) slices. Extracellular DA levels in both non-Tg and α6L9'S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6β2* nAChR activation by acetylcholine plays a role in enhancing DA levels, as α-conotoxin MII completely abolished evoked DA release in α6L9'S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9'S NAc tissue contained significantly more DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid compared to non-Tg NAc tissue. Serotonin (5-HT), 5-hydroxyindoleacetic acid, and norepinephrine (NE) were unchanged in α6L9'S compared to non-Tg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9'S NAc. Overall, these results show that enhanced α6β2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels. PMID:24266758

  17. 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. PMID:27098031

  18. Pharmacological stress is required for the anti-alcohol effect of the α3β4* nAChR partial agonist AT-1001

    PubMed Central

    Cippitelli, Andrea; Brunori, Gloria; Gaiolini, Kelly A.; Zaveri, Nurulain T.; Toll, Lawrence

    2015-01-01

    Alcohol and nicotine are often taken together. The mechanisms underlying this frequent co-abuse are not well known. Genetic and pharmacological evidence suggests that the nicotinic acetylcholine receptors (nAChRs) containing the α3 and β4 subunits play a role in alcohol as well as nicotine addiction. AT-1001 is a high affinity α3β4 nAChR partial agonist recently found to block nicotine self-administration and relapse-like behavior in rats. Here, to study the involvement of α3β4 nAChRs in the mechanisms that regulate alcohol abuse we evaluated the effects of AT-1001 on alcohol taking and seeking in Sprague-Dawley rats. AT-1001 reduced operant alcohol self-administration at the highest dose examined (3.0 mg/kg), an effect also observed for food self-administration. A dose of 1.5 mg/kg AT-1001, which had no effect on alcohol or food self-administration, essentially eliminated reinstatement of alcohol seeking induced by yohimbine (0.625 mg/kg) whereas, reinstatement induced by alcohol-associated cues was not altered, nor did AT-1001 induce reinstatement of extinguished self-administration on its own. Finally, AT-1001 showed an anxiolytic activity when measured in the presence or absence of yohimbine stress in the elevated plus maze paradigm. Together, these observations do not support a specific involvement of the α3β4 nAChR in mediating alcohol reward or cue-induced relapse to alcohol seeking but rather indicate that the α3β4 nAChR partial agonism may constitute an attractive approach for treating alcohol use disorders exacerbated by elevated stress response. PMID:25689019

  19. Pharmacological stress is required for the anti-alcohol effect of the α3β4* nAChR partial agonist AT-1001.

    PubMed

    Cippitelli, Andrea; Brunori, Gloria; Gaiolini, Kelly A; Zaveri, Nurulain T; Toll, Lawrence

    2015-06-01

    Alcohol and nicotine are often taken together. The mechanisms underlying this frequent co-abuse are not well known. Genetic and pharmacological evidence suggests that the nicotinic acetylcholine receptors (nAChRs) containing the α3 and β4 subunits play a role in alcohol as well as nicotine addiction. AT-1001 is a high affinity α3β4 nAChR partial agonist recently found to block nicotine self-administration and relapse-like behavior in rats. Here, to study the involvement of α3β4 nAChRs in the mechanisms that regulate alcohol abuse we evaluated the effects of AT-1001 on alcohol taking and seeking in Sprague-Dawley rats. AT-1001 reduced operant alcohol self-administration at the highest dose examined (3.0 mg/kg), an effect also observed for food self-administration. A dose of 1.5 mg/kg AT-1001, which had no effect on alcohol or food self-administration, essentially eliminated reinstatement of alcohol seeking induced by yohimbine (0.625 mg/kg) whereas, reinstatement induced by alcohol-associated cues was not altered, nor did AT-1001 induce reinstatement of extinguished self-administration on its own. Finally, AT-1001 showed an anxiolytic activity when measured in the presence or absence of yohimbine stress in the elevated plus maze paradigm. Together, these observations do not support a specific involvement of the α3β4 nAChR in mediating alcohol reward or cue-induced relapse to alcohol seeking but rather indicate that the α3β4 nAChR partial agonism may constitute an attractive approach for treating alcohol use disorders exacerbated by elevated stress response. PMID:25689019

  20. Antinociceptive effect of spirocyclopiperazinium salt compound LXM-15 via activating peripheral α7 nAChR and M4 mAChR in mice.

    PubMed

    Zhao, Xia; Ye, Jia; Sun, Qi; Xiong, Yulan; Li, Runtao; Jiang, Yimin

    2011-01-01

    The aim of this study was to evaluate the antinociceptive effects and potential mechanisms of the spirocyclopiperazinium compound LXM-15. We found that LXM-15 produced significant antinociceptive effects in a dose- and time-dependent manner in mice. The maximum inhibition ratio was 70% in the acetic acid writhing test; the effect started at 1.0 h, peaked at 2.0 h with the MPEs of 61%, and persisted 3.5 h in the hot-plate test; LXM-15 reduced the time spent licking or biting the injected paw remarkably with inhibitions of 53% in formalin test. LXM-15 did not affect motor coordination, spontaneous activity, body temperature, heart rate, or liver enzyme activity, the LD(50) values was 616.26 μmol/kg. The antinociceptive effect of LXM-15 was blocked by mecamylamine, hexamethonium, atropine or atropine methylnitrate, and was also blocked by MLA, tropicamide. In contrast, the effect was not blocked by naloxone. Meanwhile, competition receptor binding assays showed LXM-15 can bind to α7 nAChR or M4 mAChR. Our studies show that LXM-15 may be via activating peripheral α7 nicotnic and M4 muscarinic receptors, resulted in antinociceptive effects. PMID:21035471

  1. nAChRs Mediate Human Embryonic Stem Cell-Derived Endothelial Cells: Proliferation, Apoptosis, and Angiogenesis

    PubMed Central

    Velotta, Jeffrey B.; Huang, Mei; Li, Zongjin; Lee, Andrew; Robbins, Robert C.; Cooke, John P.; Wu, Joseph C.

    2009-01-01

    Background Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs). Methods and Results To induce endothelial cell differentiation, undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days, CD31+ cells (13.7±2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation, these hESC-ECs expressed endothelial specific markers such as vWF (96.3±1.4%), CD31 (97.2±2.5%), and VE-cadherin (93.7±2.8%), form vascular-like channels, and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward, 5×106 hESC-ECs treated for 24 hours with nicotine (10−8 M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 µg/ml) in the drinking water. Surprisingly, bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally, in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs). Conclusions This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs, and enhance their angiogenic effects in vivo. Furthermore, activation of nAChRs has anti-apoptotic, angiogenic, and proliferative effects through MAPK and Akt signaling pathways. PMID:19753305

  2. 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

  3. 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. PMID:25639674

  4. Impairment of contextual fear extinction by chronic nicotine and withdrawal from chronic nicotine is associated with hippocampal nAChR upregulation.

    PubMed

    Kutlu, Munir Gunes; Oliver, Chicora; Huang, Peng; Liu-Chen, Lee-Yuan; Gould, Thomas J

    2016-10-01

    Chronic nicotine and withdrawal from chronic nicotine have been shown to be major modulators of fear learning behavior. Moreover, recent studies from our laboratory have shown that acute nicotine impaired fear extinction and safety learning in mice. However, the effects of chronic nicotine and withdrawal on fear extinction are unknown. Therefore, the current experiments were conducted to investigate the effects of chronic nicotine as well as withdrawal from chronic nicotine on contextual fear extinction in mice. C57BL6/J mice were given contextual fear conditioning training and retention testing during chronic nicotine administration. Mice then received contextual fear extinction either during chronic nicotine or during withdrawal from chronic nicotine. Our results showed that contextual fear extinction was impaired both during chronic nicotine administration and subsequent withdrawal. However, it was also observed that the effects of prior chronic nicotine disappeared after 72 h in withdrawal, a timeline that closely matches with the timing of the chronic nicotine-induced upregulation of hippocampal nicotinic acetylcholine receptor (nAChR) density. Additional experiments found that 4 days, but not 1 day, of continuous nicotine administration upregulated hippocampal nAChRs and impaired contextual fear extinction. These effects disappeared following 72 h withdrawal. Overall, these experiments provide a potential link between nicotine-induced upregulation of hippocampal nAChRs and fear extinction deficits observed in patients with anxiety disorders, which may lead to advancements in the pharmacological treatment methods for this disorder. PMID:27378334

  5. Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

    PubMed

    Padilla-Morales, Luis F; Colón-Sáez, José O; González-Nieves, Joel E; Quesada-González, Orestes; Lasalde-Dominicci, José A

    2016-01-01

    In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30 day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30 day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex

  6. Neurobiology of nAChRs and cognition: A mini review of Dr. Jerry J. Buccafusco's contributions over a 25 year career

    PubMed Central

    Terry, Alvin V.; Decker, Michael W.

    2011-01-01

    This review highlights some of the many contributions of the late Dr. Jerry J. Buccafusco to the neurobiology of nicotinic acetylcholine receptors (nAChRs) and cognition over a 25 year period. The article is written by two of Dr. Buccafusco's professional colleagues, one from academia and one from the pharmaceutical industry. While Dr. Buccafusco's expertise in the cholinergic field was extensive, his insights into the practical relevance of his work (with a long-term goal of formulating new drug development strategies) were unique, and a great asset to both the basic science community and pharmaceutical companies. In 1988, Dr. Buccafusco's laboratory was the first to report the cognitive enhancing action of low doses of nicotine in non-human primates. Since that time he studied a large number of novel pro-cognitive agents from several pharmacological classes in rodents as well as monkeys. Based on years of observing paradoxical effects of nicotinic ligands in vitro and in vivo, Dr. Buccafusco made the provocative argument that it might be possible to develop new chemical entities (with pro-cognitive actions) that have the ability to desensitize nAChRs without producing an antecedent agonist action. Some of his more recent work focused on development of single molecular entities that act on multiple CNS targets (including nAChRs) to enhance cognition, provide neuroprotection, and/or provide additional therapeutic actions (e.g., antipsychotic effects). Dr. Buccafusco's influence will live on in the work of the numerous graduate students, postdoctoral fellows, and junior faculty that he mentored over the years who now serve in prestigious positions throughout the world. PMID:21684265

  7. A novel 4/6-type alpha-conotoxin ViIA selectively inhibits nAchR α3β2 subtype.

    PubMed

    Li, Liang; Liu, Na; Ding, Rong; Wang, Shuo; Liu, Zhuguo; Li, Haiying; Zheng, Xing; Dai, Qiuyun

    2015-12-01

    Conotoxins (CTxs) are typically small peptides composed of 12-50 amino acid residues with 2-5 disulfide bridges. Most of them potently and selectively target a wide variety of ion channels and membrane receptors. They are highly valued as neuropharmacological probes and in pharmaceutical development. In this work, a novel α4/6-CTx named ViIA (RDCCSNPPCAHNNPDC-NH2) was identified from a cDNA library of the venom ducts of Conus virgo (C. virgo). ViIA was then synthesized chemically and its disulfide connectivity was identified as 'C(1)-C(3), C(2)-C(4)'. Its molecular targets were further assessed using two-electrode voltage clamping. The results indicated that ViIA selectively inhibited nicotinic acetylcholine receptor (nAChR) α3β2 subtype with an IC50 of 845.5 nM, but did not target dorsal root ganglion sodium (Na(+))-, potassium (K(+))- or calcium (Ca(2+))-ion channels. Further structure-activity relationship analysis demonstrated that Arg(1) and His(11) but not Asp(2) were the functional residues. To the best of our knowledge, ViIA is the first 4/6 α-CTx that selectively inhibits nAChR α3β2 subtype. This finding expands the knowledge of targets of α4/6-family CTxs. PMID:26511093

  8. Marine Macrocyclic Imines, Pinnatoxins A and G: Structural Determinants and Functional Properties to Distinguish Neuronal α7 from Muscle α1(2)βγδ nAChRs.

    PubMed

    Bourne, Yves; Sulzenbacher, Gerlind; Radić, Zoran; Aráoz, Rómulo; Reynaud, Morgane; Benoit, Evelyne; Zakarian, Armen; Servent, Denis; Molgó, Jordi; Taylor, Palmer; Marchot, Pascale

    2015-06-01

    Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal α7, α4β2, α3β2, and muscle-type α12βγδ nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the α7 and α12βγδ subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low Kd values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 Å resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity. PMID:26004441

  9. Identification and pharmacological characterization of 3,6-diazabicyclo[3.1.1]heptane-3-carboxamides as novel ligands for the α4β2 and α6/α3β2β3 nicotinic acetylcholine receptors (nAChRs).

    PubMed

    Strachan, Jon-Paul; Kombo, David C; Mazurov, Anatoly; Heemstra, Ronald; Bhatti, Balwinder S; Akireddy, Rao; Murthy, Srinivasa; Miao, Lan; Jett, John E; Speake, Jason; Bencherif, Merouane

    2014-10-30

    We have synthesized a novel series of compounds, 3,6-diazabicyclo[3.1.1]heptane-3-carboxamides, targeting both the α4β2 and α6/α3β2β3 nAChRs. Members of the obtained chemical library are partial or full agonists at both the high sensitivity (α4)2(β2)3 and α6/α3β2β3 nAChRs. 3-(Cyclopropylcarbonyl)-3,6-diazabicyclo[3.1.1]heptane (TC-8831 or compound 7 herein) demonstrated a safe in vitro pharmacological profile and the potential for reducing or preventing L-dopa-induced dyskinesias (LID) in several in vivo animal models [1-4]. In vivo metabolism studies in rat and in vitro metabolism studies in liver microsomes from human, rat, dog and monkey showed TC-8831 to be relatively stable. In vivo pharmacokinetic analysis in the rat confirmed brain penetration, with an average brain:plasma ratio of approximately 0.3 across time points from 0.5 to 4 h. Docking into homology models predicted alternative binding modes for TC-8831 and highlighted the importance of the cationic center, hydrogen-bond acceptor, and hydrophobic aliphatic features in promoting binding affinity to both nAChRs. Pharmacophore elucidation confirmed the importance of these key interactions. QSAR modeling suggested that binding affinity is primarily driven by ligand shape, relative positive charge distribution onto the molecular surface, and molecular flexibility. Of the two subtypes, ligand binding to α6β2β3 appears to be more sensitive to bulkiness and flexibility. PMID:25147147

  10. 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

  11. Cloning of eight Rhopalosiphum padi (Hemiptera: Aphididae) nAChR subunit genes and mutation detection of the β1 subunit in field samples from China.

    PubMed

    Zhang, Meng; Qiao, Xianfeng; Li, Yuting; Fang, Bing; Zuo, Yayun; Chen, Maohua

    2016-09-01

    The bird cherry-oat aphid, Rhopalosiphum padi (L.), is one of the most important wheat pests. This aphid damages through direct feeding and by transmitting the Barley yellow dwarf virus (BYDV). Both types of damage significantly reduce the quality and yield of wheat crops globally. Insecticides are the primary method of controlling the bird cherry-oat aphid in China, yet this aphid species has developed resistance to different types of insecticides, especially organophosphates and carbamates. In the last decade, control of R. padi depends primarily on the spray of neonicotinoid insecticides, however, research on the resistance of R. padi to neonicotinoids has been limited. In this study, the full lengths of seven α-subunit (Rpα1, Rpα2, Rpα3, Rpα4, Rpα5, Rpα7-1, and Rpα7-2) and one β-subunit (Rpβ1) genes from R. padi were obtained with RT-PCR and RACE techniques. Sequence analysis showed that these genes had all the characteristics of the nAChR gene family and were highly homologous with the reported nAChR genes from other insects, and alternative splicing was detected in Rpα3 and Rpα5 subunits. Analysis of the cDNA sequence of the extracellular region of the nicotinic acetylcholine receptor β1 subunit gene from 120 R. padi field samples collected in 11 Provinces revealed 17 single nucleotides polymorphism (SNP) sites, of which seven were amino acid polymorphism sites (V53I, V53G, N54T, A60T, F61L, W79C, and V83I) and two were in the loop D region (W79C and V83I). The current study will facilitate further studies on the molecular mechanisms of targeted resistance of the aphid to neonicotinoid insecticides. PMID:27521918

  12. Transcriptional Changes in nAChRs, Interactive Proteins and P450s in Locusta migratoria manilensis (Orthoptera: Acrididae) CNS in Response to High and Low Oral Doses of Imidacloprid.

    PubMed

    Wang, Xin; Sun, Huahua; Zhang, Yixi; Liu, Chuanjun; Liu, Zewen

    2015-01-01

    The insect central nervous system (CNS) is the target for many insecticides, and changes in transcript levels could be expected after insecticide applications. In this study, differentially expressed genes in the locust (Locusta migratoria manilensis) CNS in response to imidacloprid treatments at low dose (LD, 10% mortality) and high dose (HD, 80% mortality) were identified. Two nicotine acetylcholine receptor (nAChR) subunits genes and 18 interacting protein genes were regulated at LD, and only one nAChR subunit gene and 11 interacting proteins were regulated at HD. Among the 110 annotated P450 unigenes, 43 unigenes were regulated at LD and 34 unigenes were regulated at HD. Most of the differentially expressed P450 unigenes were mapped to CYP4, in which most unigenes were upregulated at LD, but downregulated at HD. Totally, the numbers and regulation levels of the regulated genes were more at LD than that at HD. Seventeen unigenes were selected to test their expression changes following insecticide treatments by qRT-PCR, in which the changes in more than half of the selected genes were verified. The results revealed the variation in the response of locusts to different insecticide pressure, such as different doses. PMID:26180048

  13. Transcriptional Changes in nAChRs, Interactive Proteins and P450s in Locusta migratoria manilensis (Orthoptera: Acrididae) CNS in Response to High and Low Oral Doses of Imidacloprid

    PubMed Central

    Wang, Xin; Sun, Huahua; Zhang, Yixi; Liu, Chuanjun; Liu, Zewen

    2015-01-01

    The insect central nervous system (CNS) is the target for many insecticides, and changes in transcript levels could be expected after insecticide applications. In this study, differentially expressed genes in the locust (Locusta migratoria manilensis) CNS in response to imidacloprid treatments at low dose (LD, 10% mortality) and high dose (HD, 80% mortality) were identified. Two nicotine acetylcholine receptor (nAChR) subunits genes and 18 interacting protein genes were regulated at LD, and only one nAChR subunit gene and 11 interacting proteins were regulated at HD. Among the 110 annotated P450 unigenes, 43 unigenes were regulated at LD and 34 unigenes were regulated at HD. Most of the differentially expressed P450 unigenes were mapped to CYP4, in which most unigenes were upregulated at LD, but downregulated at HD. Totally, the numbers and regulation levels of the regulated genes were more at LD than that at HD. Seventeen unigenes were selected to test their expression changes following insecticide treatments by qRT-PCR, in which the changes in more than half of the selected genes were verified. The results revealed the variation in the response of locusts to different insecticide pressure, such as different doses. PMID:26180048

  14. Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes

    PubMed Central

    Wang, Shuo; Zhao, Cong; Liu, Zhuguo; Wang, Xuesong; Liu, Na; Du, Weihong; Dai, Qiuyun

    2015-01-01

    In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH2), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 310-helix from residues Pro7 to His10 and a type I β-turn from residues Pro14 to Cys17. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu2 by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes. PMID:26023835

  15. Further proof of the existence of a non-neuronal cholinergic system in the human Achilles tendon: Presence of the AChRα7 receptor in tendon cells and cells in the peritendinous tissue.

    PubMed

    Forsgren, Sture; Alfredson, Håkan; Andersson, Gustav

    2015-11-01

    Human tendon cells have the capacity for acetylcholine (ACh) production. It is not known if the tendon cells also have the potential for ACh breakdown, nor if they show expression of the nicotinic acetylcholine receptor AChRα7 (α7nAChR). Therefore, tendon tissue specimens from patients with midportion Achilles tendinopathy/tendinosis and from normal midportion Achilles tendons were examined. Reaction for the degradative enzyme acetylcholinesterase (AChE) was found in some tenocytes in only a few tendinopathy tendons, and was never found in those of control tendons. Tenocytes displayed more regularly α7nAChR immunoreactivity. However, there was a marked heterogeneity in the degree of this reaction within and between the specimens. α7nAChR immunoreactivity was especially pronounced for tenocytes showing an oval/widened appearance. There was a tendency that the magnitude of α7nAChR immunoreactivity was higher in tendinopathy tendons as compared to control tendons. A stronger α7nAChR immunoreactivity than seen for tenocytes was observed for the cells in the peritendinous tissue. It is likely that the α7nAChR may be an important part of an auto-and paracrine loop of non-neuronal ACh that is released from the tendon cells. The effects may be related to proliferative and blood vessel regulatory functions as well as features related to collagen deposition. ACh can furthermore be of importance in leading to anti-inflammatory effects in the peritendinous tissue, a tissue nowadays considered to be of great relevance for the tendinopathy process. Overall, the findings show that tendon tissue, a tissue known to be devoid of cholinergic innervation, is a tissue in which there is a marked non-neuronal cholinergic system. PMID:25981114

  16. Nicotinic Acetylcholine Receptors in Sensory Cortex

    ERIC Educational Resources Information Center

    Metherate, Raju

    2004-01-01

    Acetylcholine release in sensory neocortex contributes to higher-order sensory function, in part by activating nicotinic acetylcholine receptors (nAChRs). Molecular studies have revealed a bewildering array of nAChR subtypes and cellular actions; however, there is some consensus emerging about the major nAChR subtypes and their functions in…

  17. Neuroprotective effects of donepezil against Aβ42-induced neuronal toxicity are mediated through not only enhancing PP2A activity but also regulating GSK-3β and nAChRs activity.

    PubMed

    Noh, Min-Young; Koh, Seong H; Kim, Sung-Min; Maurice, Tangui; Ku, Sae-Kwang; Kim, Seung H

    2013-11-01

    The main purpose of this study was to evaluate whether donepezil, acetylcholinesterase inhibitor, shown to play a protective role through inhibiting glycogen synthesis kinase-3β (GSK-3β) activity, could also exert neuroprotective effects by stimulating protein phosphatase 2A (PP2A) activity in the amyloid-beta (Aβ)42-induced neuronal toxicity model of Alzheimer's disease. In Aβ42-induced toxic conditions, each PP2A and GSK-3β activity measured at different times showed time-dependent reverse pattern toward the direction of accelerating neuronal deaths with the passage of time. In addition, donepezil pre-treatment showed dose-dependent stepwise increase of neuronal viability and stimulation of PP2A activity. However, such effects on them were significantly reduced through the depletion of PP2A activity with either okadaic acid or PP2Ac siRNA. In spite of blocked PP2A activity in this Aβ42 insult, however, donepezil pretreatment showed additional significant recovering effect on neuronal viability when compared to the value without donepezil. Moreover, donepezil partially recovered its dephosphorylating effect on hyperphosphorylated tau induced by Aβ42. This observation led us to assume that additional mechanisms of donepezil, including its inhibitory effect on GSK-3β activity and/or the activation role of nicotinic acetylcholine receptors (nAChRs), might be involved. Taken together, our results suggest that the neuroprotective effects of donepezil against Aβ42-induced neurotoxicity are mediated through activation of PP2A, but its additional mechanisms including regulation of GSK-3β and nAChRs activity would partially contribute to its effects. We investigated neuroprotective mechanisms of donepezil against Aβ42 toxicity: Donepezil increased neuronal viability with reduced p-tau by enhancing PP2A activity. Despite of blocked PP2A activity, donepezil showed additional recovering effect on neuronal viability, which findings led us to assume that additional

  18. The role of the a7 subunit of the nicotinic acetylcholine receptor on motor coordination in mice treated with methyllcaconitine and anabasine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The adverse effects of methyllycaconitine (MLA) have been attributed to competitive antagonism of nicotinic acetylcholine receptors (nAChR). Research has indicated a correlation between the LD50 of MLA and the amount of a7 nAChR in various mouse strains, suggesting that mice with more a7 nAChR requi...

  19. Multiple Modes of α7 nAChR Noncompetitive Antagonism of Control Agonist-Evoked and Allosterically Enhanced Currents

    PubMed Central

    Peng, Can; Kimbrell, Matthew R.; Tian, Chengju; Pack, Thomas F.; Crooks, Peter A.; Fifer, E. Kim

    2013-01-01

    Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors can enhance ion channel currents and downstream effects of α7 stimulation. We investigated the approach of using noncompetitive antagonists to regulate α7 receptor function, potentially distinguishing effects requiring ion channel currents from signaling induced by nonconducting states. Three small readily reversible antagonists, (1S,2R,4R)-N,2,3,3-tetramethylbicyclo[2.2.1]heptan-2-amine (mecamylamine), N-(2.6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), and 2-(dimethylamino)ethyl 4-(butylamino)benzoate (tetracaine), as well as three large slowly reversible antagonists, bis-(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (BTMPS), 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), and 1,2,4,5-tetra-{5-[1-(3-benzyl)pyridinium]pent-1-yl}benzene tetrabromide (tkP3BzPB), were investigated for their effectiveness and voltage dependence in the inhibition of responses evoked by acetylcholine alone or augmented by the α7-selective PAM N-(5-chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596). Analyses of the small antagonists on PNU-120596–potentiated single-channel bursts indicated that each agent had a distinct mechanism of inhibition and only that of QX-314 was consistent with simple open channel block. In addition to decreasing channel open times and burst durations, mecamylamine and tetracaine induced unique subconductance states. To determine whether channel-blocking activity alone would be sufficient to prevent cell death, the antagonists were tested for their ability to protect α7-expressing cells from cytotoxic effects of the α7 agonist choline in combination with PNU-120596. Only tetracaine and tkP3BzPB, the two agents that had effects least consistent with simple ion channel block, were fully cytoprotective at concentrations that gave submaximal inhibition of macroscopic currents in oocytes. Further analyses indicated that toxicity

  20. Characterization of the human beta4 nAChR gene and polymorphisms in CHRNA3 and CHRNB4.

    PubMed

    Lev-Lehman, E; Bercovich, D; Xu, W; Stockton, D W; Beaudet, A L

    2001-01-01

    Most neuronal nicotinic acetylcholine receptors are heteropentamers, composed of alpha and beta subunits. Mice lacking the alpha3 subunit and mice lacking both the beta2 and beta4 subunits, but not mice lacking the beta2 or beta4 subunits alone, have a severe phenotype characterized by megacystis, failure of bladder strips to contract in response to nicotine, widely dilated ocular pupils, growth failure, and perinatal mortality. The deficit in bladder contraction was also found in mice lacking only the beta4 subunit, although they did not develop megacystis. The major bladder phenotype resembles the human autosomal recessive disorder of megacystis-microcolon-hypoperistalsis syndrome (MMIHS). Based on the similarity of the mouse and human phenotypes, we initiated mutation analyses in the alpha3 and beta4 genes in MMIHS families. The human gene encoding the beta4 subunit was fully characterized, including refinement of its mapping. Analysis of disease families and controls identified numerous genetic variants, including high-frequency polymorphisms in both CHRNA3 and CHRNB4. Although no loss-of-function mutations have been identified to date, these genes remain strong candidates for involvement in MMIHS, because various mutations might be obscured within the complex cluster of genes. Some of the markers presented here are valuable tools for analysis of the role of genetic variation in responses to nicotine and for characterization of various dysautonomic abnormalities. PMID:11450844

  1. Nicotinic acetylcholine receptor expression in human airway correlates with lung function.

    PubMed

    Lam, David Chi-Leung; Luo, Susan Yang; Fu, Kin-Hang; Lui, Macy Mei-Sze; Chan, Koon-Ho; Wistuba, Ignacio Ivans; Gao, Boning; Tsao, Sai-Wah; Ip, Mary Sau-Man; Minna, John Dorrance

    2016-02-01

    Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChRs) on bronchial epithelial cells, can regulate cellular signaling and inflammatory processes. Delineation of nAChR subtypes and their responses to nicotine stimulation in bronchial epithelium may provide information for therapeutic targeting in smoking-related inflammation in the airway. Expression of nAChR subunit genes in 60 bronchial epithelial biopsies and immunohistochemical staining for the subcellular locations of nAChR subunit expression were evaluated. Seven human bronchial epithelial cell lines (HBECs) were exposed to nicotine in vitro for their response in nAChR subunit gene expression to nicotine exposure and removal. The relative normalized amount of expression of nAChR α4, α5, and α7 and immunohistochemical staining intensity of nAChR α4, α5, and β3 expression showed significant correlation with lung function parameters. Nicotine stimulation in HBECs resulted in transient increase in the levels of nAChR α5 and α6 but more sustained increase in nAChR α7 expression. nAChR expression in bronchial epithelium was found to correlate with lung function. Nicotine exposure in HBECs resulted in both short and longer term responses in nAChR subunit gene expression. These results gave insight into the potential of targeting nAChRs for therapy in smoking-related inflammation in the airway. PMID:26608528

  2. 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

  3. In vivo pharmacological interactions between a type II positive allosteric modulator of α7 nicotinic ACh receptors and nicotinic agonists in a murine tonic pain model

    PubMed Central

    Freitas, K; Negus, SS; Carroll, FI; Damaj, MI

    2013-01-01

    Background and Purpose The α7 nicotinic ACh receptor subtype is abundantly expressed in the CNS and in the periphery. Recent evidence suggests that α7 nicotinic ACh receptor (nAChR) subtypes, which can be activated by an endogenous cholinergic tone comprising ACh and the α7 agonist choline, play an important role in chronic pain and inflammation. In this study, we evaluated whether type II α7 positive allosteric modulator PNU-120596 induces antinociception on its own and in combination with choline in the formalin pain model. Experimental Approach We assessed the effects of PNU-120596 and choline and the nature of their interactions in the formalin test using an isobolographic analysis. In addition, we evaluated the interaction of PNU-120596 with PHA-54613, an exogenous selective α7 nAChR agonist, in the formalin test. Finally, we assessed the interaction between PNU-120596 and nicotine using acute thermal pain, locomotor activity, body temperature and convulsing activity tests in mice. Key Results We found that PNU-120596 dose-dependently attenuated nociceptive behaviour in the formalin test after systemic administration in mice. In addition, mixtures of PNU-120596 and choline synergistically reduced formalin-induced pain. PNU-120596 enhanced the effects of nicotine and α7 agonist PHA-543613 in the same test. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, hypomotility and antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via activation of α7 nAChRs. Conclusions and Implications Our results demonstrate that type II α7 positive allosteric modulators produce antinociceptive effects in the formalin test through a synergistic interaction with the endogenous α7 agonist choline. PMID:23004024

  4. Caenorhabditis elegans nicotinic acetylcholine receptors are required for nociception

    PubMed Central

    Cohen, Emiliano; Chatzigeorgiou, Marios; Husson, Steven J.; Steuer-Costa, Wagner; Gottschalk, Alexander; Schafer, William R.; Treinin, Millet

    2014-01-01

    Polymodal nociceptors sense and integrate information on injurious mechanical, thermal, and chemical stimuli. Chemical signals either activate nociceptors or modulate their responses to other stimuli. One chemical known to activate or modulate responses of nociceptors is acetylcholine (ACh). Across evolution nociceptors express subunits of the nicotinic acetylcholine receptor (nAChR) family, a family of ACh-gated ion channels. The roles of ACh and nAChRs in nociceptor function are, however, poorly understood. Caenorhabditis elegans polymodal nociceptors, PVD, express nAChR subunits on their sensory arbor. Here we show that mutations reducing ACh synthesis and mutations in nAChR subunits lead to defects in PVD function and morphology. A likely cause for these defects is a reduction in cytosolic calcium measured in ACh and nAChR mutants. Indeed, overexpression of a calcium pump in PVD mimics defects in PVD function and morphology found in nAChR mutants. Our results demonstrate, for the first time, a central role for nAChRs and ACh in nociceptor function and suggest that calcium permeating via nAChRs facilitates activity of several signaling pathways within this neuron. PMID:24518198

  5. Insect nicotinic receptor interactions in vivo with neonicotinoid, organophosphorus, and methylcarbamate insecticides and a synergist

    PubMed Central

    Shao, Xusheng; Xia, Shanshan; Durkin, Kathleen A.; Casida, John E.

    2013-01-01

    The nicotinic acetylcholine (ACh) receptor (nAChR) is the principal insecticide target. Nearly half of the insecticides by number and world market value are neonicotinoids acting as nAChR agonists or organophosphorus (OP) and methylcarbamate (MC) acetylcholinesterase (AChE) inhibitors. There was no previous evidence for in vivo interactions of the nAChR agonists and AChE inhibitors. The nitromethyleneimidazole (NMI) analog of imidacloprid, a highly potent neonicotinoid, was used here as a radioligand, uniquely allowing for direct measurements of house fly (Musca domestica) head nAChR in vivo interactions with various nicotinic agents. Nine neonicotinoids inhibited house fly brain nAChR [3H]NMI binding in vivo, corresponding to their in vitro potency and the poisoning signs or toxicity they produced in intrathoracically treated house flies. Interestingly, nine topically applied OP or MC insecticides or analogs also gave similar results relative to in vivo nAChR binding inhibition and toxicity, but now also correlating with in vivo brain AChE inhibition, indicating that ACh is the ultimate OP- or MC-induced nAChR active agent. These findings on [3H]NMI binding in house fly brain membranes validate the nAChR in vivo target for the neonicotinoids, OPs and MCs. As an exception, the remarkably potent OP neonicotinoid synergist, O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR in vivo without the corresponding AChE inhibition, possibly via a reactive ketene metabolite reacting with a critical nucleophile in the cytochrome P450 active site and the nAChR NMI binding site. PMID:24108354

  6. Roles of nicotinic acetylcholine receptor β subunits in function of human α4-containing nicotinic receptors

    PubMed Central

    Wu, Jie; Liu, Qiang; Yu, Kewei; Hu, Jun; Kuo, Yen-Ping; Segerberg, Marsha; St John, Paul A; Lukas, Ronald J

    2006-01-01

    Naturally expressed nicotinic acetylcholine receptors (nAChR) containing α4 subunits (α4*-nAChR) in combination with β2 subunits (α4β2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. β4 subunits are also richly expressed and colocalize with α4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, α4β4-nAChR also may exist and play important functional roles. In this study, properties were determined of human α4β2- and α4β4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human α4β4-nAChR have ∼4-fold higher amplitude than those mediated via human α4β2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at α4β4-nAChR than at α4β2-nAChR. Cytisine and lobeline serve as full agonists at α4β4-nAChR but are only partial agonists at α4β2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional α4β2- and α4β4-nAChR. Whole-cell current responses show stronger inward rectification for α4β2-nAChR than for α4β4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR β2 or β4 subunits can combine with α4 subunits to generate two forms of α4*-nAChR with distinctive physiological and pharmacological features. Diversity in α4*-nAChR is of potential relevance to nervous system function, disease, and nicotine dependence. PMID:16825297

  7. Nicotinic acetylcholine receptors and cancer

    PubMed Central

    DANG, NINGNING; MENG, XIANGUANG; SONG, HAIYAN

    2016-01-01

    Nicotine, the primary addictive constituent of cigarettes, is believed to contribute to cancer promotion and progression through the activation of nicotinic acetylcholine receptors (nAChRs), which are membrane ligand-gated cation channels. nAChRs activation can be triggered by the neurotransmitter Ach, or certain other biological compounds, such as nicotine. In recent years, genome-wide association studies have indicated that allelic variation in the α5-α3-β4 nAChR cluster on chromosome 15q24-15q25.1 is associated with lung cancer risk. The role of nAChRs in other types of cancer has also been reported. The present review highlights the role of nAChRs in types of human cancer. PMID:27123240

  8. α7 Nicotinic Receptor Agonists: Potential Therapeutic Drugs for Treatment of Cognitive Impairments in Schizophrenia and Alzheimer’s Disease

    PubMed Central

    Toyohara, Jun; Hashimoto, Kenji

    2010-01-01

    Accumulating evidence suggests that α7 nicotinic receptors (α7 nAChRs), a subtype of nAChRs, play a role in the pathophysiology of neuropsychiatric diseases, including schizophrenia and Alzheimer’s disease (AD). A number of psychopharmacological and genetic studies shown that α7 nAChRs play an important role in the deficits of P50 auditory evoked potential in patients with schizophrenia, and that (α nAChR agonists would be potential therapeutic drugs for cognitive impairments associated with P50 deficits in schizophrenia. Furthermore, some studies have demonstrated that α7 nAChRs might play a key role in the amyloid-β (Aβ)-mediated pathology of AD, and that α7 nAChR agonists would be potential therapeutic drugs for Aβ deposition in the brains of patients with AD. Interestingly, the altered expression of α7 nAChRs in the postmortem brain tissues from patients with schizophrenia and AD has been reported. Based on all these findings, selective α7 nAChR agonists can be considered potential therapeutic drugs for cognitive impairments in both schizophrenia and AD. In this article, we review the recent research into the role of α7 nAChRs in the pathophysiology of these diseases and into the potential use of novel α7 nAChR agonists as therapeutic drugs. PMID:21249164

  9. Natural Compounds Interacting with Nicotinic Acetylcholine Receptors: From Low-Molecular Weight Ones to Peptides and Proteins

    PubMed Central

    Kudryavtsev, Denis; Shelukhina, Irina; Vulfius, Catherine; Makarieva, Tatyana; Stonik, Valentin; Zhmak, Maxim; Ivanov, Igor; Kasheverov, Igor; Utkin, Yuri; Tsetlin, Victor

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds. PMID:26008231

  10. The combination of memantine and galantamine improves cognition in rats: The synergistic role of the α7 nicotinic acetylcholine and NMDA receptors.

    PubMed

    Nikiforuk, Agnieszka; Potasiewicz, Agnieszka; Kos, Tomasz; Popik, Piotr

    2016-10-15

    The combination of memantine and acetylcholinesterase inhibitors (AChEIs) is used as a therapeutic strategy to improve cognition in Alzheimer's disease. Among AChEIs, galantamine, which is also a positive allosteric modulator (PAM) of nicotinic acetylcholine receptors (nAChRs), including α7-nAChRs, may be particularly beneficial. The α7-nAChR is involved in interactions between the cholinergic and glutamatergic systems. In the present study, we investigated the potential role of α7-nAChRs in the pro-cognitive effects of this drug combination. To this aim, cognitive performance in rats was assessed using the attentional set shifting task (ASST) and novel object recognition task (NORT). Co-administration of inactive doses of memantine with galantamine facilitated the rats' set-shifting performance and reversed delay-induced deficits in object recognition. These effects were blocked by the α7-nAChR antagonist methyllycaconitine, suggesting that the observed cognitive enhancement is α7-nAChR dependent. Moreover, combined administration of memantine with inactive doses of selective α7-nAChRs PAMs, CCMI and PNU-120596, also improved ASST and NORT performance in a methyllycaconitine-dependent manner. Stimulation of α7-nAChRs may underlie the pro-cognitive effects of combining memantine and galantamine. Our results suggest that memantine, when given with enhancers of α7-nAChRs, may represent an effective strategy for cognitive improvement. PMID:27435422

  11. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  12. Effect of a nicotinic acetylcholine receptor agonists and antagonists on motor function in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nicotinic acetylcholine receptors (nAChR) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChR located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The...

  13. α6* nicotinic acetylcholine receptor expression and function in a visual salience circuit

    PubMed Central

    Mackey, Elisha D.W.; Engle, Staci E.; Kim, Mi Ran; O’Neill, Heidi C.; Wageman, Charles R.; Patzlaff, Natalie E.; Wang, Ying; Grady, Sharon R.; McIntosh, J. Michael; Marks, Michael J.; Lester, Henry A.; Drenan, Ryan M.

    2012-01-01

    Nicotinic ACh receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus coeruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with GFP fused in-frame in the M3–M4 intracellular loop. Inα6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitiveα6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway. PMID:22836257

  14. Role of α5 Nicotinic Acetylcholine Receptors in Pharmacological and Behavioral Effects of Nicotine in Mice

    PubMed Central

    Marks, M. J.; Vann, R. E.; Chen, X.; Gamage, T. F.; Warner, J. A.; Damaj, M. I.

    2010-01-01

    Incorporation of the α5 nicotinic acetylcholine receptor (nAChR) subunit can greatly influence nAChR function without altering receptor number. Although few animal studies have assessed the role of the α5 nAChR in nicotine-mediated behaviors, recent evidence suggests an association between polymorphisms in the α5 nAChR gene and nicotine dependence phenotypes in humans. Thus, additional studies are imperative to elucidate the role and function of the α5 nAChR subunit in nicotine dependence. Using α5(−/−) mice, the current study aimed to examine the role of α5 nAChRs in the initial pharmacological effects of nicotine, nicotine reward using the conditioned place preference model, and the discriminative effects of nicotine using a two-lever drug discrimination model. 86Rb+ efflux and 125I-epibatidine binding assays were conducted to examine the effect of α5 nAChR subunit deletion on expression and activity of functional nAChRs. Results show that α5(−/−) mice are less sensitive to the initial effects of nicotine in antinociception, locomotor activity, and hypothermia measures and that the α5 nAChR is involved in nicotine reward. Alternatively, α5(−/−) mice did not differ from wild-type littermates in sensitivity to the discriminative stimulus effects of nicotine. Furthermore, deletion of the α5 nAChR subunit resulted in a statistically significant decrease in function in the thalamus and hindbrain, but the decreases noted in spinal cord were not statistically significant. Receptor number was unaltered in all areas tested. Taken together, results of the study suggest that α5 nAChRs are involved in nicotine-mediated behaviors relevant to development of nicotine dependence. PMID:20400469

  15. α7 Nicotinic Acetylcholine Receptors Occur at Postsynaptic Densities of AMPA Receptor-Positive and -Negative Excitatory Synapses in Rat Sensory Cortex

    PubMed Central

    Levy, Robert B.; Aoki, Chiye

    2010-01-01

    NMDA receptor (NMDAR) activation requires concurrent membrane depolarization, and glutamatergic synapses lacking AMPA receptors (AMPARs) are often considered “silent” in the absence of another source of membrane depolarization. During the second postnatal week, NMDA currents can be enhanced in rat auditory cortex through activation of the α7 nicotinic acetylcholine receptor (α7nAChR). Electrophysiological results support a mainly presynaptic role for α7nAChR at these synapses. However, immunocytochemical evidence that α7nAChR is prevalent at postsynaptic sites of glutamatergic synapses in hippocampus and neocortex, along with emerging electrophysiological evidence for postsynaptic nicotinic currents in neocortex and hippocampus, has prompted speculation that α7nAChR allows for activation of NMDAR postsynaptically at synapses lacking AMPAR. Here we used dual immunolabeling and electron microscopy to examine the distribution of α7nAChR relative to AMPAR (GluR1, GluR2, and GluR3 subunits combined) at excitatory synapses in somatosensory cortex of adult and 1-week-old rats. α7nAChR occurred discretely over most of the thick postsynaptic densities in all cortical layers of both age groups. AMPAR immunoreactivity was also detectable at most synapses; its distribution was independent of that of α7nAChR. In both age groups, approximately one-quarter of asymmetrical synapses were α7nAChR positive and AMPAR negative. The variability of postsynaptic α7nAChR labeling density was greater at postnatal day (PD) 7 than in adulthood, and PD 7 neuropil contained a subset of small AMPA receptor-negative synapses with a high density of α7nAChR immunoreactivity. These observations support the idea that acetylcholine receptors can aid in activating glutamatergic synapses and work together with AMPA receptors to mediate postsynaptic excitation throughout life. PMID:12077196

  16. N-glycosylation sites on the nicotinic ACh receptor subunits regulate receptor channel desensitization and conductance.

    PubMed

    Nishizaki, Tomoyuki

    2003-06-10

    The present study investigated the effects of N-glycosylation sites on Torpedo acetylcholine (ACh) receptors expressed in Xenopus oocytes by monitoring whole-cell membrane currents and single-channel currents from excised patches. Receptors with the mutant subunit at the asparagine residue on the conserved N-glycosylation site (mbetaN141D, mgammaN141D, or mdeltaN143D) or the serine/threonine residue (mbetaT143A, mgammaS143A, or mdeltaS145A) delayed the rate of current decay as compared with wild-type receptors, and the most striking effect was found with receptors with mbetaT143A or mgammaS143A. For wild-type receptors, the lectin concanavalin A, that binds to glycosylated membrane proteins with high affinity, mimicked this effect. Receptors with mbetaN141D or mdeltaN143D exhibited lower single-channel conductance, but those with mbetaT143A, mgammaS143A, or mdeltaS145A otherwise revealed higher conductance than wild-type receptors. Mean opening time of single-channel currents was little affected by the mutation. N-glycosylation sites, thus, appear to play a role in the regulation of ACh receptor desensitization and ion permeability. PMID:12829329

  17. The role of the a7 subunit of the nicotinic acetylcholine receptor in the acute toxicosis of methyllycaconitine in mice.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research demonstrated a correlation between the LD50 of MLA and the amount of a7 nAChR in various mouse strains, suggesting that mice...

  18. Putative nicotinic acetylchloline receptor subunits express differentially through life cycle of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nicotinic acetylcholine receptors (nAChRs) are the targets of neonicotinoids and spinosads, two insecticides used in orchards to effectively control codling moth, Cydia pomonella (L.)(Lepidoptera: Tortricidae). The nAChRs mediate the fast actions of the neurotransmitter acetylcholine in synaptic tr...

  19. Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors.

    PubMed

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Shenkarev, Zakhar O; Kasheverov, Igor E; Chugunov, Anton O; Kulbatskii, Dmitrii S; Myshkin, Mikhail Yu; Utkin, Yuri N; Efremov, Roman G; Tsetlin, Victor I; Arseniev, Alexander S; Kirpichnikov, Mikhail P; Dolgikh, Dmitry A

    2016-09-01

    'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs. PMID:27343701

  20. Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain.

    PubMed

    Wu, Jie; Liu, Qiang; Tang, Pei; Mikkelsen, Jens D; Shen, Jianxin; Whiteaker, Paul; Yakel, Jerrel L

    2016-07-01

    The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co-assembled with β2 subunits to form a novel type of α7β2 nAChR. Interestingly, the α7β2 nAChR exhibits distinctive function and pharmacology from traditional homomeric α7 nAChRs. We review recent advances in probing the distribution, function, pharmacology, pathophysiology, and stoichiometry of the heteromeric α7β2 nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling. PMID:27179601

  1. α4β2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief.

    PubMed

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

    2011-07-27

    Nicotine is the primary psychoactive substance in tobacco, and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the α4β2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal as well as nicotine-induced behaviors. Although α4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addiction, mental illness, and movement control in humans. We developed a unique model system to examine the role of α4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the α4 subunit from dopaminergic neurons in mice. The loss α4 mRNA and α4β2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of α4β2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. α4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. α4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze, and elimination of α4β2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of α4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression; however, nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine-related behaviors. PMID:21795541

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

    PubMed Central

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

    2012-01-01

    Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addition, mental illness and movement control in humans. We developed a unique model system to examine the role of alpha4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the alpha4 subunit from dopaminergic neurons in mice. The loss alpha4 mRNA and alpha4beta2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of alpha4beta2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. Alpha4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. Alpha4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze and elimination of alpha4-beta2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of alpha4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression, however nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine behaviors. PMID:21795541

  3. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 due to the binding, activation, and prolonged desensitization of fetal muscle-type nAChR which re...

  4. Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

    PubMed Central

    Murray, Teresa A; Liu, Qiang; Whiteaker, Paul; Wu, Jie; Lukas, Ronald J

    2009-01-01

    Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors. Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion. PMID:19498423

  5. Allosteric modifiers of neuronal nicotinic acetylcholine receptors: new methods, new opportunities.

    PubMed

    Moaddel, Ruin; Jozwiak, Krzysztof; Wainer, Irving W

    2007-09-01

    Allosteric, non-competitive inhibitors (NCIs) of neuronal nicotinic acetylcholine receptors (nAChRs) have been shown to produce a wide variety of clinically relevant responses. Many of the observed effects are desired as the nAChR is the therapeutic target, while others are undesired consequences due to off-target binding at the nAChR. Thus, the determination of whether or not a lead drug candidate is an NCI should play an important role in drug discovery programs. However, the current experimental techniques used to identify NCIs are challenging, expensive, and time consuming. This review focuses on an alternative approach to the investigation of interactions between test compounds and nAChRs based upon liquid chromatographic stationary phases containing cellular fragments from cell lines expressing nAChRs. The development and validation of these phases as well as their use in drug discovery and pharmacophore modeling are discussed. PMID:17238157

  6. AzoCholine Enables Optical Control of Alpha 7 Nicotinic Acetylcholine Receptors in Neural Networks.

    PubMed

    Damijonaitis, Arunas; Broichhagen, Johannes; Urushima, Tatsuya; Hüll, Katharina; Nagpal, Jatin; Laprell, Laura; Schönberger, Matthias; Woodmansee, David H; Rafiq, Amir; Sumser, Martin P; Kummer, Wolfgang; Gottschalk, Alexander; Trauner, Dirk

    2015-05-20

    Nicotinic acetylcholine receptors (nAChRs) are essential for cellular communication in higher organisms. Even though a vast pharmacological toolset to study cholinergic systems has been developed, control of endogenous neuronal nAChRs with high spatiotemporal precision has been lacking. To address this issue, we have generated photoswitchable nAChR agonists and re-evaluated the known photochromic ligand, BisQ. Using electrophysiology, we found that one of our new compounds, AzoCholine, is an excellent photoswitchable agonist for neuronal α7 nAChRs, whereas BisQ was confirmed to be an agonist for the muscle-type nAChR. AzoCholine could be used to modulate cholinergic activity in a brain slice and in dorsal root ganglion neurons. In addition, we demonstrate light-dependent perturbation of behavior in the nematode, Caenorhabditis elegans. PMID:25741856

  7. Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology.

    PubMed

    Niessen, K V; Muschik, S; Langguth, F; Rappenglück, S; Seeger, T; Thiermann, H; Worek, F

    2016-04-15

    Organophosphorus compounds (OPC), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). Inhibited AChE results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nAChR) in the postsynaptic membrane is provoked. Direct targeting of nAChR to reduce receptor desensitisation might be an alternative therapeutic approach. For drug discovery, functional properties of potent therapeutic candidates need to be investigated in addition to affinity properties. Solid supported membrane (SSM)-based electrophysiology is useful for functional characterisation of ligand-gated ion channels like nAChRs, as charge translocations via capacitive coupling of the supporting membrane can be measured. By varying the agonist (carbamoylcholine) concentration, different functional states of the nAChR were initiated. Using plasma membrane preparations obtained from Torpedo californica electric organ, functional properties of selected nAChR ligands and non-oxime bispyridinium compounds were investigated. Depending on overall-size, the bispyridinium compounds enhanced or inhibited cholinergic signals induced by 100μM carbamoylcholine. Applying excessive concentrations of the agonist carbamoylcholine provoked desensitisation of the nAChRs, whereas addition of bispyridinium compounds bearing short alkyl linkers exhibited functional recovery of previously desensitised nAChRs. The results suggest that these non-oxime bispyridinium compounds possibly interacted with nAChR subtypes in a manner of a positive allosteric modulator (PAM). The described newly developed functional assay is a valuable tool for the assessment of functional properties of potential compounds such as nAChR modulating ligands, which might be a promising approach in the therapeutically treatment of OPC-poisonings. PMID:26851639

  8. Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen).

    PubMed

    Bao, Haibo; Sun, Huahua; Xiao, Youxin; Zhang, Yixi; Wang, Xin; Xu, Xiaoyong; Liu, Zewen; Fang, Jichao; Li, Zhong

    2015-01-01

    Associated proteins are important for the correct functioning of nicotinic acetylcholine receptors (nAChRs). In the present study, a neonicotinoid-agarose affinity column was used to isolate related proteins from a solubilized membrane preparation from the nervous system of Locusta migratoria manilensis (Meyen). 1530 peptides were identified and most of them were involved in the membranous structure, molecular interaction and cellular communication. Among these peptides, Na(+)/K(+) ATPase had the highest MASCOT score and were involved in the molecular interaction, which suggested that Na(+)/K(+) ATPase and nAChRs might have strong and stable interactions in insect central nervous system. In the present study, functional interactions between nAChRs and Na(+)/K(+) ATPase were examined by heterologous expression in Xenopus oocytes. The results showed that the activated nAChRs increased pump currents of Na(+)/K(+) ATPase, which did not require current flow through open nAChRs. In turn, Na(+)/K(+) ATPase significantly increased agonist sensitivities of nAChRs in a pump activity-independent manner and reduced the maximum current (Imax) of nAChRs. These findings provide novel insights concerning the functional interactions between insect nAChRs and Na(+)/K(+) ATPase. PMID:25743085

  9. Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen)

    PubMed Central

    Bao, Haibo; Sun, Huahua; Xiao, Youxin; Zhang, Yixi; Wang, Xin; Xu, Xiaoyong; Liu, Zewen; Fang, Jichao; Li, Zhong

    2015-01-01

    Associated proteins are important for the correct functioning of nicotinic acetylcholine receptors (nAChRs). In the present study, a neonicotinoid-agarose affinity column was used to isolate related proteins from a solubilized membrane preparation from the nervous system of Locusta migratoria manilensis (Meyen). 1530 peptides were identified and most of them were involved in the membranous structure, molecular interaction and cellular communication. Among these peptides, Na+/K+ ATPase had the highest MASCOT score and were involved in the molecular interaction, which suggested that Na+/K+ ATPase and nAChRs might have strong and stable interactions in insect central nervous system. In the present study, functional interactions between nAChRs and Na+/K+ ATPase were examined by heterologous expression in Xenopus oocytes. The results showed that the activated nAChRs increased pump currents of Na+/K+ ATPase, which did not require current flow through open nAChRs. In turn, Na+/K+ ATPase significantly increased agonist sensitivities of nAChRs in a pump activity-independent manner and reduced the maximum current (Imax) of nAChRs. These findings provide novel insights concerning the functional interactions between insect nAChRs and Na+/K+ ATPase. PMID:25743085

  10. Analysis of free ACh and 5-HT in milk from four different species and their bioactivity on 5-HT(3) and nACh receptors.

    PubMed

    Gallegos-Perez, Jose-Luis; Limon, Agenor; Reyes-Ruiz, Jorge M; Alshanqeeti, Ali S; Aljohi, Mohammad A; Miledi, Ricardo

    2014-07-25

    Milk is one of the most beneficial aliments and is highly recommended in normal conditions; however, in certain disorders, like irritable bowel syndrome, cow milk and dairy products worsen the gastric symptoms and their use is not recommended. Among the most recognized milk-induced gatrointestinal symptoms are abdominal pain, nausea and vomiting, which are processes controlled by cholinergic and serotonergic transmission. Whether the presence of bioavailable ACh and 5-HT in milk may contribute to normal peristalsis, or to the developing of these symptoms, is not known. In this work we attempt to determine whether the content of free ACh and 5-HT is of physiological significance in milk from four different species: cow (bovine), goat, camel and human. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to identify and quantify free ACh and 5-HT in milk, and activation of the serotonergic and cholinergic ionotropic receptors was investigated using electrophysiological experiments. Our principal hypothesis was that milk from these four species had sufficient free ACh and 5-HT to activate their correspondent receptors expressed in a heterologous system. Our results showed a more complex picture, in which free ACh and 5-HT and their ability to activate cholinergic and serotonergic receptors are not correlated. This work is a first step to elucidate whether 5-HT and ACh, at the concentrations present in the milk, can be associated to a direct function in the GI. PMID:24820623

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

    PubMed

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

    2016-06-01

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

  12. Going up in Smoke? A Review of nAChRs-based Treatment Strategies for Improving Cognition in Schizophrenia

    PubMed Central

    Boggs, Douglas L.; Carlson, Jon; Cortes-Briones, Jose; Krystal, John H.; D’Souza, D. Cyril

    2015-01-01

    Cognitive impairment is known to be a core deficit in schizophrenia. Existing treatments for schizophrenia have limited efficacy against cognitive impairment. The ubiquitous use of nicotine in this population is thought to reflect an attempt by patients to self-medicate certain symptoms associated with the illness. Concurrently there is evidence that nicotinic receptors that have lower affinity for nicotine are more important in cognition. Therefore, a number of medications that target nicotinic acetylcholine receptors (nAChRs) have been tested or are in development. In this article we summarize the clinical evidence of nAChRs dysfunction in schizophrenia and review clinical studies testing either nicotine or nicotinic medications for the treatment of cognitive impairment in schizophrenia. Some evidence suggests beneficial effects of nAChRs based treatments for the attentional deficits associated with schizophrenia. Standardized cognitive test batteries have failed to capture consistent improvements from drugs acting at nAChRs. However, more proximal measures of brain function, such as ERPs relevant to information processing impairments in schizophrenia, have shown some benefit. Further work is necessary to conclude that nAChRs based treatments are of clinical utility in the treatment of cognitive deficits of schizophrenia. PMID:24345265

  13. New quinoline derivatives as nicotinic receptor modulators.

    PubMed

    Manetti, Dina; Bellucci, Cristina; Dei, Silvia; Teodori, Elisabetta; Varani, Katia; Spirova, Ekaterina; Kudryavtsev, Denis; Shelukhina, Irina; Tsetlin, Victor; Romanelli, Maria Novella

    2016-03-01

    As a continuation of previous work on quinoline derivatives, which showed some preference (2-3 times) for the α7 with respect to α4β2 acetylcholine nicotinic receptors (nAChRs), we synthesized a series of novel azabicyclic or diazabicyclic compounds carrying a quinoline or isoquinoline ring, with the aim of searching for more selective α7 nAChR compounds. Radioligand binding studies on α7* and α4β2* nAChRs (rat brain homogenate) revealed one compound (7) with a 2-fold higher affinity for the α4β2*-subtype, and four compounds (11, 13, 14 and 16) with at least 3-fold higher affinity for α7* nAChR. The most promising was 11, showing Ki∼100 nM and over 10-fold selectivity for α7* nAChR. Compounds 7, 11, 13 and 16 at 50 μM suppressed ion currents induced in the rat α4β2 nAChR and the chimeric nAChR composed of the ligand-binding domain of the chick α7 and transmembrane domain of the α1 glycine receptor, expressed in Xenopus oocytes. Calcium imaging experiments on the human α7 nAChR expressed in the Neuro2a cells and potentiated by PNU-120596 confirmed the antagonistic activity for 7; on the contrary, 11, 13 and 16 were agonists with the EC50 values in the range of 1.0-1.6 μM. Thus, the introduced modifications allowed us to enhance the selectivity of quinolines towards α7 nAChR and to get novel compounds with agonistic activity. PMID:26840365

  14. Glutamatergic synapse formation is promoted by α7-containing nicotinic acetylcholine receptors.

    PubMed

    Lozada, Adrian F; Wang, Xulong; Gounko, Natalia V; Massey, Kerri A; Duan, Jingjing; Liu, Zhaoping; Berg, Darwin K

    2012-05-30

    Glutamate is the primary excitatory transmitter in adult brain, acting through synapses on dendritic spines and shafts. Early in development, however, when glutamatergic synapses are only beginning to form, nicotinic cholinergic excitation is already widespread; it is mediated by acetylcholine activating nicotinic acetylcholine receptors (nAChRs) that generate waves of activity across brain regions. A major class of nAChRs contributing at this time is a species containing α7 subunits (α7-nAChRs). These receptors are highly permeable to calcium, influence a variety of calcium-dependent events, and are diversely distributed throughout the developing CNS. Here we show that α7-nAChRs unexpectedly promote formation of glutamatergic synapses during development. The dependence on α7-nAChRs becomes clear when comparing wild-type (WT) mice with mice constitutively lacking the α7-nAChR gene. Ultrastructural analysis, immunostaining, and patch-clamp recording all reveal synaptic deficits when α7-nAChR input is absent. Similarly, nicotinic activation of α7-nAChRs in WT organotypic culture, as well as cell culture, increases the number of glutamatergic synapses. RNA interference demonstrates that the α7-nAChRs must be expressed in the neuron being innervated for normal innervation to occur. Moreover, the deficits persist throughout the developmental period of major de novo synapse formation and are still fully apparent in the adult. GABAergic synapses, in contrast, are undiminished in number under such conditions. As a result, mice lacking α7-nAChRs have an altered balance in the excitatory/inhibitory input they receive. This ratio represents a fundamental feature of neural networks and shows for the first time that endogenous nicotinic cholinergic signaling plays a key role in network construction. PMID:22649244

  15. α6β2*-subtype nicotinic acetylcholine receptors are more sensitive than α4β2*-subtype receptors to regulation by chronic nicotine administration

    PubMed Central

    Marks, MJ; Grady, SR; Salminen, O; Paley, MA; Wageman, CR; McIntosh, JM; Whiteaker, P

    2014-01-01

    Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where * indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*-nAChR are downregulated following chronic nicotine exposure (unlike other subtypes that have been investigated – most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose-responses and quantitative ligand-binding autoradiography were used to define nicotine sensitivity of changes in α4β2*-nAChR and α6β2*-nAChR expression. α6β2*-nAChR downregulation by chronic nicotine exposure in dopaminergic and optic-tract nuclei was ≈three-fold more sensitive than upregulation of α4β2*-nAChR. In contrast, nAChR-mediated [3H]-dopamine release from dopamine-terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, while dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR-mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [3H]-DA release are primarily due to changes in nAChR, rather than in dopaminergic, function. PMID:24661093

  16. Therapeutic Potential of α7 Nicotinic Acetylcholine Receptors.

    PubMed

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

    2015-10-01

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

  17. The Conserved RIC-3 Coiled-Coil Domain Mediates Receptor-specific Interactions with Nicotinic Acetylcholine Receptors

    PubMed Central

    Biala, Yoav; Liewald, Jana F.; Ben-Ami, Hagit Cohen; Gottschalk, Alexander

    2009-01-01

    RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation. PMID:19116311

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

    PubMed

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

    2016-03-01

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

  19. Heterogeneous Inhibition in Macroscopic Current Responses of Four Nicotinic Acetylcholine Receptor Subtypes by Cholesterol Enrichment.

    PubMed

    Báez-Pagán, Carlos A; Del Hoyo-Rivera, Natalie; Quesada, Orestes; Otero-Cruz, José David; Lasalde-Dominicci, José A

    2016-08-01

    The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane. PMID:27116687

  20. Molecular Mechanisms of Cross-inhibition Between Nicotinic Acetylcholine Receptors and P2X Receptors in Myenteric Neurons and HEK-293 cells

    PubMed Central

    Decker, Dima A.; Galligan, James J.

    2010-01-01

    Background P2X2 and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and α3β4subunit-containing nAChRs. Methods The function of P2X2 and α3β4 nAChRs expressed by HEK-293 cells and guinea pig ileum myenteric neurons in culture was studied using whole-cell patch clamp techniques. Results In HEK-293 cells expressing α3β4 nAChRs and P2X2 receptors, co-application of ATP and ACh caused inward currents that were 56 ± 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs and a C-terminal tail truncated P2X2 receptor (P2X2TR)(P >0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2 receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK-293 cells expressing both receptors. Constitutive inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs transfected with P2X2TR. Currents caused by low (≤30 μM), but not high (≥100 μM) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co-expression with α3β4 nAChRs. Conclusions The C-terminal tail of P2X2 receptors mediates cross-inhibition between α3β4 nAChR-P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co-transmitters. PMID:20426799

  1. Nicotine Inhibits Cisplatin-Induced Apoptosis via Regulating α5-nAChR/AKT Signaling in Human Gastric Cancer Cells.

    PubMed

    Jia, Yanfei; Sun, Haiji; Wu, Hongqiao; Zhang, Huilin; Zhang, Xiuping; Xiao, Dongjie; Ma, Xiaoli; Wang, Yunshan

    2016-01-01

    Gastric cancer incidence demonstrates a strong etiologic association with smoking. Nicotine, the major component in tobacco, is a survival agonist that inhibits apoptosis induced by certain chemotherapeutic agents, but the precise mechanisms involved remain largely unknown. Recently studies have indicated that α5-nicotinic acetylcholine receptor (α5-nAChR) is highly associated with lung cancer risk and nicotine dependence. Nevertheless, no information has been available about whether nicotine also affects proliferation of human gastric cancer cells through regulation of α5-nAChR. To evaluate the hypothesis that α5-nAChR may play a role in gastric cancer, we investigated its expression in gastric cancer tissues and cell lines. The expression of α5-nAChR increased in gastric cancer tissue compared with para-carcinoma tissues. In view of the results, we proceeded to investigate whether nicotine inhibits cisplatin-induced apoptosis via regulating α5-nAChR in gastric cancer cell. The results showed that nicotine significantly promoted cell proliferation in a dose and time-dependent manner through α5-nAChR activation in human gastric cells. Furthermore, nicotine inhibited apoptosis induced by cisplatin. Silence of α5-nAChR ablated the protective effects of nicotine. However, when co-administrating LY294002, an inhibitor of PI3K/AKT pathway, an increased apoptosis was observed. This effect correlated with the induction of Bcl-2, Bax, Survivin and Caspase-3 by nicotine in gastric cell lines. These results suggest that exposure to nicotine might negatively impact the apoptotic potential of chemotherapeutic drugs and that α5-nAChR/AKT signaling plays a key role in the anti-apoptotic activity of nicotine induced by cisplatin. PMID:26909550

  2. Expression patterns of nicotinic subunits α2, α7, α8, and β1 affect the kinetics and pharmacology of ACh-induced currents in adult bee olfactory neuropiles.

    PubMed

    Dupuis, Julien Pierre; Gauthier, Monique; Raymond-Delpech, Valérie

    2011-10-01

    Acetylcholine (ACh) is the main excitatory neurotransmitter of the insect brain, where nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee Apis mellifera, nAChRs are expressed in diverse structures including the primary olfactory centers of the brain, the antennal lobes (ALs) and the mushroom bodies (MBs), where they participate in olfactory information processing. To understand the nature and properties of the nAChRs involved in these processes, we performed a pharmacological and molecular characterization of nAChRs on cultured Kenyon cells of the MBs, using whole cell patch-clamp recordings combined with single-cell RT-PCR. In all cells, applications of ACh as well as nicotinic agonists such as nicotine and imidacloprid induced inward currents with fast desensitization. These currents were fully blocked by saturating doses of the antagonists α-bungarotoxin (α-BGT), dihydroxy-β-erythroidine (DHE), and methyllycaconitine (MLA) (MLA ≥ α-BGT ≥ DHE). Molecular analysis of ACh-responding cells revealed that of the 11 nicotinic receptor subunits encoded within the honeybee genome, α2, α8, and β1 subunits were expressed in adult Kenyon cells. Comparison with the expression pattern of adult AL cells revealed the supplementary presence of subunit α7, which could be responsible for the kinetic and pharmacological differences observed when comparing ACh-induced currents from AL and Kenyon cells. Together, our data demonstrate the existence of functional nAChRs on adult MB Kenyon cells that differ from nAChRs on AL cells in both their molecular composition and pharmacological properties, suggesting that changing receptor subsets could mediate different processing functions depending on the brain structure within the olfactory pathway. PMID:21734106

  3. Acetylcholine Acts through Nicotinic Receptors to Enhance the Firing Rate of a Subset of Hypocretin Neurons in the Mouse Hypothalamus through Distinct Presynaptic and Postsynaptic Mechanisms1,2

    PubMed Central

    Zhou, Wen-Liang; Gao, Xiao-Bing

    2015-01-01

    Abstract Hypocretin/orexin neurons regulate many behavioral functions, including addiction. Nicotine acts through nicotinic acetylcholine receptors (nAChRs) to alter firing rate of neurons throughout the brain, leading to addiction-related behaviors. While nAChRs are expressed in the hypothalamus and cholinergic fibers project to this structure, it is unclear how acetylcholine modulates the activity of hypocretin neurons. In this study, we stimulated hypocretin neurons in mouse brain slices with ACh in the presence of atropine to dissect presynaptic and postsynaptic modulation of these neurons through nAChRs. Approximately one-third of tested hypocretin neurons responded to pressure application of ACh (1 mM) with an increase in firing frequency. Stimulation of postsynaptic nAChRs with ACh or nicotine resulted in a highly variable inward current in approximately one-third of hypocretin neurons. In contrast, ACh or nicotine (1 µM) reliably decreased the frequency of miniature EPSCs (mEPSCs). Antagonism of nAChRs with mecamylamine also suppressed mEPSC frequency, suggesting that an endogenous, tonic activation of presynaptic nAChRs might be required for maintaining functional mEPSC frequency. Antagonism of heteromeric (α4β2) or homomeric (α7) nAChRs alone suppressed mEPSCs to a lesser extent. Finally, blocking internal calcium release reduced the frequency of mEPSCs, occluding the suppressive effect of presynaptic ACh. Taken together, these data provide a mechanism by which phasic ACh release enhances the firing of a subset of hypocretin neurons through postsynaptic nAChRs, but disrupts tonic, presynaptic nAChR-mediated glutamatergic inputs to the overall population of hypocretin neurons, potentially enhancing the signal-to-noise ratio during the response of the nAChR-positive subset of neurons. PMID:26322330

  4. Imidacloprid and thiacloprid neonicotinoids bind more favourably to cockroach than to honeybee α6 nicotinic acetylcholine receptor: insights from computational studies.

    PubMed

    Selvam, Balaji; Graton, Jérôme; Laurent, Adèle D; Alamiddine, Zakaria; Mathé-Allainmat, Monique; Lebreton, Jacques; Coqueret, Olivier; Olivier, Christophe; Thany, Steeve H; Le Questel, Jean-Yves

    2015-02-01

    The binding interactions of two neonicotinoids, imidacloprid (IMI) and thiacloprid (THI) with the extracellular domains of cockroach and honeybee α6 nicotinic acetylcholine receptor (nAChR) subunits in an homomeric receptor have been studied through docking and molecular dynamics (MD) simulations. The binding mode predicted for the two neonicotinoids is validated through the good agreement observed between the theoretical results with the crystal structures of the corresponding complexes with Ac-AChBP, the recognized structural surrogate for insects nAChR extracellular ligand binding domain. The binding site of the two insect α6 receptors differs by only one residue of loop D, a serine residue (Ser83) in cockroach being replaced by a lysine residue (Lys108) in honeybee. The docking results show very close interactions for the two neonicotinoids with both α6 nAChR models, in correspondence to the trends observed in the experimental neonicotinoid-Ac-AChBP complexes. However, the docking parameters (scores and energies) are not significantly different between the two insect α6 nAChRs to draw clear conclusions. The MD results bring distinct trends. The analysis of the average interaction energies in the two insects α6 nAChRs shows indeed better affinity of neonicotinoids bound to α6 cockroach compared to honeybee nAChR. This preference is explained by tighter contacts with aromatic residues (Trp and Tyr) of the binding pocket. Interestingly, the non-conserved residue Lys108 of loop D of α6 honeybee nAChR interacts through van der Waals contacts with neonicotinoids, which appear more favourable than the direct or water mediated hydrogen-bond interaction between the OH group of Ser83 of α6 cockroach nAChR and the electronegative terminal group of the two neonicotinoids (nitro in IMI and cyano in THI). Finally, in both insects nAChRs, THI is consistently found to bind more favourably than IMI. PMID:25424654

  5. Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor

    PubMed Central

    Schrage, R; Seemann, WK; Klöckner, J; Dallanoce, C; Racké, K; Kostenis, E; De Amici, M; Holzgrabe, U; Mohr, K

    2013-01-01

    Background and Purpose Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such ‘superagonism’ has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a ‘superagonist’. Experimental Approach Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. Key Results In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi/Gs signalling competence. In the orthosteric loss-of-function mutant M2-Y1043.33A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. ‘Superagonism’ is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure–signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that ‘superagonism’ of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. Conclusion and Implications Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR ‘superagonism’ is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators. Linked Article This article is commented on by Langmead and Christopoulos, pp. 353–356 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12142 PMID:23062057

  6. Cholinergic receptors as target for cancer therapy in a systems medicine perspective.

    PubMed

    Russo, P; Del Bufalo, A; Milic, M; Salinaro, G; Fini, M; Cesario, A

    2014-01-01

    Epithelial cells not innervated by cholinergic neurons express nicotinic and muscarinic acetylcholine (ACh) receptors (nAChR, mAChR). nAChR and mAChR are components of the auto-/paracrine-regulatory loop of non-neuronal ACh release. The cholinergic control of non-neuronal cells may be mediated by different effects (synergistic, additive, or reciprocal) triggered by these receptors. The ionic events (Ca(+2) influx) are generated by the ACh-opening of nAChR channels, while the metabolic events by ACh-binding to G-proteincoupled mAChR. Effective inter- and intracellular signaling is crucial for valuable cancer cells proliferation and survival. Depending on cancer cell type, different AChR have been identified. The proliferation of airways epithelial cancer cells and pancreatic cancer cells may be under the control of α7-nAChR and M3-mAChR, while breast cancer cells and colon cancer cells are regulated by α9-nAChR, and M3-mAChR, respectively. In turn, these receptors may activate different pathways (Ras-Raf-1-Erk-AKT) as well as other receptors (β- adrenergicR). nAChR or mAChR antagonists may inhibit cancer growth. Inhibition of M3 by antisense or antagonists (Darifenacin, Tiotropium) reduces lung or colon cancer proliferation, as well as inhibition of α9- nAChR [polyphenol (-)-epigallocatechin-3-gallate] diminishes breast cancer cells growth. α7-nAChR silencing inhibits lung cancer proliferation. Moreover, inhibition of the nAChR-β-adrenergicR pathway (β-blockers) could be also useful. This review will describe the future translational perspectives of cholinergic receptors druginhibition in a complex disease such as cancer that poses compelling treatment challenges. Cancer happens as consequence of disease-perturbed molecular networks in relevant organ cells that change during progression. The framework for approaching these challenges is a systems approach. PMID:25324001

  7. Habenula cholinergic neurons regulate anxiety during nicotine withdrawal via nicotinic acetylcholine receptors.

    PubMed

    Pang, Xueyan; Liu, Liwang; Ngolab, Jennifer; Zhao-Shea, Rubing; McIntosh, J Michael; Gardner, Paul D; Tapper, Andrew R

    2016-08-01

    Cholinergic neurons in the medial habenula (MHb) modulate anxiety during nicotine withdrawal although the molecular neuroadaptation(s) within the MHb that induce affective behaviors during nicotine cessation is largely unknown. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their behavioral role as autoreceptors in these neurons has not been described. To test the hypothesis that nAChR signaling in MHb cholinergic neurons could modulate anxiety, we expressed novel "gain of function" nAChR subunits selectively in MHb cholinergic neurons of adult mice. Mice expressing these mutant nAChRs exhibited increased anxiety-like behavior that was alleviated by blockade with a nAChR antagonist. To test the hypothesis that anxiety induced by nicotine withdrawal may be mediated by increased MHb nicotinic receptor signaling, we infused nAChR subtype selective antagonists into the MHb of nicotine naïve and withdrawn mice. While antagonists had little effect on nicotine naïve mice, blocking α4β2 or α6β2, but not α3β4 nAChRs in the MHb alleviated anxiety in mice undergoing nicotine withdrawal. Consistent with behavioral results, there was increased functional expression of nAChRs containing the α6 subunit in MHb neurons that also expressed the α4 subunit. Together, these data indicate that MHb cholinergic neurons regulate nicotine withdrawal-induced anxiety via increased signaling through nicotinic receptors containing the α6 subunit and point toward nAChRs in MHb cholinergic neurons as molecular targets for smoking cessation therapeutics. PMID:27020042

  8. Nicotinic Receptors in Neurodegeneration

    PubMed Central

    Posadas, Inmaculada; López-Hernández, Beatriz; Ceña, Valentín

    2013-01-01

    Many studies have focused on expanding our knowledge of the structure and diversity of peripheral and central nicotinic receptors. Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of pentameric ligand-gated ion channels, which include GABA (A and C), serotonin, and glycine receptors. Currently, 9 alpha (α2-α10) and 3 beta (β2-β4) subunits have been identified in the central nervous system (CNS), and these subunits assemble to form a variety of functional nAChRs. The pentameric combination of several alpha and beta subunits leads to a great number of nicotinic receptors that vary in their properties, including their sensitivity to nicotine, permeability to calcium and propensity to desensitize. In the CNS, nAChRs play crucial roles in modulating presynaptic, postsynaptic, and extrasynaptic signaling, and have been found to be involved in a complex range of CNS disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), schizophrenia, Tourette´s syndrome, anxiety, depression and epilepsy. Therefore, there is growing interest in the development of drugs that modulate nAChR functions with optimal benefits and minimal adverse effects. The present review describes the main characteristics of nAChRs in the CNS and focuses on the various compounds that have been tested and are currently in phase I and phase II trials for the treatment of neurodegenerative diseases including PD, AD and age-associated memory and mild cognitive impairment. PMID:24179465

  9. The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies

    PubMed Central

    Muldoon, P P; Jackson, K J; Perez, E; Harenza, J L; Molas, S; Rais, B; Anwar, H; Zaveri, N T; Maldonado, R; Maskos, U; McIntosh, J M; Dierssen, M; Miles, M F; Chen, X; De Biasi, M; Damaj, M I

    2014-01-01

    BACKGROUND AND PURPOSE Recent data have indicated that α3β4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. EXPERIMENTAL APPROACHES To assess the role of α3β4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. KEY RESULTS BXD recombinant mouse lines demonstrated an increased expression of α3, β4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and β4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3β4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. CONCLUSION AND IMPLICATIONS Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence. PMID:24750073

  10. Neuronal Nicotinic Acetylcholine Receptors: Common Molecular Substrates of Nicotine and Alcohol Dependence

    PubMed Central

    Hendrickson, Linzy M.; Guildford, Melissa J.; Tapper, Andrew R.

    2013-01-01

    Alcohol and nicotine are often co-abused. As many as 80–95% of alcoholics are also smokers, suggesting that ethanol and nicotine, the primary addictive component of tobacco smoke, may functionally interact in the central nervous system and/or share a common mechanism of action. While nicotine initiates dependence by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels normally activated by endogenous acetylcholine (ACh), ethanol is much less specific with the ability to modulate multiple gene products including those encoding voltage-gated ion channels, and excitatory/inhibitory neurotransmitter receptors. However, emerging data indicate that ethanol interacts with nAChRs, both directly and indirectly, in the mesocorticolimbic dopaminergic (DAergic) reward circuitry to affect brain reward systems. Like nicotine, ethanol activates DAergic neurons of the ventral tegmental area (VTA) which project to the nucleus accumbens (NAc). Blockade of VTA nAChRs reduces ethanol-mediated activation of DAergic neurons, NAc DA release, consumption, and operant responding for ethanol in rodents. Thus, ethanol may increase ACh release into the VTA driving activation of DAergic neurons through nAChRs. In addition, ethanol potentiates distinct nAChR subtype responses to ACh and nicotine in vitro and in DAergic neurons. The smoking cessation therapeutic and nAChR partial agonist, varenicline, reduces alcohol consumption in heavy drinking smokers and rodent models of alcohol consumption. Finally, single nucleotide polymorphisms in nAChR subunit genes are associated with alcohol dependence phenotypes and smoking behaviors in human populations. Together, results from pre-clinical, clinical, and genetic studies indicate that nAChRs may have an inherent role in the abusive properties of ethanol, as well as in nicotine and alcohol co-dependence. PMID:23641218

  11. The role of the M4 lipid-sensor in the folding, trafficking, and allosteric modulation of nicotinic acetylcholine receptors.

    PubMed

    Hénault, Camille M; Sun, Jiayin; Therien, J P Daniel; daCosta, Corrie J B; Carswell, Casey L; Labriola, Jonathan M; Juranka, Peter F; Baenziger, John E

    2015-09-01

    With the availability of high resolution structural data, increasing attention has focused on the mechanisms by which drugs and endogenous compounds allosterically modulate nicotinic acetylcholine receptor (nAChR) function. Lipids are potent modulators of the nAChR from Torpedo. Membrane lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of pre-existing resting, open, desensitized, and uncoupled conformations, as well as influencing the transitions between these conformational states. Structural and functional data highlight a role for the lipid-exposed M4 transmembrane α-helix of each subunit in lipid sensing, and suggest that lipids influence gating by altering the binding of M4 to the adjacent transmembrane α-helices, M1 and M3. M4 has also been implicated in both the folding and trafficking of nAChRs to the cell surface, as well as in the potentiation of nAChR gating by neurosteroids. Here, we discuss the roles of M4 in the folding, trafficking, and allosteric modulation of nAChRs. We also consider the hypothesis that variable chemistry at the M4-M1/M3 transmembrane α-helical interface in different nAChR subunits governs the capacity for potentiation by activating lipids. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25433148

  12. Deletion of the alpha 7 nicotinic acetylcholine receptor gene improves cognitive deficits and synaptic pathology in a mouse model of Alzheimer's disease.

    PubMed

    Dziewczapolski, Gustavo; Glogowski, Carolina M; Masliah, Eliezer; Heinemann, Stephen F

    2009-07-01

    It has been recently shown that the Alzheimer's disease (AD) pathogenic peptide amyloid beta(1-42) (Abeta(1-42)) binds to the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) with high affinity and the alpha7nAChR and Abeta(1-42) are both found colocalized in neuritic plaques of human brains with AD. Moreover, the intraneuronal accumulation of Abeta(1-42) was shown to be facilitated by its high-affinity binding to the alpha7nAChR, and alpha7nAChR activation mediates Abeta-induced tau protein phosphorylation. To test the hypothesis that alpha7nAChRs are involved in AD pathogenesis, we used a transgenic mouse model of AD overexpressing a mutated form of the human amyloid precursor protein (APP) and lacking the alpha7nAChR gene (APPalpha7KO). We have shown that, despite the presence of high amounts of APP and amyloid deposits, deleting the alpha7nAChR subunit in the mouse model of AD leads to a protection from the dysfunction in synaptic integrity (pathology and plasticity) and learning and memory behavior. Specifically, APPalpha7KO mice express APP and Abeta at levels similar to APP mice, and yet they were able to solve a cognitive challenge such as the Morris water maze test significantly better than APP, with performances comparable to control groups. Moreover, deleting the alpha7nAChR subunit protected the brain from loss of the synaptic markers synaptophysin and MAP2, reduced the gliosis, and preserved the capacity to elicit long-term potentiation otherwise deficient in APP mice. These results are consistent with the hypothesis that the alpha7nAChR plays a role in AD and suggest that interrupting alpha7nAChR function could be beneficial in the treatment of AD. PMID:19587288

  13. Voltage-dependent interaction between the muscarinic ACh receptor and proteins of the exocytic machinery.

    PubMed Central

    Linial, M; Ilouz, N; Parnas, H

    1997-01-01

    1. Release of neurotransmitter into the synaptic cleft is the last step in the chain of molecular events following the arrival of an action potential at the nerve terminal. The neurotransmitter exerts negative feedback on its own release. This inhibition would be most effective if exerted on the first step in this chain of events, i.e. a step that is mediated by membrane depolarization. Indeed, in numerous studies feedback inhibition was found to be voltage dependent. 2. The purpose of this study is to investigate whether the mechanism underlying feedback inhibition of transmitter release resides in interaction between the presynaptic autoreceptors and the exocytic apparatus, specifically the soluble NSF-attachment protein receptor (SNARE) complex. 3. Using rat synaptosomes we show that the muscarinic ACh autoreceptor (mAChR) is an integral component of the exocytic machinery. It interacts with syntaxin, synaptosomal-associated protein of 25 kDa (SNAP-25), vesicle-associated membrane protein (VAMP) and synaptotagmin as shown using both cross-linking and immunoprecipitation. 4. The interaction between mAChRs and both syntaxin and SNAP-25 is modulated by depolarization levels; binding is maximal at resting potential and disassembly occurs at higher depolarization. 5. This voltage-dependent interaction of mAChRs with the secretory core complex appears suitable for controlling the rapid, synchronous neurotransmitter release at nerve terminals. Images Figure 2 Figure 3 PMID:9365901

  14. The open duration of fetal ACh receptor-channel changes during mouse muscle development

    PubMed Central

    Grassi, Francesca; Epifano, Olga; Mileo, Anna Maria; Barabino, Benedetta; Eusebi, Fabrizio

    1998-01-01

    We performed an RNase protection assay on cultured C2C12 mouse myotubes, demonstrating that the γ subunit of the fetal muscle acetylcholine receptor (AChR) exists as two splice variants, which differ in the presence of the amino terminal exon 5. We studied unitary ACh-evoked events in fibres acutely dissociated from the hindlimb flexor digitorum brevis muscle of BALB/C mice aged between embryonic day 16 (E16) and postnatal day 6 (P6). At all ages, the channel conductance was about 30 pS, typical of the fetal form of the AChR. The mean open time increased significantly from 6 ms at E16 to 9 ms at E19, then decreased to about 5 ms during the first postnatal week. The lengthening of the open time was considerably delayed in hypothyroid mice. Data were recorded at 24-26 °C. On the basis of previously reported experiments in heterologous expression systems, we suggest that the modulation of channel open time is related to the expression of the AChR incorporating the γs subunit. These events might be coupled to the crucial modifications in muscle innervation that take place during the same developmental period. PMID:9508804

  15. Transmembrane potential polarization, calcium influx, and receptor conformational state modulate the sensitivity of the imidacloprid-insensitive neuronal insect nicotinic acetylcholine receptor to neonicotinoid insecticides.

    PubMed

    Bodereau-Dubois, Béatrice; List, Olivier; Calas-List, Delphine; Marques, Olivier; Communal, Pierre-Yves; Thany, Steeve H; Lapied, Bruno

    2012-05-01

    Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs). Recent studies revealed that their efficiency was altered by the phosphorylation/dephosphorylation process and the intracellular signaling pathway involved in the regulation of nAChRs. Using whole-cell patch-clamp electrophysiology adapted for dissociated cockroach dorsal unpaired median (DUM) neurons, we demonstrated that intracellular factors involved in the regulation of nAChR function modulated neonicotinoid sensitivity. DUM neurons were known to express two α-bungarotoxin-insensitive nAChR subtypes: nAChR1 and nAChR2. Whereas nAChR1 was sensitive to imidacloprid, nAChR2 was insensitive to this insecticide. Here, we demonstrated that, like nicotine, acetamiprid and clothianidin, other types of neonicotinoid insecticides, acted as agonists on the nAChR2 subtype. Using acetamiprid, we revealed that both steady-state depolarization and hyperpolarization affected nAChR2 sensitivity. The measurement of the input membrane resistance indicated that change in the acetamiprid-induced agonist activity was related to the receptor conformational state. Using cadmium chloride, ω-conotoxin GVIA, and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-acetamide (LOE 908), we found that inhibition of calcium influx through high voltage-activated calcium channels and transient receptor potential γ (TRPγ) activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to acetamiprid. Finally, using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), forskolin, and cAMP, we demonstrated that adenylyl cyclase sensitive to the calcium/calmodulin complex regulated internal cAMP concentration, which in turn modulated TRPγ function and nAChR2 sensitivity to acetamiprid. Similar TRPγ-induced modulatory effects were also obtained when clothianidin was tested. These findings bring insights into the signaling pathway modulating

  16. Mouse muscle denervation increases expression of an α7 nicotinic receptor with unusual pharmacology

    PubMed Central

    Tsuneki, Hiroshi; Salas, Ramiro; Dani, John A

    2003-01-01

    Neuronal nicotinic α7 subunits have been found in chick and rat skeletal muscle during development and denervation. In the present study, reverse transcriptase-polymerase chain reaction was used to detect α7 subunit mRNA in denervated mouse muscle. To determine whether the α7 subunit forms functional nicotinic acetylcholine receptors (nAChRs) in muscle, choline was used to induce a membrane depolarization because choline has been considered a specific agonist of α7-containing (α7*) nAChRs. We found, however, that choline (3–10 mm) also weakly activates muscle nAChRs. After inhibiting muscle nAChRs with a specific muscle nAChR inhibitor, α-conotoxin GI (αCTxGI), choline was used to activate the α7* nAChRs on muscle selectively. Four weeks after denervation, rapid application of choline (10 mm) elicited a substantial depolarization in the presence of αCTxGI (0.1 µm). This component of the depolarization was never present in denervated muscles obtained from mutant mice lacking the α7 subunit (i.e. α7-null mice). The depolarization component that is resistant to αCTxGI was antagonized by pancuronium (3–10 µm) and by a 4-oxystilbene derivative (F3, 0.1–0.5 µm) at concentrations considered highly specific for α7* nAChRs. Another selective α7 antagonist, methyllycaconitine (0.05–5 µm), did not strongly inhibit this choline-induced depolarization. Furthermore, the choline-sensitive nAChRs showed little desensitization over 10 s of application with choline (10–30 mm). These results indicate that functional α7* nAChRs are significantly present on denervated muscle, and that these receptors display unusual functional and pharmacological characteristics. PMID:12562921

  17. Nicotine Dependence Reveals Distinct Responses from Neurons and Their Resident Nicotinic Receptors in Medial Habenula.

    PubMed

    Shih, Pei-Yu; McIntosh, J Michael; Drenan, Ryan M

    2015-12-01

    Nicotinic acetylcholine receptors (nAChRs) are the molecular target of nicotine. nAChRs in the medial habenula (MHb) have recently been shown to play a role in nicotine dependence, but it is not clear which nAChR subtypes or MHb neuron types are most important. To identify MHb nAChRs and/or cell types that play a role in nicotine dependence, we studied these receptors and cells with brain slice electrophysiology using both acute and chronic nicotine application. Cells in the ventroinferior (MHbVI) and ventrolateral MHb (MHbVL) subregions expressed functional nAChRs with different pharmacology. Further, application of nicotine to cells in these subregions led to different action potential firing patterns. The latter result was correlated with a differing ability of nicotine to induce nAChR desensitization. Chronic nicotine caused functional upregulation of nAChRs selectively in MHbVI cells, but did not change nAChR function in MHbVL. Importantly, firing responses were also differentially altered in these subregions following chronic nicotine. MHbVI neurons treated chronically with nicotine exhibited enhanced basal pacemaker firing but a blunted nicotine-induced firing response. MHbVL neurons did not change their firing properties in response to chronic nicotine. Together, these results suggest that acute and chronic nicotine differentially affect nAChR function and output of cells in MHb subregions. Because the MHb extensively innervates the interpeduncular nucleus, an area critical for both affective and somatic signs of withdrawal, these results could reflect some of the neurophysiological changes thought to occur in the MHb to the interpeduncular nucleus circuit in human smokers. PMID:26429939

  18. Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

    PubMed

    Mousavi, M; Hellström-Lindahl, E; Guan, Z Z; Bednar, I; Nordberg, A

    2001-12-21

    Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent. PMID:11811902

  19. α-Bungarotoxin Binding to Acetylcholine Receptor Membranes Studied by Low Angle X-Ray Diffraction

    PubMed Central

    Young, Howard S.; Herbette, Leo G.; Skita, Victor

    2003-01-01

    The nicotinic acetylcholine receptor (nAChR) carries two binding sites for snake venom neurotoxins. α-Bungarotoxin from the Southeast Asian banded krait, Bungarus multicinctus, is a long neurotoxin which competitively blocks the nAChR at the acetylcholine binding sites in a relatively irreversible manner. Low angle x-ray diffraction was used to generate electron density profile structures at 14-Å resolution for Torpedo californica nAChR membranes in the absence and presence of α-bungarotoxin. Analysis of the lamellar diffraction data indicated a 452-Å lattice spacing between stacked nAChR membrane pairs. In the presence of α-bungarotoxin, the quality of the diffraction data and the lamellar lattice spacing were unchanged. In the plane of the membrane, the nAChRs packed together with a nearest neighbor distance of 80 Å, and this distance increased to 85 Å in the presence of toxin. Electron density profile structures were calculated in the absence and presence of α-bungarotoxin, revealing a location for the toxin binding sites. In native, fully-hydrated nAChR membranes, α-bungarotoxin binds to the nAChR outer vestibule and contacts the surface of the membrane bilayer. PMID:12885641

  20. Requisite Role of the Cholinergic α7 Nicotinic Acetylcholine Receptor Pathway in Suppressing Gram-Negative Sepsis-Induced Acute Lung Inflammatory Injury

    PubMed Central

    Su, Xiao; Matthay, Michael A.; Malik, Asrar B.

    2010-01-01

    Although activation of the α7 nicotinic acetylcholine receptor (α7 nAChR) modulates the response to sepsis, the role of this pathway in the development of sepsis-induced acute lung injury (ALI) is not known. In this study, we addressed the contribution of α7 nAChR in mediating endotoxin- and live Escherichia coli–induced ALI in mice. Because we found that α7 nAChR+ alveolar macrophages and neutrophils were present in bronchoalveolar lavage and injured lungs of mice, we tested whether acetylcholine released by lung vagal innervation stimulated these effector cells and thereby down-regulated proinflammatory chemokine/cytokine generation. Administration of α7 nAChR agonists reduced bronchoalveolar lavage MIP-2 production and transalveolar neutrophil migration and reduced mortality in E. coli pneumonia mice, whereas vagal denervation increased MIP-2 production and airway neutrophil accumulation and increased mortality. In addition, α7 nAChR−/− mice developed severe lung injury and had higher mortality compared with α7 nAChR+/+ mice. The immunomodulatory cholinergic α7 nAChR pathway of alveolar macrophages and neutrophils blocked LPS- and E. coli–induced ALI by reducing chemokine production and transalveolar neutrophil migration, suggesting that activation of α7 nAChR may be a promising strategy for treatment of sepsis-induced ALI. PMID:19949071

  1. N-Benzylpiperidine Derivatives as α7 Nicotinic Receptor Antagonists.

    PubMed

    Criado, Manuel; Mulet, José; Sala, Francisco; Sala, Salvador; Colmena, Inés; Gandía, Luis; Bautista-Aguilera, Oscar M; Samadi, Abdelouahid; Chioua, Mourad; Marco-Contelles, José

    2016-08-17

    A series of multitarget directed propargylamines, as well as other differently susbstituted piperidines have been screened as potential modulators of neuronal nicotinic acetylcholine receptors (nAChRs). Most of them showed antagonist actions on α7 nAChRs. Especially, compounds 13, 26, and 38 displayed submicromolar IC50 values on homomeric α7 nAChRs, whereas they were less effective on heteromeric α3β4 and α4β2 nAChRs (up to 20-fold higher IC50 values in the case of 13). Antagonism was concentration dependent and noncompetitive, suggesting that these compounds behave as negative allosteric modulators of nAChRs. Upon the study of a series of less complex derivatives, the N-benzylpiperidine motif, common to these compounds, was found to be the main pharmacophoric group. Thus, 2-(1-benzylpiperidin-4-yl)-ethylamine (48) showed an inhibitory potency comparable to the one of the previous compounds and also a clear preference for α7 nAChRs. In a neuroblastoma cell line, representative compounds 13 and 48 also inhibited, in a concentration-dependent manner, cytosolic Ca(2+) signals mediated by nAChRs. Finally, compounds 38 and 13 inhibited 5-HT3A serotonin receptors whereas they had no effect on α1 glycine receptors. Given the multifactorial nature of many pathologies in which nAChRs are involved, these piperidine antagonists could have a therapeutic potential in cases where cholinergic activity has to be negatively modulated. PMID:27254782

  2. [125I]AT-1012, a New High Affinity Radioligand for the α3β4 Nicotinic Acetylcholine Receptors

    PubMed Central

    Wu, Jinhua; Perry, David C.; Bupp, James E.; Jiang, Faming; Polgar, Willma E.; Toll, Lawrence; Zaveri, Nurulain T.

    2013-01-01

    Recent genetic and pharmacological studies have implicated the α3, β4 and a5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [125I]-radiolabeled analog of a high affinity, selective small-molecule α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [125I]AT-1012 was characterized at the rat α3β4- and α4β2 nAChR transfected into HEK cells as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [125I]AT-1012 at the rat α3β4 nAChR was 1.4 nM, with a Bmax of 10.3 pmol/mg protein, similar to what was determined using [3H]epibatidine. Saturation isotherms suggested that [125I]AT-1012 binds to a single site on the α3β4 nAChR. Similar high binding affinity was also observed for [125I]AT-1012 at human α3β4α5 nAChR in a human α3β4aα nAChR transfected cell line. [125I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells, and [3H]epibatidine binding studies showed that AT-1012 had over 100-fold binding selectivity for the α3β4 over α4β2 nAChR. Ki values determined for known nAChR compounds using [125I]AT-1012 as radioligand were comparable to those obtained with [3H]epibatidine. [125I]AT-1012 was also used to label the α3β4 nAChR in rat brain slices in vitro using autoradiography which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3β4 nAChR. We

  3. Expression and Functional Role of α7 Nicotinic Receptor in Human Cytokine-stimulated Natural Killer (NK) Cells.

    PubMed

    Zanetti, Samanta R; Ziblat, Andrea; Torres, Nicolás I; Zwirner, Norberto W; Bouzat, Cecilia

    2016-08-01

    The homomeric α7 nicotinic receptor (nAChR) is one of the most abundant nAChRs in the central nervous system where it contributes to cognition, attention, and working memory. α7 nAChR is also present in lymphocytes, dendritic cells (DCs), and macrophages and it is emerging as an important drug target for intervention in inflammation and sepsis. Natural killer (NK) cells display cytotoxic activity against susceptible target cells and modulate innate and adaptive immune responses through their interaction with DCs. We here show that human NK cells also express α7 nAChR. α7 nAChR mRNA is detected by RT-PCR and cell surface expression of α7 nAChR is detected by confocal microscopy and flow cytometry using α-bungarotoxin, a specific antagonist. Both mRNA and protein levels increase during NK stimulation with cytokines (IL-12, IL-18, and IL-15). Exposure of cytokine-stimulated NK cells to PNU-282987, a specific α7 nAChR agonist, increases intracellular calcium concentration ([Ca(2+)]i) mainly released from intracellular stores, indicating that α7 nAChR is functional. Moreover, its activation by PNU-282987 plus a specific positive allosteric modulator greatly enhances the Ca(2+) responses in NK cells. Stimulation of NK cells with cytokines and PNU-282987 decreases NF-κB levels and nuclear mobilization, down-regulates NKG2D receptors, and decreases NKG2D-dependent cell-mediated cytotoxicity and IFN-γ production. Also, such NK cells are less efficient to trigger DC maturation. Thus, our results demonstrate the anti-inflammatory role of α7 nAChR in NK cells and suggest that modulation of its activity in these cells may constitute a novel target for regulation of the immune response. PMID:27284006

  4. Orthosteric and Allosteric Ligands of Nicotinic Acetylcholine Receptors for Smoking Cessation

    PubMed Central

    Mohamed, Tasnim S.; Jayakar, Selwyn S.; Hamouda, Ayman K.

    2015-01-01

    Nicotine addiction, the result of tobacco use, leads to over six million premature deaths world-wide per year, a number that is expected to increase by a third within the next two decades. While more than half of smokers want and attempt to quit, only a small percentage of smokers are able to quit without pharmacological interventions. Therefore, over the past decades, researchers in academia and the pharmaceutical industry have focused their attention on the development of more effective smoking cessation therapies, which is now a growing 1.9 billion dollar market. Because the role of neuronal nicotinic acetylcholine receptors (nAChR) in nicotine addiction is well established, nAChR based therapeutics remain the leading strategy for smoking cessation. However, the development of neuronal nAChR drugs that are selective for a nAChR subpopulation is challenging, and only few neuronal nAChR drugs are clinically available. Among the many neuronal nAChR subtypes that have been identified in the brain, the α4β2 subtype is the most abundant and plays a critical role in nicotine addiction. Here, we review the role of neuronal nAChRs, especially the α4β2 subtype, in the development and treatment of nicotine addiction. We also compare available smoking cessation medications and other nAChR orthosteric and allosteric ligands that have been developed with emphasis on the difficulties faced in the development of clinically useful compounds with high nAChR subtype selectivity. PMID:26635524

  5. Lynx1 and Aβ1-42 bind competitively to multiple nicotinic acetylcholine receptor subtypes.

    PubMed

    Thomsen, Morten S; Arvaniti, Maria; Jensen, Majbrit M; Shulepko, Mikhail A; Dolgikh, Dmitry A; Pinborg, Lars H; Härtig, Wolfgang; Lyukmanova, Ekaterina N; Mikkelsen, Jens D

    2016-10-01

    Lynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification to demonstrate that a water-soluble variant of human Lynx1 (Ws-Lynx1) isolates α3, α4, α5, α6, α7, β2, and β4 nAChR subunits from human and rat cortical extracts, and rat midbrain and olfactory bulb extracts, suggesting that Lynx1 forms complexes with multiple nAChR subtypes in the human and rodent brain. Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal-regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4β2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4β2 nAChR subtypes. We further show that Lynx1 and oligomeric β-amyloid1-42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents β-amyloid1-42-induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant β-amyloid and tau pathology. Our data suggest that Lynx1 binds to multiple nAChR subtypes in the brain and that this interaction might have functional and pathophysiological implications in relation to Alzheimer's disease. PMID:27460145

  6. Nicotinic receptor modulation to treat alcohol and drug dependence

    PubMed Central

    Rahman, Shafiqur; Engleman, Eric A.; Bell, Richard L.

    2015-01-01

    Alcohol and drug dependence are serious public health problems worldwide. The prevalence of alcohol and drug dependence in the United States and other parts of the world is significant. Given the limitations in the efficacy of current pharmacotherapies to treat these disorders, research in developing alternative pharmacotherapies continues. Preclinical and clinical evidence thus far has indicated that brain nicotinic acetylcholine receptors (nAChRs) are important pharmacological targets for the development of medications to treat alcohol and drug dependence. The nAChRs are a super family of ligand gated ion channels, and are expressed throughout the brain with twelve neuronal nAChR subunits (α2–α10 and β2–β4) identified. Here, we review preclinical and clinical evidence involving a number of nAChR ligands that target different nAChR subtypes in alcohol and nicotine addiction. The important ligands include cytisine, lobeline, mecamylamine, varenicline, sazetidine A and others that target α4β2* nAChR subtypes as small molecule modulators of the brain nicotinic cholinergic system are also discussed. Taken together, both preclinical and clinical data exist that support nAChR–based ligands as promising therapeutic agents for the treatment of alcohol and drug dependence. PMID:25642160

  7. The α3β4* nicotinic acetylcholine receptor subtype mediates nicotine reward and physical nicotine withdrawal signs independently of the α5 subunit in the mouse

    PubMed Central

    Jackson, Kia J.; Sanjakdar, Sarah S.; Muldoon, Pretal P.; McIntosh, J. Michael; Damaj, M. Imad

    2013-01-01

    The 15q25 gene cluster contains genes that code for the α5, α3, and β4 nicotinic acetylcholine receptor (nAChRs) subunits, and in human genetic studies, has shown the most robust association with smoking behavior and nicotine dependence to date. The limited available animal studies implicate a role for the α5 and β4 nAChR subunits in nicotine dependence and withdrawal; however studies focusing on the behavioral role of the α3β4* nAChR receptor subtype in nicotine dependence are lacking. Because of the apparent role of the α3β4* nAChR subtype in nicotine dependence, the goal of the current study was to better evaluate the involvement of this subtype in nicotine mediated behavioral responses. Using the selective α3β4* nAChR antagonist, α-conotoxin AuIB, we assessed the role of α3β4* nAChRs in acute nicotine, nicotine reward, and physical and affective nicotine withdrawal. Because α5 has also been implicated in nicotine dependence behaviors in mice and can form functional receptors with α3β4*, we also evaluated the role of the α3β4α5* nAChR subtype in nicotine reward and somatic nicotine withdrawal signs by blocking the α3β4* nAChR subtype in α5 nAChR knockout mice with AuIB. AuIB had no significant effect on acute nicotine behaviors, but dose-dependently attenuated nicotine reward and physical withdrawal signs, with no significant effect in affective withdrawal measures. Interestingly, AuIB also attenuated nicotine reward and somatic signs in α5 nAChR knockout mice. This study shows that α3β4* nAChRs mediate nicotine reward and physical nicotine withdrawal, but not acute nicotine behaviors or affective nicotine withdrawal signs in mice. The α5 subunit is not required in the receptor assembly to mediate these effects. Our findings suggest an important role for the α3β4* nAChR subtype in nicotine reward and physical aspects of the nicotine withdrawal syndrome. PMID:23416040

  8. Atypical α-Conotoxin LtIA from Conus litteratus Targets a Novel Microsite of the α3β2 Nicotinic Receptor*

    PubMed Central

    Luo, Sulan; Akondi, Kalyana Bharati; Zhangsun, Dongting; Wu, Yong; Zhu, Xiaopeng; Hu, Yuanyan; Christensen, Sean; Dowell, Cheryl; Daly, Norelle L.; Craik, David J.; Wang, Ching-I. Anderson; Lewis, Richard J.; Alewood, Paul F.; Michael McIntosh, J.

    2010-01-01

    Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. α-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of α-conotoxins may serve as a platform for development of new therapies. Previously characterized α-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel α-conotoxin LtIA, which lacks this highly conserved motif but potently blocked α3β2 nAChRs with a 9.8 nm IC50 value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing α-conotoxin MII. Nevertheless, pre-block of α3β2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR β subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for α3β2 versus α3β4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC50 value on α3β2(F119Q) versus wild type nAChRs, whereas T59K and V111I β2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the α3β2 nAChR that includes β2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes. PMID:20145249

  9. Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

    PubMed Central

    Cheng, Hao; Fan, Chen; Zhang, Si-wei; Wu, Zhong-shan; Cui, Zhi-cheng; Melcher, Karsten; Zhang, Cheng-hai; Jiang, Yi; Cong, Yao; Xu, H Eric

    2015-01-01

    Aim: To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target. Methods: α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis. Results: Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel. Conclusion: We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis. PMID:26073323

  10. Nicotinic α7 receptors enhance NMDA cognitive circuits in dorsolateral prefrontal cortex

    PubMed Central

    Yang, Yang; Paspalas, Constantinos D.; Jin, Lu E.; Picciotto, Marina R.; Arnsten, Amy F. T.; Wang, Min

    2013-01-01

    The cognitive function of the highly evolved dorsolateral prefrontal cortex (dlPFC) is greatly influenced by arousal state, and is gravely afflicted in disorders such as schizophrenia, where there are genetic insults in α7 nicotinic acetylcholine receptors (α7-nAChRs). A recent behavioral study indicates that ACh depletion from dlPFC markedly impairs working memory [Croxson PL, Kyriazis DA, Baxter MG (2011) Nat Neurosci 14(12):1510–1512]; however, little is known about how α7-nAChRs influence dlPFC cognitive circuits. Goldman-Rakic [Goldman-Rakic (1995) Neuron 14(3):477–485] discovered the circuit basis for working memory, whereby dlPFC pyramidal cells excite each other through glutamatergic NMDA receptor synapses to generate persistent network firing in the absence of sensory stimulation. Here we explore α7-nAChR localization and actions in primate dlPFC and find that they are enriched in glutamate network synapses, where they are essential for dlPFC persistent firing, with permissive effects on NMDA receptor actions. Blockade of α7-nAChRs markedly reduced, whereas low-dose stimulation selectively enhanced, neuronal representations of visual space. These findings in dlPFC contrast with the primary visual cortex, where nAChR blockade had no effect on neuronal firing [Herrero JL, et al. (2008) Nature 454(7208):1110–1114]. We additionally show that α7-nAChR stimulation is needed for NMDA actions, suggesting that it is key for the engagement of dlPFC circuits. As ACh is released in cortex during waking but not during deep sleep, these findings may explain how ACh shapes differing mental states during wakefulness vs. sleep. The results also explain why genetic insults to α7-nAChR would profoundly disrupt cognitive experience in patients with schizophrenia. PMID:23818597

  11. Nicotinic receptor involvement in regulation of functions of mouse neutrophils from inflammatory site.

    PubMed

    Safronova, Valentina G; Vulfius, Catherine A; Shelukhina, Irina V; Mal'tseva, Valentina N; Berezhnov, Alexey V; Fedotova, Eugeniya I; Miftahova, Regina G; Kryukova, Elena V; Grinevich, Andrey A; Tsetlin, Victor I

    2016-07-01

    Participation of nicotinic acetylcholine receptors (nAChRs) in functioning of polymorphonuclear neutrophils (PMNs) isolated from inflammatory site of mice and expression of different nAChR subunits were studied. Nicotine and acetylcholine (ACh) modified respiratory burst induced by a chemotactic peptide N-formyl-MLF in neutrophils of male (but not female) mice. Antagonists of nAChRs α-cobratoxin (αCTX), α-conotoxins MII and [A10L]PnIA at concentrations of 0.01-5μM, 0.2μM and 1μM, respectively, eliminated nAChR agonist effects. ACh also affected adhesion of PMNs, this effect was also prevented by αCTX (100nM) and MII (1nM). Neutrophils of female mice after chronic nicotine consumption acquired sensitivity to nAChR agonists. Changes of free intracellular Ca(2+) concentration in neutrophils under the action of nAChR ligands were analyzed. In cells with no Ca(2+) oscillations and relatively low resting level of intracellular Ca(2+), nicotine triggered Ca(2+)-spikes, the lag of the response shortened with increasing nicotine concentration. A nicotinic antagonist caramiphen strongly decreased the effect of nicotine. RT-PCR analysis revealed mRNAs of α2, α3, α4, α5, α6, α7, α9, β2, β3, and β4 nAChR subunits. Specific binding of [(125)I]-α-bungarotoxin was demonstrated. Thus in view of the effects and binding characteristics the results obtained suggest a regulatory role of α7, α3β2 or α6* nAChR types in specific functions of PMNs. PMID:26965141

  12. Otilonium: a potent blocker of neuronal nicotinic ACh receptors in bovine chromaffin cells.

    PubMed Central

    Gandía, L.; Villarroya, M.; Lara, B.; Olmos, V.; Gilabert, J. A.; López, M. G.; Martínez-Sierra, R.; Borges, R.; García, A. G.

    1996-01-01

    1. Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide-spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells. 2. 45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 microM. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 microM). 3. Whole-cell Ba2+ currents (IBa) through Ca2+ channels of voltage-clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 microM, very close to that of K(+)-evoked 45Ca2+ uptake. Blockade developed in 10-20 s, almost as a single step and was rapidly and almost fully reversible. 4. Whole-cell nicotinic AChR-mediated currents (250 ms pulses of 100 microM DMPP) applied at 30 s intervals were blocked by otilonium in a concentration-dependent manner, showing an IC50 of 0.36 microM. Blockade was induced in a step-wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps. 5. In experiments with recordings in the same cells of whole-cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 microM otilonium blocked 87% IDMPP, 7% INa and 13% ICa. 6. Otilonium inhibited the K(+)-evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 microM, very close to the IC50 for blockade of K(+)-induced 45Ca2+ uptake and IBa. 7. Otilonium inhibited the secretory responses induced by 10 s pulses of 50 microM DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP-evoked responses with an IC50 of 29.8 microM, 4,000 fold higher than that of otilonium. 8. In conclusion, otilonium is a potent blocker of nicotinic AChR-mediated responses. The drugs also blocked various subtypes of neuronal voltage-dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by

  13. A study of brain insulin receptors, AChE activity and oxidative stress in rat model of ICV STZ induced dementia.

    PubMed

    Agrawal, Rahul; Tyagi, Ethika; Shukla, Rakesh; Nath, Chandishwar

    2009-03-01

    In the present study, role of brain insulin receptors (IRs) in memory functions and its correlation with acetylcholinesterase (AChE) activity and oxidative stress in different brain regions were investigated in intracerebroventricular (ICV) streptozotocin (STZ) induced dementia model. Rats were treated with STZ (3 mg/kg, ICV) on day 1 and 3. Donepezil (5 mg/kg po) and melatonin (20 mg/kg ip) were administered in pre- and post-treatment schedules. Morris water maze test was done on day 14 and animals were sacrificed on day 21 from 1st STZ injection. Memory deficit was found in STZ group as indicated by no significant decrease in latency time antagonized by donepezil and melatonin. IR protein level was found significantly increased in trained group as compared to control, whereas STZ decreased IR level significantly as compared to trained rats in hippocampus which indicates that IR is associated with memory functions. STZ induced decrease in IR was reversed by melatonin but not by donepezil. Melatonin per se did not show any significant change in IR level as compared to control. AChE activity (DS and SS fraction) was found to be increased in hippocampus in STZ group as compared to trained which was inhibited by donepezil and melatonin. Increase in MDA level and decrease in GSH level were obtained in STZ group indicating oxidative stress, which was attenuated by donepezil and melatonin. Effectiveness of antioxidant, melatonin but not of anti-cholinesterase, donepezil against STZ induced changes in IR indicates that IR is more affected with oxidative stress than cholinergic changes. PMID:19705549

  14. Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

    SciTech Connect

    Ma, Xiaoli; Jia, Yanfei; Zu, Shanshan; Li, Ruisheng; Jia, Ying; Zhao, Yun; Xiao, Dongjie; Dang, Ningning; Wang, Yunshan

    2014-07-15

    By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels of α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P < 0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer. - Highlights

  15. Nicotinic acetylcholine receptor-mediated responses in medial vestibular and prepositus hypoglossi nuclei neurons showing distinct neurotransmitter phenotypes.

    PubMed

    Zhang, Yue; Yanagawa, Yuchio; Saito, Yasuhiko

    2016-06-01

    Cholinergic transmission in both the medial vestibular nucleus (MVN) and prepositus hypoglossi nucleus (PHN) plays an important role in horizontal eye movements. We previously demonstrated that the current responses mediated via nicotinic acetylcholine receptors (nAChRs) were larger than those mediated via muscarinic acetylcholine receptors (mAChRs) in cholinergic MVN and PHN neurons that project to the cerebellum. In this study, to clarify the predominant nAChR responses and the expression patterns of nAChRs in MVN and PHN neurons that exhibit distinct neurotransmitter phenotypes, we identified cholinergic, inhibitory, and glutamatergic neurons using specific transgenic rats and investigated current responses to the application of acetylcholine (ACh) using whole cell recordings in brain stem slices. ACh application induced larger nAChR-mediated currents than mAChR-mediated currents in every neuronal phenotype. In the presence of an mAChR antagonist, we found three types of nAChR-mediated currents that exhibited different rise and decay times and designated these as fast (F)-, slow (S)-, and fast and slow (FS)-type currents. F-type currents were the predominant response in inhibitory MVN neurons, whereas S-type currents were observed in the majority of glutamatergic MVN and PHN neurons. No dominant response type was observed in cholinergic neurons. Pharmacological analyses revealed that the F-, S-, and FS-type currents were mainly mediated by α7, non-α7, and both α7 and non-α7 nAChRs, respectively. These findings suggest that cholinergic responses in the major neuronal populations of the MVN and PHN are predominantly mediated by nAChRs and that the expression of α7 and non-α7 nAChRs differ among the neuronal phenotypes. PMID:26936981

  16. Coantagonism of Glutamate Receptors and Nicotinic Acetylcholinergic Receptors Disrupts Fear Conditioning and Latent Inhibition of Fear Conditioning

    ERIC Educational Resources Information Center

    Gould, Thomas J.; Lewis, Michael C.

    2005-01-01

    The present study investigated the hypothesis that both nicotinic acetylcholinergic receptors (nAChRs) and glutamate receptors ([alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) and N-methyl-D-aspartate glutamate receptors (NMDARs)) are involved in fear conditioning, and may modulate similar processes. The effects of the…

  17. Nicotinic receptors in addiction pathways.

    PubMed

    Leslie, Frances M; Mojica, Celina Y; Reynaga, Daisy D

    2013-04-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that consist of pentameric combinations of α and β subunits. These receptors are widely distributed throughout the brain and are highly expressed in addiction circuitry. The role of nAChRs in regulating neuronal activity and motivated behavior is complex and varies both in and among brain regions. The rich diversity of central nAChRs has hampered the characterization of their structure and function with use of classic pharmacological techniques. However, recent molecular approaches using null mutant mice with specific regional lentiviral re-expression, in combination with neuroanatomical and electrophysiological techniques, have allowed the elucidation of the influence of different nAChR types on neuronal circuit activity and behavior. This review will address the influence of nAChRs on limbic dopamine circuitry and the medial habenula-interpeduncular nucleus complex, which are critical mediators of reinforced behavior. Characterization of the mechanisms underlying regulation of addiction pathways by endogenous cholinergic transmission and by nicotine may lead to the identification of new therapeutic targets for treating tobacco dependence and other addictions. PMID:23247824

  18. α4α6β2* Nicotinic Acetylcholine Receptor Activation on Ventral Tegmental Area Dopamine Neurons Is Sufficient to Stimulate a Depolarizing Conductance and Enhance Surface AMPA Receptor Function

    PubMed Central

    Engle, Staci E.; Shih, Pei-Yu; McIntosh, J. Michael

    2013-01-01

    Tobacco addiction is a serious threat to public health in the United States and abroad, and development of new therapeutic approaches is a major priority. Nicotine activates and/or desensitizes nicotinic acetylcholine receptors (nAChRs) throughout the brain. nAChRs in ventral tegmental area (VTA) dopamine (DA) neurons are crucial for the rewarding and reinforcing properties of nicotine in rodents, suggesting that they may be key mediators of nicotine’s action in humans. However, it is unknown which nAChR subtypes are sufficient to activate these neurons. To test the hypothesis that nAChRs containing α6 subunits are sufficient to activate VTA DA neurons, we studied mice expressing hypersensitive, gain-of-function α6 nAChRs (α6L9′S mice). In voltage-clamp recordings in brain slices from adult mice, 100 nM nicotine was sufficient to elicit inward currents in VTA DA neurons via α6β2* nAChRs. In addition, we found that low concentrations of nicotine could act selectively through α6β2* nAChRs to enhance the function of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) receptors on the surface of these cells. In contrast, α6β2* activation did not enhance N-methyl-d-aspartic acid receptor function. Finally, AMPA receptor (AMPAR) function was not similarly enhanced in brain slices from α6L9′S mice lacking α4 nAChR subunits, suggesting that α4α6β2* nAChRs are important for enhancing AMPAR function in VTA DA neurons. Together, these data suggest that activation of α4α6β2* nAChRs in VTA DA neurons is sufficient to support the initiation of cellular changes that play a role in addiction to nicotine. α4α6β2* nAChRs may be a promising target for future smoking cessation pharmacotherapy. PMID:23788655

  19. Nicotine Effects in Adolescence and Adulthood on Cognition and α4β2-Nicotinic Receptors in the Neonatal Ventral Hippocampal Lesion Rat Model of Schizophrenia

    PubMed Central

    Berg, Sarah A.; Sentir, Alena M.; Bell, Richard L.; Engleman, Eric A.; Chambers, R. Andrew

    2014-01-01

    Rational Nicotine use in schizophrenia has traditionally been explained as ‘self-medication’ of cognitive and/or nicotinic acetylcholinergic receptor (nAChR) abnormalities. Objectives We test this hypothesis in a neurodevelopmental rat model of schizophrenia that shows increased addiction behaviors including enhanced nicotine reinforcement and drug-seeking. Methods Nicotine transdermal patch (5 mg/kg/day vs. placebo × 10 days in adolescence or adulthood) effects on subsequent radial-arm maze learning (15 sessions) and frontal-cortical-striatal nAChR densities (α4β2; [3H]-epibatidine binding) were examined in neonatal ventral hippocampal lesion (NVHL) and SHAM-operated rats. Results NVHL cognitive deficits were not differentially affected by nicotine history compared to SHAMs. Nicotine history produced minimal cognitive effects while increasing food–reward consumption on the maze, compounding with NVHL-induced overconsumption. Acute nicotine (0.5 mg/kg) delivered before the final maze sessions produced modest improvements in maze performance in rats with nicotine patch histories only, but not differentially so in NVHLs. Consistent with in vivo neuroimaging of β2 nAChR binding in schizophrenia smokers vs. non-smokers and healthy controls, adult NVHLs showed 12% reductions in nAChR binding in MPFC (p<0.05) but not ventral striatum (<5% changes, p>.40), whereas nicotine history elevated nAChRs across both regions (>30%, p<0.001) without interacting with NVHLs. Adolescent vs. adult nicotine exposure did not alter nAChRs differentially. Conclusions Although replicating nicotine-induced up-regulation of nAChRs in human smokers and demonstrating NVHL validity in terms of schizophrenia-associated nAChR density patterns, these findings do not support hypotheses explaining increased nicotine use in schizophrenia as reflecting illness-specific effects of nicotine to therapeutically alter cognition or nAChR densities. PMID:25388292

  20. The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

    PubMed Central

    2012-01-01

    Background Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. Results A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular

  1. Nicotinic Acetylcholine Receptors in the Pathophysiology of Al zheimer's Disease: The Role of Protein-Protein Interactions in Current and Future Treatment.

    PubMed

    Thomsen, Morten Skøtt; Andreasen, Jesper Tobias; Arvaniti, Maria; Kohlmeier, Kristi Anne

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) have been pursued for decades as potential molecular targets to treat cognitive dysfunction in Alzheimer's disease (AD) due to their positioning within regions of the brain critical in learning and memory, such as the prefrontal cortex and hippocampus, and their demonstrated role in processes underlying cognition such as synaptic facilitation, and theta and gamma wave activity. Historically, activity at these receptors is facilitated in AD by use of drugs that increase the levels of their endogenous agonist acetylcholine, and more recently nAChR selective ligands have undergone clinical trials. Here we discuss recent findings suggesting that the expression and function of nAChRs in AD may be regulated by direct interactions with specific proteins, including Lynx proteins, NMDA-receptors and the Wnt/β-catenin pathway, as well as β-amyloid. The ability of protein interactions to modify nAChR function adds a new level of complexity to cholinergic signaling in the brain that may be specifically altered in AD. It is currently not known to what degree current nAChR ligands affect these interactions, and it is possible that the difference in the clinical effect of nAChR ligands in AD is related to differences in their ability to modulate nAChR protein interactions, rather than their effects on ion flow through the receptors. Drugs designed to target these interactions may thus provide a new avenue for drug development to ameliorate cognitive symptoms in AD. Notably, the development of experimental drugs that specifically modulate these interactions may provide the opportunity to selectively affect those aspects of nAChR function that are affected in AD. PMID:26818866

  2. Rational design of alpha-conotoxin analogues targeting alpha7 nicotinic acetylcholine receptors: improved antagonistic activity by incorporation of proline derivatives.

    PubMed

    Armishaw, Christopher; Jensen, Anders A; Balle, Thomas; Clark, Richard J; Harpsøe, Kasper; Skonberg, Christian; Liljefors, Tommy; Strømgaard, Kristian

    2009-04-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that belong to the superfamily of Cys loop receptors. Valuable insight into the orthosteric ligand binding to nAChRs in recent years has been obtained from the crystal structures of acetylcholine-binding proteins (AChBPs) that share significant sequence homology with the amino-terminal domains of the nAChRs. alpha-Conotoxins, which are isolated from the venom of carnivorous marine snails, selectively inhibit the signaling of neuronal nAChR subtypes. Co-crystal structures of alpha-conotoxins in complex with AChBP show that the side chain of a highly conserved proline residue in these toxins is oriented toward the hydrophobic binding pocket in the AChBP but does not have direct interactions with this pocket. In this study, we have designed and synthesized analogues of alpha-conotoxins ImI and PnIA[A10L], by introducing a range of substituents on the Pro(6) residue in these toxins to probe the importance of this residue for their binding to the nAChRs. Pharmacological characterization of the toxin analogues at the alpha(7) nAChR shows that although polar and charged groups on Pro(6) result in analogues with significantly reduced antagonistic activities, analogues with aromatic and hydrophobic substituents in the Pro(6) position exhibit moderate activity at the receptor. Interestingly, introduction of a 5-(R)-phenyl substituent at Pro(6) in alpha-conotoxin ImI gives rise to a conotoxin analogue with a significantly higher binding affinity and antagonistic activity at the alpha(7) nAChR than those exhibited by the native conotoxin. PMID:19131337

  3. A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Jin, Zhuang; Norleans, Jack; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-11-27

    Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs. PMID:26432642

  4. Identifying the binding site of novel methyllycaconitine (MLA) analogs at α4β2 nicotinic acetylcholine receptors.

    PubMed

    Quek, Gracia X J; Lin, Diana; Halliday, Jill I; Absalom, Nathan; Ambrus, Joseph I; Thompson, Andrew J; Lochner, Martin; Lummis, Sarah C R; McLeod, Malcolm D; Chebib, Mary

    2010-12-15

    Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the α7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat α7, α4β2, and α3β4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC(50)) within an IC(50) range of 2.3-26.6 μM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the α7 nAChR. At α4β2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of α4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of α4β2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6' and 13' in a state-dependent manner, but the reactive probe only bonded with 13' in the open state. The data suggest that the 13' position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (α4)(3)(β2)(2) homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker. PMID:22778816

  5. The multiple roles of the α7 nicotinic acetylcholine receptor in modulating glutamatergic systems in the normal and diseased nervous system.

    PubMed

    Koukouli, Fani; Maskos, Uwe

    2015-10-15

    Neuronal nicotinic acetylcholine receptors (nAChRs) play an important role in a variety of modulatory and regulatory processes including neurotransmitter release and synaptic transmission in various brain regions of the central nervous system (CNS). Glutamate is the principal excitatory neurotransmitter in the brain and the glutamatergic system participates in the pathophysiology of several neuropsychiatric disorders. Underpinning the importance of nAChRs, many studies demonstrated that nAChRs containing the α7 subunit facilitate glutamate release. Here, we review the currently available body of experimental evidence pertaining to α7 subunit containing nAChRs in their contribution to the modulation of glutamatergic neurotransmission, and we highlight the role of α7 in synaptic plasticity, the morphological and functional maturation of the glutamatergic system and therefore its important contribution in the modulation of neural circuits of the CNS. PMID:26206184

  6. Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors.

    PubMed

    Ton, Hoai T; Smart, Amanda E; Aguilar, Brittany L; Olson, Thao T; Kellar, Kenneth J; Ahern, Gerard P

    2015-08-01

    The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action. PMID:25964258

  7. Drug binding to the acetylcholine receptor: Nitroxide analogs of phencyclidine and a local anesthetic

    SciTech Connect

    Palma, A.L.

    1988-01-01

    The interaction of noncompetitive inhibitors (NCIs) with Torpedo californica native nicotinic acetylcholine receptor (nAChR) membranes was examined primarily by the technique of electron paramagnetic resonance (EPR) spectroscopy. The goal of this work being to define some of the physical characteristics for the site(s) of association between an NCI and the nAChR membrane. A nitroxide labeled analog of a quaternary amine local anesthetic, 2-(N,N-dimethyl-N-4-(2,2,6,6-tetramethylpiperidinoxyl)amino)-ethyl 4-hexyloxybenzoate iodide (C6SLMeI), displays a strongly immobilized EPR component when added to nAChR membranes in the presence of carbamylcholine (carb). To further this work, a nitroxide labeled analog of phencyclidine (PCP), a potent NCI, was synthesized. 4-phenyl-4-(1-piperidinyl)-2,2,6,6-tetramethylpiperidinoxyl (PPT) exhibited one-third the potency of PCP in inhibiting nAChR mediated ion flux, and from competition binding studies with ({sup 3}H)PCP displayed a K{sub D} of 0.21 {mu}M towards a carb desensitized nAChR and a K{sub 0.5} of 18 {mu}M for a resting {alpha}-bungarotoxin treated nAChR.

  8. Serotoninergic dorsal raphe neurons possess functional postsynaptic nicotinic acetylcholine receptors.

    PubMed

    Galindo-Charles, Luis; Hernandez-Lopez, Salvador; Galarraga, Elvira; Tapia, Dagoberto; Bargas, José; Garduño, Julieta; Frías-Dominguez, Carmen; Drucker-Colin, René; Mihailescu, Stefan

    2008-08-01

    Very few neurons in the telencephalon have been shown to express functional postsynaptic nicotinic acetylcholine receptors (nAChRs), among them, the noradrenergic and dopaminergic neurons. However, there is no evidence for postsynaptic nAChRs on serotonergic neurons. In this study, we asked if functional nAChRs are present in serotonergic (5-HT) and nonserotonergic (non-5-HT) neurons of the dorsal raphe nucleus (DRN). In rat midbrain slices, field stimulation at the tegmental pedunculopontine (PPT) nucleus evoked postsynaptic currents (eEPSCs) with different components in DRN neurons. After blocking the glutamatergic and GABAergic components, the remaining eEPSCs were blocked by mecamylamine and reduced by either the selective alpha7 nAChR antagonist methyllycaconitine (MLA) or the selective alpha4beta2 nAChR antagonist dihydro-beta-eritroidine (DHbetaE). Simultaneous addition of MLA and DHbetaE blocked all eEPSCs. Integrity of the PPT-DRN pathway was assessed by both anterograde biocytin tracing and antidromic stimulation from the DRN. Inward currents evoked by the direct application of acetylcholine (ACh), in the presence of atropine and tetrodotoxin, consisted of two kinetically different currents: one was blocked by MLA and the other by DHbetaE; in both 5-HT and non-5-HT DR neurons. Analysis of spontaneous (sEPSCs) and evoked (eEPSCs) synaptic events led to the conclusion that nAChRs were located at the postsynaptic membrane. The possible implications of these newly described nAChRs in various physiological processes and behavioral events, such as the wake-sleep cycle, are discussed. PMID:18512214

  9. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    PubMed Central

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs. PMID:27485575

  10. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    NASA Astrophysics Data System (ADS)

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-08-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

  11. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors.

    PubMed

    Lyukmanova, E N; Shulepko, M A; Shenkarev, Z O; Bychkov, M L; Paramonov, A S; Chugunov, A O; Kulbatskii, D S; Arvaniti, M; Dolejsi, Eva; Schaer, T; Arseniev, A S; Efremov, R G; Thomsen, M S; Dolezal, V; Bertrand, D; Dolgikh, D A; Kirpichnikov, M P

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs. PMID:27485575

  12. Evaluation of the Nicotinic Acetylcholine Receptor-Associated Proteome at Baseline and Following Nicotine Exposure in Human and Mouse Cortex.

    PubMed

    McClure-Begley, Tristan D; Esterlis, Irina; Stone, Kathryn L; Lam, TuKiet T; Grady, Sharon R; Colangelo, Christopher M; Lindstrom, Jon M; Marks, Michael J; Picciotto, Marina R

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein-protein interactions of high-affinity nAChRs containing the β2 subunit (β2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the β2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets. PMID:27559543

  13. Evaluation of the Nicotinic Acetylcholine Receptor-Associated Proteome at Baseline and Following Nicotine Exposure in Human and Mouse Cortex

    PubMed Central

    Esterlis, Irina; Stone, Kathryn L.; Grady, Sharon R.; Lindstrom, Jon M.; Marks, Michael J.

    2016-01-01

    Abstract Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein–protein interactions of high-affinity nAChRs containing the β2 subunit (β2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the β2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets. PMID:27559543

  14. Activation of α7 Nicotinic Acetylcholine Receptor Decreases On-site Mortality in Crush Syndrome through Insulin Signaling-Na/K-ATPase Pathway

    PubMed Central

    Fan, Bo-Shi; Zhang, En-Hui; Wu, Miao; Guo, Jin-Min; Su, Ding-Feng; Liu, Xia; Yu, Jian-Guang

    2016-01-01

    On-site mortality in crush syndrome remains high due to lack of effective drugs based on definite diagnosis. Anisodamine (Ani) is widely used in China for treatment of shock, and activation of α7 nicotinic acetylcholine receptor (α7nAChR) mediates such antishock effect. The present work was designed to test whether activation of α7nAChR with Ani decreased mortality in crush syndrome shortly after decompression. Sprague-Dawley rats and C57BL/6 mice with crush syndrome were injected with Ani (20 mg/kg and 28 mg/kg respectively, i.p.) 30 min before decompression. Survival time, serum potassium, insulin, and glucose levels were observed shortly after decompression. Involvement of α7nAChR was verified with methyllycaconitine (selective α7nAChR antagonist) and PNU282987 (selective α7nAChR agonist), or in α7nAChR knockout mice. Effect of Ani was also appraised in C2C12 myotubes. Ani reduced mortality and serum potassium and enhanced insulin sensitivity shortly after decompression in animals with crush syndrome, and PNU282987 exerted similar effects. Such effects were counteracted by methyllycaconitine or in α7nAChR knockout mice. Mortality and serum potassium in rats with hyperkalemia were also reduced by Ani. Phosphorylation of Na/K-ATPase was enhanced by Ani in C2C12 myotubes. Inhibition of tyrosine kinase on insulin receptor, phosphoinositide 3-kinase, mammalian target of rapamycin, signal transducer and activator of transcription 3, and Na/K-ATPase counteracted the effect of Ani on extracellular potassium. These findings demonstrated that activation of α7nAChR could decrease on-site mortality in crush syndrome, at least in part based on the decline of serum potassium through insulin signaling-Na/K-ATPase pathway. PMID:27065867

  15. Diversity of native nicotinic receptor subtypes in mammalian brain.

    PubMed

    Zoli, Michele; Pistillo, Francesco; Gotti, Cecilia

    2015-09-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are a heterogeneous family of pentameric ligand-gated cation channels that are expressed throughout the brain and involved in a wide range of physiological and pathophysiological processes. The nAChR subtypes share a common basic structure, but their biophysical and pharmacological properties depend on their subunit composition, which is therefore central to understanding their function in the nervous system and discovering new subtype selective drugs. The development of new technologies and the generation of mice carrying deletions or the expression of gain-of-function nAChR subunits, or GFP-tagged receptor genes has allowed the in vivo identification of complex subtypes and to study the role of individual subtypes in specific cells and complex neurobiological systems but much less is known about which native nAChR subtypes are involved in specific physiological functions and pathophysiological conditions in human brain. We briefly review some recent findings concerning the structure and function of native nAChRs, focussing on the subtypes identified in the rodent habenulo-interpeduncular pathway, a pathway involved in nicotine reinforcement and withdrawal. We also discuss recent findings concerning the expression of native subtypes in primate brain. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25460185

  16. Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Tochitsky, Ivan; Banghart, Matthew R.; Mourot, Alexandre; Yao, Jennifer Z.; Gaub, Benjamin; Kramer, Richard H.; Trauner, Dirk

    2012-02-01

    Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3β4 and α4β2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.

  17. Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

    PubMed Central

    Tochitsky, Ivan; Banghart, Matthew R.; Mourot, Alexandre; Yao, Jennifer Z.; Gaub, Benjamin

    2016-01-01

    Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3β4 and α4β2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels. PMID:22270644

  18. Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum.

    PubMed

    Nakajima, Hidemitsu; Goto, Hiroto; Azuma, Yasu-Taka; Fujita, Akikazu; Takeuchi, Tadayoshi

    2007-12-01

    The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca2+-activated K+ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the alpha3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the alpha3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs. PMID:17953675

  19. cGMP/cGMP-dependent protein kinase pathway modulates nicotine-induced currents through the activation of α-bungarotoxin-insensitive nicotinic acetylcholine receptors from insect neurosecretory cells.

    PubMed

    Mannai, Safa; Bitri, Lofti; Thany, Steeve H

    2016-06-01

    Insect neurosecretory cells, called dorsal unpaired median neurons, are known to express two α-bungarotoxin-insensitive nicotinic acetylcholine receptor (nAChR) subtypes, nAChR1 and nAChR2. It was demonstrated that nAChR1 was sensitive to cAMP/cAMP-dependent protein kinase (PKA) regulation, resulting in a modulation of nicotine currents. In this study, we show that cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway modulates nicotine-induced currents, as increased cGMP affects the second compound of the biphasic current-voltage curve, corresponding to the nAChR2 receptors. Indeed, maintaining the guanosine triphosphate level with 100 μM guanosine triphosphate-γ-S increased nicotine currents through nAChR2. We also demonstrated that inhibition of PKG activity with 0.2 μM (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one (KT5823), a PKG specific inhibitor, reduced nicotine-induced current amplitudes. KT5823 effect on nicotine currents is associated with calcium (Ca(2+) ) activity because inhibition of Ca(2+) concentration with cadmium chloride (CdCl2 ) abolished KT5823-induced inhibition mediated by nAChR2. However, specific inhibition of nitric oxide-guanylyl cyclase (GC) complex by 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) significantly increased nicotine-induced current amplitudes on both nAChR1 and nAChR2. These results suggest that nicotine-induced currents mediated by both α-bungarotoxin-insensitive nAChR1 and nAChR2 are coupled to the cGMP/PKG pathway. We propose that nicotinic acetylcholine receptor activation induces an increase in intracellular calcium (Ca(2+) ) concentration. Elevation of intracellular Ca(2+) results in the formation of Ca(2+) -calmodulin (CaM) complex, which activates guanylyl cyclase (GC) and/or adenylyl cyclase (AC). Ca(2+) -CaM complex could activate Ca(2+) calmodulin kinase II which

  20. A Case Report of Congenital Fiber Type Disproportion with an Increased Level of Anti-ACh Receptor Antibodies.

    PubMed

    Kimura, Shigemi; Ozasa, Shiro; Nomura, Keiko; Kosuge, Hirofumi; Yoshioka, Kowasi

    2013-01-01

    Congenital fiber type disproportion (CFTD) is a form of congenital myopathy, which is defined by type 1 myofibers that are 12% smaller than type 2 myofibers, as well as a general predominance of type 1 myofibers. Conversely, myasthenia gravis (MG) is an acquired immune-mediated disease, in which the acetylcholine receptor (AChR) of the neuromuscular junction is blocked by antibodies. Thus, the anti-AChR antibody is nearly specific to MG. Herein, we report on a case of CFTD with increased anti-AChR antibody levels. A 23-month-old boy exhibited muscle hypotonia and weakness. Although he could walk by himself, he easily fell down and could not control his head for a long time. His blood test was positive for the anti-AChR antibody, while a muscle biopsy revealed characteristics of CFTD. We could not explain the relationship between MG and CFTD. However, we considered different diagnoses aside from MG, even when the patient's blood is positive for the anti-AChR antibody. PMID:23762716

  1. Selective actions of Lynx proteins on different nicotinic acetylcholine receptors in the locust, Locusta migratoria manilensis.

    PubMed

    Wang, Xin; Bao, Haibo; Sun, Huahua; Zhang, Yixi; Fang, Jichao; Liu, Qinghong; Liu, Zewen

    2015-08-01

    Nicotinic acetylcholine receptors (nAChRs) are major neurotransmitter receptors and targets of neonicotinoid insecticides in the insect nervous system. The full function of nAChRs is often dependent on associated proteins, such as chaperones, regulators and modulators. Here, three Lynx (Ly-6/neurotoxin) proteins, Loc-lynx1, Loc-lynx2 and Loc-lynx3, were identified in the locust, Locusta migratoria manilensis. Co-expression with Lynx resulted in a dramatic increase in agonist-evoked macroscopic currents on nAChRs Locα1/β2 and Locα2/β2 in Xenopus oocytes, but no changes in agonist sensitivity. Loc-lynx1 and Loc-lynx3 only modulated nAChRs Locα1/β2 while Loc-lynx2 modulated Locα2/β2 specifically. Meanwhile, Loc-lynx1 induced a more significant increase in currents evoked by imidacloprid and epibatidine than Loc-lynx3, and the effects of Loc-lynx1 on imidacloprid and epibatidine were significantly higher than those on acetylcholine. Among three lynx proteins, only Loc-lynx1 significantly increased [(3) H]epibatidine binding on Locα1/β2. The results indicated that Loc-lynx1 had different modulation patterns in nAChRs compared to Loc-lynx2 and Loc-lynx3. Taken together, these findings indicated that three Lynx proteins were nAChR modulators and had selective activities in different nAChRs. Lynx proteins might display their selectivities from three aspects: nAChR subtypes, various agonists and different modulation patterns. Insect Lynx (Ly-6/neurotoxin) proteins act as the allosteric modulators on insect nicotinic acetylcholine receptors (nAChRs), the important targets of insecticides. We found that insect lynx proteins showed their selectivities from at least three aspects: nAChR subtypes, various agonists and different modulation patterns. PMID:25951893

  2. Activation of α2A-Containing Nicotinic Acetylcholine Receptors Mediates Nicotine-Induced Motor Output in Embryonic Zebrafish

    PubMed Central

    Menelaou, Evdokia; Udvadia, Ava J.; Tanguay, Robert L.; Svoboda, Kurt R.

    2014-01-01

    It is well established that cholinergic signaling has critical roles during central nervous system development. In physiological and behavioral studies, activation of nicotinic acetylcholine receptors has been implicated in mediating cholinergic signaling. In developing spinal cord, cholinergic transmission is associated with neural circuits responsible for producing locomotor behaviors. In this study, we investigated the expression pattern of the α2A nAChR subunit as evidence from others suggested it could be expressed by spinal neurons. In situ hybridization and immunohistochemistry revealed that the α2A nAChR subunits are expressed in spinal Rohon-Beard (RB) neurons and olfactory sensory neurons in young embryos. In order to examine the functional role of the α2A nAChR subunit during embryogenesis, we blocked its expression using antisense modified oligonucleotides. Blocking the expression of α2A nAChR subunits had no effect on spontaneous motor activity. However, it did alter the embryonic nicotine-induced motor output. This reduction in motor activity was not accompanied by defects in neuronal and muscle elements associated with the motor output. Moreover, the anatomy and functionality of RB neurons was normal even in the absence of the α2A nAChR subunit. Thus, we propose that α2A-containing nAChR are dispensable for normal RB development. However, in the context of nicotine-induced motor output, α2A-containing nAChRs on RB neurons provide the substrate that nicotine acts upon to induce the motor output. These findings also indicate that functional neuronal nAChRs are present within spinal cord at the time when locomotor output in zebrafish first begins to manifest itself. PMID:24738729

  3. Functional Upregulation of α4* Nicotinic Acetylcholine Receptors in VTA GABAergic Neurons Increases Sensitivity to Nicotine Reward

    PubMed Central

    Ngolab, Jennifer; Liu, Liwang; Zhao-Shea, Rubing; Gao, Guangping; Gardner, Paul D.

    2015-01-01

    Chronic nicotine exposure increases sensitivity to nicotine reward during a withdrawal period, which may facilitate relapse in abstinent smokers, yet the molecular neuroadaptation(s) that contribute to this phenomenon are unknown. Interestingly, chronic nicotine use induces functional upregulation of nicotinic acetylcholine receptors (nAChRs) in the mesocorticolimbic reward pathway potentially linking upregulation to increased drug sensitivity. In the ventral tegmental area (VTA), functional upregulation of nAChRs containing the α4 subunit (α4* nAChRs) is restricted to GABAergic neurons. To test the hypothesis that increased functional expression of α4* nAChRs in these neurons modulates nicotine reward behaviors, we engineered a Cre recombinase-dependent gene expression system to selectively express α4 nAChR subunits harboring a “gain-of-function” mutation [a leucine mutated to a serine residue at the 9′ position (Leu9′Ser)] in VTA GABAergic neurons of adult mice. In mice expressing Leu9′Ser α4 nAChR subunits in VTA GABAergic neurons (Gad2VTA:Leu9′Ser mice), subreward threshold doses of nicotine were sufficient to selectively activate VTA GABAergic neurons and elicit acute hypolocomotion, with subsequent nicotine exposures eliciting tolerance to this effect, compared to control animals. In the conditioned place preference procedure, nicotine was sufficient to condition a significant place preference in Gad2VTA:Leu9′Ser mice at low nicotine doses that failed to condition control animals. Together, these data indicate that functional upregulation of α4* nAChRs in VTA GABAergic neurons confers increased sensitivity to nicotine reward and points to nAChR subtypes specifically expressed in GABAergic VTA neurons as molecular targets for smoking cessation therapeutics. PMID:26041923

  4. Propofol and AZD3043 Inhibit Adult Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

    PubMed Central

    Jonsson Fagerlund, Malin; Krupp, Johannes; Dabrowski, Michael A.

    2016-01-01

    Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs) has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1β1δε, α3β2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1β1δε, α3β2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses. PMID:26861354

  5. Role of the nicotinic acetylcholine receptor in Alzheimer's disease pathology and treatment.

    PubMed

    Lombardo, Sylvia; Maskos, Uwe

    2015-09-01

    Alzheimer's Disease (AD) is the major form of senile dementia, characterized by neuronal loss, extracellular deposits, and neurofibrillary tangles. It is accompanied by a loss of cholinergic tone, and acetylcholine (ACh) levels in the brain, which were hypothesized to be responsible for the cognitive decline observed in AD. Current medication is restricted to enhancing cholinergic signalling for symptomatic treatment of AD patients. The nicotinic acetylcholine receptor family (nAChR) and the muscarinic acetylcholine receptor family (mAChR) are the target of ACh in the brain. Both families of receptors are affected in AD. It was demonstrated that amyloid beta (Aβ) interacts with nAChRs. Here we discuss how Aβ activates or inhibits nAChRs, and how this interaction contributes to AD pathology. We will discuss the potential role of nAChRs as therapeutic targets. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25514383

  6. Effect of nicotinic acetylcholine receptor alpha 1 (nAChRα1) peptides on rabies virus infection in neuronal cells.

    PubMed

    Sajjanar, Basavaraj; Saxena, Shikha; Bisht, Deepika; Singh, Arvind Kumar; Manjunatha Reddy, G B; Singh, Rajendra; Singh, R P; Kumar, Satish

    2016-06-01

    Rabies virus (RABV) is neurotropic and causes acute progressive encephalitis. Herein, we report the interaction of nAChRα1-subunit peptides with RABV and the effect of these peptides on RABV infection in cultured neuronal cells. Peptide sequences derived from torpedo, bovine, human and rats were synthesized and studied for their interactions with RABV using virus capture ELISA and peptide immunofluorescence. The results showed specific binding of the nAChRα1-subunit peptides to the RABV. In the virus adsorption assay, these peptides were found to inhibit the attachment of the RABV to the neuronal cells. The nAChRα1-subunit peptides inhibited the RABV infection and reduced viral gene expression in the cultured neuroblastoma (N2A) cells. Torpedo peptide sequence (T-32) had highest antiviral effect (IC50=14±3.01μM) compared to the other peptides studied. The results of the study indicated that nAChRα1-subunit peptides may act as receptor decoy molecules and inhibit the binding of virus to the native host cell receptors and hence may reduce viral infection. PMID:26656837

  7. A pharmacodynamic comparison of piperidine and pyridine alkaloid actions at fetal muscle-type nicotinic acetylcholine receptors (nAChR)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Piperidine and pyridine alkaloids are found in many species of plants including Lobelia spp., Conium spp., Nicotiana spp., and Lupinus spp. Some of these alkaloids cause multiple congenital contracture deformities (MCC) and cleft palates in cattle, pigs, sheep, and goats. The mechanism behind MCC ...

  8. Nicotinic Acetylcholine Receptors containing the α6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons

    PubMed Central

    Liu, Liwang; Zhao-Shea, Rubing; McIntosh, J. Michael; Tapper, Andrew R.

    2013-01-01

    Nicotine and alcohol are often co-abused suggesting a common mechanism of action may underlie their reinforcing properties. Both drugs acutely increase activity of ventral tegmental area (VTA) dopaminergic (DAergic) neurons, a phenomenon associated with reward behavior. Recent evidence indicates that nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels activated by ACh and nicotine, may contribute to ethanol-mediated activation of VTA DAergic neurons although the nAChR subtype(s) involved has not been fully elucidated. Here we show that expression and activation of nAChRs containing the α6 subunit contribute to ethanol-induced activation of VTA DAergic neurons. In wild-type (WT) mouse midbrain sections that contain the VTA, ethanol (50 or 100 mM) significantly increased firing frequency of DAergic neurons. In contrast, ethanol did not significantly increase activity of VTA DAergic neurons in mice that do not express CHRNA6, the gene encoding the α6 nAChR subunit (α6 knock-out (KO) mice). Ethanol-induced activity in WT slices was also reduced by pre-application of the α6 subtype-selective nAChR antagonist, α-conotoxin MII[E11A]. When co-applied, ethanol potentiated the response to ACh in WT DAergic neurons; whereas co-application of ACh and ethanol failed to significantly increase activity of DAergic neurons in α6 KO slices. Finally, pre-application of α-conotoxin MII[E11A] in WT slices reduced ethanol potentiation of ACh responses. Together our data indicate that α6-subunit containing nAChRs may contribute to ethanol activation of VTA DAergic neurons. These receptors are predominantly expressed in DAergic neurons and known to be critical for nicotine reinforcement, providing a potential common therapeutic molecular target to reduce nicotine and alcohol co-abuse. PMID:23811312

  9. Genetic knockout of the α7 nicotinic acetylcholine receptor gene alters hippocampal long-term potentiation in a background strain-dependent manner.

    PubMed

    Freund, Ronald K; Graw, Sharon; Choo, Kevin S; Stevens, Karen E; Leonard, Sherry; Dell'Acqua, Mark L

    2016-08-01

    Reduced α7 nicotinic acetylcholine receptor (nAChR) function is linked to impaired hippocampal-dependent sensory processing and learning and memory in schizophrenia. While knockout of the Chrna7 gene encoding the α7nAChR on a C57/Bl6 background results in changes in cognitive measures, prior studies found little impact on hippocampal synaptic plasticity in these mice. However, schizophrenia is a multi-genic disorder where complex interactions between specific genetic mutations and overall genetic background may play a prominent role in determining phenotypic penetrance. Thus, we compared the consequences of knocking out the α7nAChR on synaptic plasticity in C57/Bl6 and C3H mice, which differ in their basal α7nAChR expression levels. Homozygous α7 deletion in C3H mice, which normally express higher α7nAChR levels, resulted in impaired long-term potentiation (LTP) at hippocampal CA1 synapses, while C3H α7 heterozygous mice maintained robust LTP. In contrast, homozygous α7 deletion in C57 mice, which normally express lower α7nAChR levels, did not alter LTP, as had been previously reported for this strain. Thus, the threshold of Chrna7 expression required for LTP may be different in the two strains. Measurements of auditory gating, a hippocampal-dependent behavioral paradigm used to identify schizophrenia-associated sensory processing deficits, was abnormal in C3H α7 knockout mice confirming that auditory gating also requires α7nAChR expression. Our studies highlight the importance of genetic background on the regulation of synaptic plasticity and could be relevant for understanding genetic and cognitive heterogeneity in human studies of α7nAChR dysfunction in mental disorders. PMID:27233215

  10. Nicotinic Receptor Antagonists as Treatments for Nicotine Abuse

    PubMed Central

    Crooks, Peter A.; Bardo, Michael T.; Dwoskin, Linda P.

    2014-01-01

    Despite the proven efficacy of current pharmacotherapies for tobacco dependence, relapse rates continue to be high, indicating that novel medications are needed. Currently, several smoking cessation agents are available, including varenicline (Chantix®), bupropion (Zyban®), and cytisine (Tabex®). Varenicline and cytisine are partial agonists at the α4β2* nicotinic acetylcholine receptor (nAChR). Bupropion is an antidepressant but is also an antagonist at α3β2* ganglionic nAChRs. The rewarding effects of nicotine are mediated, in part, by nicotine-evoked dopamine (DA) release leading to sensitization, which is associated with repeated nicotine administration and nicotine addiction. Receptor antagonists that selectivity target central nAChR subtypes mediating nicotine-evoked DA release should have efficacy as tobacco use cessation agents with the therapeutic advantage of a limited side-effect profile. While α-conotoxin MII (α-CtxMII)-insensitive nAChRs (e.g., α4β2*) contribute to nicotine-evoked DA release, these nAChRs are widely distributed in the brain, and inhibition of these receptors may lead to nonselective and untoward effects. In contrast, α-CtxMII-sensitive nAChRs mediating nicotine-evoked DA release offer an advantage as targets for smoking cessation, due to their more restricted localization primarily to dopaminergic neurons. Small drug-like molecules that are selective antagonists at α-CtxMII-sensitive nAChR subtypes that contain α6 and β2 subunits have now been identified. Early research identified a variety of quaternary ammonium analogs that were potent and selective antagonists at nAChRs mediating nicotine-evoked DA release. More recent data have shown that novel, non-quaternary bis-1,2,5,6-tetrahydropyridine analogs potently inhibit (IC50<1 nM) nicotine-evoked DA release in vitro by acting as antagonists at α-CtxMII-sensitive nAChR subtypes; these compounds also decrease NIC self-administration in rats. PMID:24484986

  11. Conformational mobility of immobilized alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nicotinic acetylcholine receptors.

    PubMed

    Moaddel, Ruin; Jozwiak, Krzysztof; Whittington, Kevin; Wainer, Irving W

    2005-02-01

    Four affinity chromatography stationary phases have been developed based upon immobilized nicotinic acetylcholine receptor (nAChR) subtypes, the alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nAChRs. The stationary phases were created using membranes from cell lines expressing the subtypes and an immobilized artificial membrane stationary phase. The immobilized nAChRs were characterized using frontal chromatography with the agonist epibatidine as the marker. The observed binding affinities for the agonists epibatidine, nicotine, and cytisine were consistent with reported values, indicating that the nAChRs retained their ability to bind agonists. The noncompetitive inhibitors (NCIs) of the nAChR (R)- and (S)-mecamylamine, phencylcidine, dextromethoprphan, and levomethorphan were also chromatographed on the columns using nonlinear chromatography techniques. The studies were carried out before and after exposure of the columns to epibatidine. The NCI retention times increased after exposure to epibtatidine as did the enantioselective separation of mecamylamine and methorphan. The results indicate that the immobilized nAChRs retained their ability to undergo agonist-induced conformational change from the resting to the desensitized states. The columns provide a unique ability to study the interactions of NCIs with both of these conformational states. PMID:15679359

  12. The Neurotoxic Effect of 13,19-Didesmethyl and 13-Desmethyl Spirolide C Phycotoxins Is Mainly Mediated by Nicotinic Rather Than Muscarinic Acetylcholine Receptors.

    PubMed

    Aráoz, Romulo; Ouanounou, Gilles; Iorga, Bogdan I; Goudet, Amélie; Alili, Doria; Amar, Muriel; Benoit, Evelyne; Molgó, Jordi; Servent, Denis

    2015-09-01

    Spirolides are a large family of lipophilic marine toxins produced by dinoflagellates that have been detected in contaminated shellfish. Among them, 13,19-didesmethyl and 13-desmethyl spirolide C phycotoxins are widely distributed and their mode of action needs to be clearly defined. In order to further characterize the pharmacological profiles of these phycotoxins on various nicotinic acetylcholine receptor (nAChR) subtypes and to examine whether they act on muscarinic receptors (mAChRs), functional electrophysiological studies and competition binding experiments have been performed. While 13-desmethyl spirolide C interacted efficiently with sub-nanomolar affinities and low selectivity with muscular and neuronal nAChRs, 13,19-didesmethyl spirolide C was more selective of muscular and homopentameric α7 receptors and recognized only weakly neuronal heteropentameric receptors, especially the α4β2 subtype. Thus, the presence of an additional methyl group on the tetrahydropyran ring significantly modified the pharmacological profile of 13-desmethyl spirolide C by notably increasing its affinity on certain neuronal nAChRs. Structural explanations of this selectivity difference are proposed, based on molecular docking experiments modeling different spirolide-receptor complexes. In addition, the 2 spirolides interacted only with low micromolar affinities with the 5 mAChRs, highlighting that the toxicity of the spirolide C analogs is mainly due to their high inhibition potency on various peripheral and central nAChRs and not to their low ability to interact with mAChR subtypes. PMID:26063663

  13. 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

  14. Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

    PubMed Central

    Krause, R M; Hamann, M; Bader, C R; Liu, J H; Baroffio, A; Bernheim, L

    1995-01-01

    1. Fusion of myogenic cells is important for muscle growth and repair. The aim of this study was to examine the possible involvement of nicotinic acetylcholine receptors (nAChR) in the fusion process of myoblasts derived from postnatal human satellite cells. 2. Acetylcholine-activated currents (ACh currents) were characterized in pure preparations of freshly isolated satellite cells, proliferating myoblasts, myoblasts triggered to fuse and myotubes, using whole-cell and single-channel voltage clamp recordings. Also, the effect of cholinergic agonists on myoblast fusion was tested. 3. No nAChR were observed in freshly isolated satellite cells. nAChR were first observed in proliferating myoblasts, but ACh current densities increased markedly only just before fusion. At that time most mononucleated myoblasts had ACh current densities similar to those of myotubes. ACh channels had similar properties at all stages of myoblast maturation. 4. The fraction of myoblasts that did not fuse under fusion-promoting conditions had no ACh current and thus resembled freshly isolated satellite cells. 5. The rate of myoblast fusion was increased by carbachol, an effect antagonized by alpha-bungarotoxin, curare and decamethonium, but not by atropine, indicating that nAChR were involved. Even though a prolonged exposure to carbachol led to desensitization, a residual ACh current persisted after several days of exposure to the nicotinic agonist. 6. Our observations suggest that nAChR play a role in myoblast fusion and that part of this role is mediated by the flow of ions through open ACh channels. Images Figure 1 Figure 2 Figure 3 PMID:8788942

  15. Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus☆

    PubMed Central

    Lees, Kristin; Jones, Andrew K.; Matsuda, Kazuhiko; Akamatsu, Miki; Sattelle, David B.; Woods, Debra J.; Bowman, Alan S.

    2014-01-01

    Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect α1 nAChR group and has been named Rsanα1. Rsanα1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsanα1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken β2 nAChR, Rsanα1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 μM) and choline (100 μM). Rsanα1/β2 was insensitive to both imidacloprid (100 μM) and spinosad (100 μM). The unreliable expression of Rsanα1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides. PMID:24291321

  16. Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids.

    PubMed

    Green, Benedict T; Lee, Stephen T; Welch, Kevin D; Cook, Daniel; Kem, William R

    2016-01-01

    Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic. PMID:27384586

  17. Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids

    PubMed Central

    Green, Benedict T.; Lee, Stephen T.; Welch, Kevin D.; Cook, Daniel; Kem, William R.

    2016-01-01

    Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic. PMID:27384586

  18. Water-soluble LYNX1 Residues Important for Interaction with Muscle-type and/or Neuronal Nicotinic Receptors*

    PubMed Central

    Lyukmanova, Ekaterina N.; Shulepko, Mikhail A.; Buldakova, Svetlana L.; Kasheverov, Igor E.; Shenkarev, Zakhar O.; Reshetnikov, Roman V.; Filkin, Sergey Y.; Kudryavtsev, Denis S.; Ojomoko, Lucy O.; Kryukova, Elena V.; Dolgikh, Dmitry A.; Kirpichnikov, Mikhail P.; Bregestovski, Piotr D.; Tsetlin, Victor I.

    2013-01-01

    Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618–10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its “non-classical” binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and

  19. Evaluation of Ca2+ permeability of nicotinic acetylcholine receptors in hypothalamic histaminergic neurons

    PubMed Central

    Uteshev, Victor V.

    2010-01-01

    Hypothalamic histaminergic tuberomammillary (TM) neurons express nicotinic acetylcholine receptors (nAChRs) with kinetic and pharmacological properties resembling those of highly Ca2+ permeable α7 nAChRs. However, the Ca2+ permeability of TM nAChR channels has not been determined. To directly evaluate the Ca2+ permeability of TM nAChRs, patch-clamp recordings were conducted using non-cultured acutely dissociated TM neurons and external solutions containing low (2 mM) and high (20 mM) concentrations of Ca2+. A shift in the reversal potentials was determined from the current–voltage relationships and the permeability ratio, PCa/PNa, was estimated within the Goldman-Hodgkin-Katz constant field approximation. TM nAChRs were found to be highly Ca2+ permeable with the permeability ratio, PCa/PNa(nAChR) being ∼5.9 and the fractional Ca2+ current, Pf(nAChR) being ∼10.1% at −60 mV. As a positive control for the applied methods and analysis, the permeability ratio, PCa/PNa(NMDAR) being ∼8.3 and the fractional Ca2+ current, Pf(NMDAR) being ∼13.6% at −60 mV for NMDA receptors were determined using non-cultured acutely dissociated hippocampal pyramidal neurons and found similar to previously reported values. Therefore, these results demonstrate that native TM nAChRs are highly Ca2+ permeable, but ∼1.4 fold less permeable to Ca2+ than native hippocampal pyramidal NMDA receptors. PMID:20043042

  20. Phosphocholine – an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors

    PubMed Central

    Richter, K.; Mathes, V.; Fronius, M.; Althaus, M.; Hecker, A.; Krasteva-Christ, G.; Padberg, W.; Hone, A. J.; McIntosh, J. M.; Zakrzewicz, A.; Grau, V.

    2016-01-01

    We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions. PMID:27349288

  1. Phosphocholine - an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors.

    PubMed

    Richter, K; Mathes, V; Fronius, M; Althaus, M; Hecker, A; Krasteva-Christ, G; Padberg, W; Hone, A J; McIntosh, J M; Zakrzewicz, A; Grau, V

    2016-01-01

    We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions. PMID:27349288

  2. Presynaptic P2X1-3 and α3-containing nicotinic receptors assemble into functionally interacting ion channels in the rat hippocampus.

    PubMed

    Rodrigues, Ricardo J; Almeida, Teresa; Díaz-Hernández, Miguel; Marques, Joana M; Franco, Rafael; Solsona, Carles; Miras-Portugal, María Teresa; Ciruela, Francisco; Cunha, Rodrigo A

    2016-06-01

    Previous studies documented a cross-talk between purinergic P2X (P2XR) and nicotinic acetylcholine receptors (nAChR) in heterologous expression systems and peripheral preparations. We now investigated if this occurred in native brain preparations and probed its physiological function. We found that P2XR and nAChR were enriched in hippocampal terminals, where both P2X1-3R and α3, but not α4, nAChR subunits were located in the active zone and in dopamine-β-hydroxylase-positive hippocampal terminals. Notably, P2XR ligands displaced nAChR binding and nAChR ligands displaced P2XR binding to hippocampal synaptosomes. In addition, a negative P2XR/nAChR cross-talk was observed in the control of the evoked release of noradrenaline from rat hippocampal synaptosomes, characterized by a less-than-additive facilitatory effect upon co-activation of both receptors. This activity-dependent cross-inhibition was confirmed in Xenopus oocytes transfected with P2X1-3Rs and α3β2 (but not α4β2) nAChR. Besides, P2X2 co-immunoprecipitated α3β2 (but not α4β2) nAChR, both in HEK cells and rat hippocampal membranes indicating that this functional interaction is supported by a physical association between P2XR and nAChR. Moreover, eliminating extracellular ATP with apyrase in hippocampal slices promoted the inhibitory effect of the nAChR antagonist tubocurarine on noradrenaline release induced by high- but not low-frequency stimulation. Overall, these results provide integrated biochemical, pharmacological and functional evidence showing that P2X1-3R and α3β2 nAChR are physically and functionally interconnected at the presynaptic level to control excessive noradrenergic terminal activation upon intense synaptic firing in the hippocampus. PMID:26801076

  3. Nitrosamines as nicotinic receptor ligands.

    PubMed

    Schuller, Hildegard M

    2007-05-30

    Nitrosamines are carcinogens formed in the mammalian organism from amine precursors contained in food, beverages, cosmetics and drugs. The potent carcinogen, NNK, and the weaker carcinogen, NNN, are nitrosamines formed from nicotine. Metabolites of the nitrosamines react with DNA to form adducts responsible for genotoxic effects. We have identified NNK as a high affinity agonist for the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) whereas NNN bound with high affinity to epibatidine-sensitive nAChRs. Diethylnitrosamine (DEN) bound to both receptors but with lower affinity. High levels of the alpha7nAChR were expressed in human small cell lung cancer (SCLC) cell lines and in hamster pulmonary neuroendocrine cells (PNECs), which serve as a model for the cell of origin of human SCLC. Exposure of SCLC or PNECs to NNK or nicotine increased expression of the alpha7nAChR and caused influx of Ca(2+), activation of PKC, Raf-1, ERK1/2, and c-myc, resulting in the stimulation of cell proliferation. Signaling via the alpha7nAChR was enhanced when cells were maintained in an environment of 10-15% CO(2) similar to that in the diseased lung. Hamsters with hyperoxia-induced pulmonary fibrosis developed neuroendocrine lung carcinomas similar to human SCLC when treated with NNK, DEN, or nicotine. The development of the NNK-induced tumors was prevented by green tea or theophylline. The beta-adrenergic receptor agonist, isoproterenol or theophylline blocked NNK-induced cell proliferation in vitro. NNK and nicotine-induced hyperactivity of the alpha7nAChR/RAF/ERK1/2 pathway thus appears to play a crucial role in the development of SCLC in smokers and could be targeted for cancer prevention. PMID:17459420

  4. A novel α-conotoxin MII-sensitive nicotinic acetylcholine receptor modulates [(3) H]-GABA release in the superficial layers of the mouse superior colliculus.

    PubMed

    McClure-Begley, Tristan D; Wageman, Charles R; Grady, Sharon R; Marks, Michael J; McIntosh, J Michael; Collins, Allan C; Whiteaker, Paul

    2012-07-01

    Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [(125) I]-Epibatidine competition-binding studies revealed that the α3β2* and α6β2* nicotinic subtype-selective peptide α-conotoxin MII-blocked binding to 40 ± 5% of SuSC nAChRs. Acetylcholine-evoked [(3) H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [(3) H]-GABA release is inhibited by α-conotoxin MII. However, the highly α6β2*-subtype-selective α-conotoxin PIA did not affect [(3) H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [(3) H]-GABA release is entirely β2 subunit-dependent. α4 subunit deletion decreased total function by >90%, and eliminated α-conotoxin MII-resistant release. ACh-stimulated SuSC [(3) H]-GABA release was unaffected by β3, α5 or α6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, α-conotoxin MII-sensitive α3α4β2 nAChR. The remaining α-conotoxin MII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via α4β2* subtype nAChRs. PMID:22506481

  5. The linoleic acid derivative DCP-LA increases membrane surface localization of the α7 ACh receptor in a protein 4.1N-dependent manner.

    PubMed

    Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

    2013-03-01

    In yeast two-hybrid screening, protein 4.1N, a scaffolding protein, was identified as a binding partner of the α7 ACh (acetylcholine) receptor. For rat hippocampal slices, the linoleic acid derivative DCP-LA {8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid} increased the association of the α7 ACh receptor with 4.1N, and the effect was inhibited by GF109203X, an inhibitor of PKC (protein kinase C), although DCP-LA did not induce PKC phosphorylation of 4.1N. For PC-12 cells, the presence of the α7 ACh receptor in the plasma membrane fraction was significantly suppressed by knocking down 4.1N. DCP-LA increased the presence of the α7 ACh receptor in the plasma membrane fraction, and the effect was still inhibited by knocking down 4.1N. In the monitoring of α7 ACh receptor mobilization, DCP-LA enhanced signal intensities for the α7 ACh receptor at the membrane surface in PC-12 cells, which was clearly prevented by knocking down 4.1N. Taken together, the results of the present study show that 4.1N interacts with the α7 ACh receptor and participates in the receptor tethering to the plasma membrane. The results also indicate that DCP-LA increases membrane surface localization of the α7 ACh receptor in a 4.1N-dependent manner under the control of PKC, but without phosphorylating 4.1N. PMID:23256752

  6. Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

    SciTech Connect

    Resende, R.R.; Alves, A.S.; Britto, L.R.G; Ulrich, H.

    2008-04-15

    Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G{alpha}{sub q/11}-coupled M{sub 1}, M{sub 3} and M{sub 5} receptors and intracellular calcium stores, whereas G{alpha}{sub i/o}-protein coupled M{sub 2} receptor activity mediated neuronal differentiation.

  7. Functional interaction between Lypd6 and nicotinic acetylcholine receptors.

    PubMed

    Arvaniti, Maria; Jensen, Majbrit M; Soni, Neeraj; Wang, Hong; Klein, Anders B; Thiriet, Nathalie; Pinborg, Lars H; Muldoon, Pretal P; Wienecke, Jacob; Imad Damaj, M; Kohlmeier, Kristi A; Gondré-Lewis, Marjorie C; Mikkelsen, Jens D; Thomsen, Morten S

    2016-09-01

    Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain-containing 6 (Lypd6) with nAChRs in human brain extracts, identifying Lypd6 as a novel regulator of nAChR function. Using protein cross-linking and affinity purification from human temporal cortical extracts, we demonstrate that Lypd6 is a synaptically enriched membrane-bound protein that binds to multiple nAChR subtypes in the human brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine-induced hippocampal inward currents in rat brain slices and decreases nicotine-induced extracellular signal-regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit nAChR-mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post-natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx protein Lypd6 binds to nAChRs in human brain extracts, and that recombinant Lypd6 decreases nicotine-induced ERK phosphorylation and attenuates nicotine-induced hippocampal inward currents. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain. PMID:27344019

  8. The dual-acting H3 receptor antagonist and AChE inhibitor UW-MD-71 dose-dependently enhances memory retrieval and reverses dizocilpine-induced memory impairment in rats.

    PubMed

    Khan, Nadia; Saad, Ali; Nurulain, Syed M; Darras, Fouad H; Decker, Michael; Sadek, Bassem

    2016-01-15

    Both the histamine H3 receptor (H3R) and acetylcholine esterase (AChE) are involved in the regulation of release and metabolism of acetylcholine and several other central neurotransmitters. Therefore, dual-active H3R antagonists and AChE inhibitors (AChEIs) have shown in several studies to hold promise to treat cognitive disorders like Alzheimer's disease (AD). The novel dual-acting H3R antagonist and AChEI 7-(3-(piperidin-1-yl)propoxy)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline (UW-MD-71) with excellent selectivity profiles over both the three other HRs as well as the AChE's isoenzyme butyrylcholinesterase (BChE) shows high and balanced in vitro affinities at both H3R and AChE with IC50 of 33.9nM and hH3R antagonism with Ki of 76.2nM, respectively. In the present study, the effects of UW-MD-71 (1.25-5mg/kg, i.p.) on acquisition, consolidation, and retrieval in a one-trial inhibitory avoidance task in male rats were investigated applying donepezil (DOZ) and pitolisant (PIT) as reference drugs. Furthermore, the effects of UW-MD-71 on memory deficits induced by the non-competitive N-methyl-d-aspartate (NMDA) antagonist dizocilpine (DIZ) were tested. Our results indicate that administration of UW-MD-71 before the test session dose-dependently increased performance and enhanced procognitive effect on retrieval. However neither pre- nor post-training acute systemic administration of UW-MD-71 facilitated acquisition or consolidation. More importantly, UW-MD-71 (2.5mg/kg, i.p.) ameliorated the DIZ-induced amnesic effects. Furthermore, the procognitive activity of UW-MD-71 in retrieval was completely reversed and partly abrogated in DIZ-induced amnesia when rats were pretreated with the centrally-acting H2R antagonist zolantidine (ZOL), but not with the CNS penetrant H1R antagonist pyrilamine (PYR). These results demonstrate the procognitive effects of UW-MD-71 in two in vivo memory models, and are to our knowledge the first demonstration in vivo that a potent dual

  9. Identifying Barbiturate Binding Sites in a Nicotinic Acetylcholine Receptor with [3H]Allyl m-Trifluoromethyldiazirine Mephobarbital, a Photoreactive Barbiturate

    PubMed Central

    Hamouda, Ayman K.; Stewart, Deirdre S.; Chiara, David C.; Savechenkov, Pavel Y.; Bruzik, Karol S.

    2014-01-01

    At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [3H]R-mTFD-MPAB ([3H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [3H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [3H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [3H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ–α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ–α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators. PMID:24563544

  10. Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine.

    PubMed

    Alberola-Die, Armando; Fernández-Ballester, Gregorio; González-Ros, José M; Ivorra, Isabel; Morales, Andrés

    2016-01-01

    Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (I ACh ) in a dose-dependent manner (IC 50 close to 70 μM), but unlike lidocaine, DEA did not affect I ACh desensitization. I ACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced I ACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and

  11. The twin drug approach for novel nicotinic acetylcholine receptor ligands.

    PubMed

    Tomassoli, Isabelle; Gündisch, Daniela

    2015-08-01

    The association of two pharmacophoric entities generates so-called 'twin drugs' or dimer derivatives. We applied this approach for the design of a small compound library for the interaction with α4β2(∗) nicotinic acetylcholine receptors (nAChRs). In this compound series, the nAChR ligand N,N-dimethyl-2-(pyridin-3-yloxy)ethan-1-amine 9 served as one pharmacological entity and it was initially kept constant as one part of the 'twin' compound. 'Twin' compounds with identical or non-identical entities using the 'no linker mode' or 'overlap' mode were synthesized and evaluated for their nAChR affinities. Compound 17a showed the highest affinity for the α4β2(∗) nAChR subtype (Ki=0.188 nM) and its (di)fluoro analogs could retain nanomolar affinities, when replacing pyridine as the hydrogen bond acceptor system by mono- or difluoro-phenyls. The 'twin drug' approach proved to provide compounds with high affinity and subtype selectivity for α4β2(∗) nAChRs. PMID:26142318

  12. 6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

    PubMed Central

    Kasheverov, Igor E.; Shelukhina, Irina V.; Kudryavtsev, Denis S.; Makarieva, Tatyana N.; Spirova, Ekaterina N.; Guzii, Alla G.; Stonik, Valentin A.; Tsetlin, Victor I.

    2015-01-01

    6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes. PMID:25775422

  13. Evaluating the role of the alpha-7 nicotinic acetylcholine receptor in the pathophysiology and treatment of schizophrenia

    PubMed Central

    YOUNG, Jared W; GEYER, Mark A

    2013-01-01

    The group of schizophrenia disorders affects approximately 1% of the population and has both genetic and environmental etiologies. Sufferers report various behavioral abnormalities including hallucinations and delusions (positive symptoms), reduced joy and amotivation (negative symptoms), plus inattention and poor learning (cognitive deficits). Despite the heterogeneous symptoms experienced, most patients smoke. The self-medication hypothesis posits that patients smoke to alleviate symptoms, consistent with evidence for nicotine-induced enhancement of cognition. While nicotine acts on multiple nicotinic acetylcholine receptors (nAChRs), the primary target of research is often the homomeric α7 nAChR. Given genetic linkages between schizophrenia and this receptor, its association with P50 sensory gating deficits, and its reduced expression in post-mortem brains, many have attempted to develop α7 nAChR ligands for treating schizophrenia. Recent evidence that ligands can be orthosteric agonists or positive allosteric modulators (PAMs) has revitalized the hope for treatment discovery. Herein, we present evidence regarding: 1) Pathophysiological alterations of α7 nAChRs that might occur in patients; 2) Mechanistic evidence for the normal action of α7 nAChRs; 3) Preclinical studies using α7 nAChR orthosteric agonists and type I/II PAMs; and 4) Where successful translational testing has occurred for particular compounds, detailing what is still required. We report that the accumulating evidence is positive, but that greater work is required using positron emission tomography to understand current alterations in α7 nAChR expression and their relationship to symptoms. Finally, cross-species behavioral tasks should be used more regularly to determine the predictive efficacy of treatments. PMID:23856289

  14. Effects of nicotinic acetylcholine receptor agonists on cognition in rhesus monkeys with a chronic cocaine self-administration history.

    PubMed

    Gould, Robert W; Garg, Pradeep K; Garg, Sudha; Nader, Michael A

    2013-01-01

    Cocaine use is associated with impaired cognitive function, which may negatively impact treatment outcomes. One pharmacological strategy to improve cognition involves nicotinic acetylcholine receptor (nAChR) stimulation. However, the effects of chronic cocaine exposure on nAChR distribution and function have not been characterized. Thus, one goal of this study was to examine nAChR availability in rhesus monkeys with an extensive cocaine self-administration history (n = 4; ~6 years, mean intake, 1463 mg/kg) compared to age-matched cocaine-naive control monkeys (n = 5). Using [¹¹C]-nicotine and positron emission tomography (PET) imaging, cocaine-experienced monkeys showed significantly higher receptor availability in the hippocampus compared to cocaine-naive monkeys. A second goal was to examine the effects of nAChR agonists on multiple domains of cognitive performance in these same monkeys. For these studies, working memory was assessed using a delayed match-to-sample (DMS) task, associative learning and behavioral flexibility using stimulus discrimination and reversal learning tasks. When administered acutely, the nonselective high-efficacy agonist nicotine, the low-efficacy α4β2* subtype-selective agonist varenicline and the high-efficacy α7 subtype-selective agonist, PNU-282987 significantly improved DMS performance in both cocaine-naive and cocaine-experienced monkeys. Individual doses of nicotine and varenicline that engendered maximum cognitive enhancing effects on working memory did not affect discrimination or reversal learning, while PNU-282987 disrupted reversal learning in the cocaine-naive monkeys. These findings indicate that a cocaine self-administration history influenced nAChR distribution and the effects of nAChR agonists on cognitive performance, including a reduced sensitivity to the disrupting effects on reversal learning. The cognitive enhancing effects of nAChR agonists may be beneficial in combination with behavioral treatments for

  15. 6-bromohypaphorine from marine nudibranch mollusk Hermissenda crassicornis is an agonist of human α7 nicotinic acetylcholine receptor.

    PubMed

    Kasheverov, Igor E; Shelukhina, Irina V; Kudryavtsev, Denis S; Makarieva, Tatyana N; Spirova, Ekaterina N; Guzii, Alla G; Stonik, Valentin A; Tsetlin, Victor I

    2015-03-01

    6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes. PMID:25775422

  16. Agonist actions of clothianidin on synaptic and extrasynaptic nicotinic acetylcholine receptors expressed on cockroach sixth abdominal ganglion.

    PubMed

    Thany, Steeve H

    2009-11-01

    Clothianidin is new neonicotinoid insecticide acting selectively on insect nicotinic acetylcholine receptors (nAChRs). Its effects on nAChRs expressed on cercal afferent/giant interneuron synapses and DUM neurons have been studied using mannitol-gap and whole-cell patch-clamp techniques, respectively. Bath-application of clothianidin-induced dose-dependent depolarizations of cockroach cercal afferent/giant interneuron synapses which were not reversed after wash-out suggesting a strong desensitization of postsynaptic interneurons at the 6th abdominal ganglion (A6). Clothinidin activity on the nerve preparation was characterized by an increased firing rate of action potentials which then ceased when the depolarization reached a peak. Clothianidin responses were insensitive to all muscarinic antagonists tested but were blocked by co-application of specific nicotinic antagonists methyllicaconitine, alpha-bungarotoxin and d-tubocurarine. In a second round of experiment, clothianidin actions were tested on DUM neurons isolated from the A6. There was a strong desensitization of nAChRs which was not affected by muscarinic antagonists, pirenzepine and atropine, but was reduced with nicotinic antagonist alpha-bungarotoxin. In addition, clothianidin-induced currents were completely blocked by methyllicaconitine suggesting that (1) clothianidin acted as a specific agonist of nAChR subtypes and (2) a small proportion of receptors blocked by MLA was insensitive to alpha-bungarotoxin. Moreover, because clothianidin currents were blocked by d-tubocurarine and mecamylamine, we provided that clothianidin was an agonist of both nAChRs: imidacloprid-sensitive nAChR1 and -insensitive nAChR2 subtypes. PMID:19583978

  17. PACAP induces plasticity at autonomic synapses by nAChR-dependent NOS1 activation and AKAP-mediated PKA targeting.

    PubMed

    Jayakar, Selwyn S; Pugh, Phyllis C; Dale, Zack; Starr, Eric R; Cole, Samantha; Margiotta, Joseph F

    2014-11-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide found at synapses throughout the central and autonomic nervous system. We previously found that PACAP engages a selective G-protein coupled receptor (PAC1R) on ciliary ganglion neurons to rapidly enhance quantal acetylcholine (ACh) release from presynaptic terminals via neuronal nitric oxide synthase (NOS1) and cyclic AMP/protein kinase A (PKA) dependent processes. Here, we examined how PACAP stimulates NO production and targets resultant outcomes to synapses. Scavenging extracellular NO blocked PACAP-induced plasticity supporting a retrograde (post- to presynaptic) NO action on ACh release. Live-cell imaging revealed that PACAP stimulates NO production by mechanisms requiring NOS1, PKA and Ca(2+) influx. Ca(2+)-permeable nicotinic ACh receptors composed of α7 subunits (α7-nAChRs) are potentiated by PKA-dependent PACAP/PAC1R signaling and were required for PACAP-induced NO production and synaptic plasticity since both outcomes were drastically reduced following their selective inhibition. Co-precipitation experiments showed that NOS1 associates with α7-nAChRs, many of which are perisynaptic, as well as with heteromeric α3*-nAChRs that generate the bulk of synaptic activity. NOS1-nAChR physical association could facilitate NO production at perisynaptic and adjacent postsynaptic sites to enhance focal ACh release from juxtaposed presynaptic terminals. The synaptic outcomes of PACAP/PAC1R signaling are localized by PKA anchoring proteins (AKAPs). PKA regulatory-subunit overlay assays identified five AKAPs in ganglion lysates, including a prominent neuronal subtype. Moreover, PACAP-induced synaptic plasticity was selectively blocked when PKA regulatory-subunit binding to AKAPs was inhibited. Taken together, our findings indicate that PACAP/PAC1R signaling coordinates nAChR, NOS1 and AKAP activities to induce targeted, retrograde plasticity at autonomic synapses. Such

  18. Characterization of the retina in the alpha7 nicotinic acetylcholine receptor knockout mouse

    NASA Astrophysics Data System (ADS)

    Smith, Marci L.

    Acetylcholine receptors (AChRs) are involved in visual processing and are expressed by inner retinal neurons in all species studied to date (Keyser et al., 2000; Dmitrieva et al., 2007; Liu et al., 2009), but their distribution in the mouse retina remains unknown. Reductions in alpha7 nicotinic AChRs (nAChRs) are thought to contribute to memory and visual deficits observed in Alzheimer's and schizophrenia (Coyle et al., 1983; Nordberg et al., 1999; Leonard et al., 2006). However, the alpha7 nAChR knockout (KO) mouse has a mild phenotype (Paylor et al., 1998; Fernandes et al., 2006; Young et al., 2007; Origlia et al., 2012). The purpose of this study was to determine the expression of AChRs in wildtype (WT) mouse retina and to assess whether up-regulation of other AChRs in the alpha7 nAChR KO retina may explain the minimal deficits described in the KO mouse. Reverse-transcriptase PCR (RT-PCR) showed that mRNA transcripts for alpha2-7, alpha 9, alpha10, beta2-4 nAChR subunits and m1-m5 muscarinic AChR (mAChR) subtypes were present in WT murine retina. Western blot analysis confirmed the presence of alpha3-5, alpha9, and m1-m5 AChR proteins and immunohistochemical analysis demonstrated nAChR and mAChR proteins expressed by subsets of bipolar, amacrine and ganglion cells. This is the first reported expression of alpha9 and alpha10 nAChR transcripts and alpha9 nAChR proteins in the retina of any species. Quantitative RT-PCR (qPCR) showed changes in AChR transcript expression in the alpha7 nAChR KO mouse retina relative to WT. Within whole retina alpha2, alpha9, alpha10, beta4, m1 and m4 AChR transcripts were up-regulated, while alpha5 nAChR transcripts were down-regulated. However, cell populations showed subtle differences; m4 mAChR transcripts were up-regulated in the ganglion cell layer and outer portion of the inner nuclear layer (oINL),while beta4 nAChR transcript up-regulation was limited to the oINL. Surprisingly, alpha2, alpha9, beta4, m2 and m4 transcripts were

  19. Association between Anti-Ganglionic Nicotinic Acetylcholine Receptor (gAChR) Antibodies and HLA-DRB1 Alleles in the Japanese Population

    PubMed Central

    Maeda, Yasuhiro; Migita, Kiyoshi; Higuchi, Osamu; Mukaino, Akihiro; Furukawa, Hiroshi; Komori, Atsumasa; Nakamura, Minoru; Hashimoto, Satoru; Nagaoka, Shinya; Abiru, Seigo; Yatsuhashi, Hiroshi; Matsuo, Hidenori; Kawakami, Atsushi; Yasunami, Michio; Nakane, Shunya

    2016-01-01

    Background/Aims Anti-ganglionic nicotinic acetylcholine receptor (gAChR) antibodies are observed in autoimmune diseases, as well as in patients with autoimmune autonomic ganglionopathy. However, the genetic background of anti-gAChR antibodies is unclear. Here, we investigated HLA alleles in autoimmune hepatitis (AIH) patients with or without anti-gAChR antibodies. Methodology/Principal Findings Genomic DNA from 260 patients with type-1 autoimmune hepatitis (AIH) were genotyped for HLA-A, B, DRB1, and DQB1 loci. Anti-gAChR antibodies in the sera form AIH patients were measured using the luciferase immunoprecipitation system, and examined allelic association in patients with or without anti-gAChR antibodies. Methodology/ Methods We detected anti-α3 or -β4 gAChR antibodies in 11.5% (30/260) of patients with AIH. Among AIH patients there was no significant association between HLA-A, B DQB1 alleles and the positivity for anti-gAChR antibodies. Whereas the HLA-DRB1*0403 allele showed a significantly increased frequency in AIH patients with anti-gAChR antibodies compared with those without anti-gAChR antibodies. Conclusions/Significance The frequency of the HLA-DRB1*0403 allele differed among Japanese patients with AIH according to the presence or absence of anti-gAChR antibodies. Our findings suggest that particular HLA class II molecules might control the development of anti-gAChR antibodies in the autoimmune response to gAChR. PMID:26807576

  20. Decreased nicotinic receptor availability in smokers with slow rates of nicotine metabolism

    PubMed Central

    Dubroff, Jacob G.; Doot, Robert K.; Falcone, Mary; R, Robert A. Schnoll; Ray, Riju; Tyndale, Rachel F.; Brody, Arthur L.; Hou, Catherine; Schmitz, Alexander; Lerman, Caryn

    2015-01-01

    The nicotine metabolite ratio (NMR), a stable measure of hepatic nicotine metabolism via the CYP2A6 pathway and total nicotine clearance, is a predictive biomarker of response to nicotine replacement therapy, with increased quit rates in slower metabolizers. Nicotine binds directly to nicotinic acetylcholine receptors (nAChRs) to exert its psychoactive effects. This study examined the relationship between NMR and nAChR availability (α4β2* subtype) using positron emission tomography (PET) imaging of the radiotracer 2-18F-FA-85380 (2-18F-FA). Methods Twenty four smokers, 12 slow metabolizers (NMR <0.26) and 12 normal metabolizers (NMR ≥0.26), underwent 2-18F-FA-PET brain imaging following overnight nicotine abstinence (18 hours prior to scanning), using a validated bolus plus infusion protocol. Availability of nAChRs was compared between NMR groups in a priori volumes of interest (VOIs), with total distribution volume (VT/fP) being the measure of nAChR availability. Cravings to smoke were assessed prior to and following the scans. Results Thalamic nAChR α4β2* availability was significantly reduced in slow (versus normal) nicotine metabolizers (P=0.04). Slow metabolizers exhibited greater reductions in craving than normal metabolizers from pre- to post-scanning; however, craving was unrelated to availability. Conclusion The rate of nicotine metabolism is associated with thalamic nAChR availability. Additional studies could examine whether altered nAChR availability underlies differences in treatment response between slow and normal metabolizers of nicotine. PMID:26272810

  1. Galantamine-induced amyloid-{beta} clearance mediated via stimulation of microglial nicotinic acetylcholine receptors.

    PubMed

    Takata, Kazuyuki; Kitamura, Yoshihisa; Saeki, Mana; Terada, Maki; Kagitani, Sachiko; Kitamura, Risa; Fujikawa, Yasuhiro; Maelicke, Alfred; Tomimoto, Hidekazu; Taniguchi, Takashi; Shimohama, Shun

    2010-12-17

    Reduction of brain amyloid-β (Aβ) has been proposed as a therapeutic target for Alzheimer disease (AD), and microglial Aβ phagocytosis is noted as an Aβ clearance system in brains. Galantamine is an acetylcholinesterase inhibitor approved for symptomatic treatment of AD. Galantamine also acts as an allosterically potentiating ligand (APL) for nicotinic acetylcholine receptors (nAChRs). APL-binding site is located close to but distinct from that for acetylcholine on nAChRs, and FK1 antibody specifically binds to the APL-binding site without interfering with the acetylcholine-binding site. We found that in human AD brain, microglia accumulated on Aβ deposits and expressed α7 nAChRs including the APL-binding site recognized with FK1 antibody. Treatment of rat microglia with galantamine significantly enhanced microglial Aβ phagocytosis, and acetylcholine competitive antagonists as well as FK1 antibody inhibited the enhancement. Thus, the galantamine-enhanced microglial Aβ phagocytosis required the combined actions of an acetylcholine competitive agonist and the APL for nAChRs. Indeed, depletion of choline, an acetylcholine-competitive α7 nAChR agonist, from the culture medium impeded the enhancement. Similarly, Ca(2+) depletion or inhibition of the calmodulin-dependent pathways for the actin reorganization abolished the enhancement. These results suggest that galantamine sensitizes microglial α7 nAChRs to choline and induces Ca(2+) influx into microglia. The Ca(2+)-induced intracellular signaling cascades may then stimulate Aβ phagocytosis through the actin reorganization. We further demonstrated that galantamine treatment facilitated Aβ clearance in brains of rodent AD models. In conclusion, we propose a further advantage of galantamine in clinical AD treatment and microglial nAChRs as a new therapeutic target. PMID:20947502

  2. Effect of α{sub 7} nicotinic acetylcholine receptor agonists and antagonists on motor function in mice

    SciTech Connect

    Welch, Kevin D.; Green, Benedict T.; Gardner, Dale R.

    2013-02-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChRs located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The toxicity and teratogenicity of many plants (which results in millions of dollars in losses annually to the livestock industry) are due to various toxins that bind to nAChRs including deltaline and methyllycaconitine (MLA) from larkspur (Delphinium) species, and nicotine and anabasine from tobacco (Nicotiana) species. The primary result of the actions of these alkaloids at nAChRs is neuromuscular paralysis and respiratory failure. The objective of this study was to further characterize the motor coordination deficiencies that occur upon exposure to a non-lethal dose of nAChR antagonists MLA and deltaline as well as nAChR agonists nicotine and anabasine. We evaluated the effect of nAChR agonists and antagonists on the motor function and coordination in mice using a balance beam, grip strength meter, rotarod, open field analysis and tremor monitor. These analyses demonstrated that within seconds after treatment the mice had significant loss of motor function and coordination that lasted up to 1 min, followed by a short period of quiescence. Recovery to normal muscle coordination was rapid, typically within approximately 10 min post-dosing. However, mice treated with the nAChR agonist nicotine and anabasine required a slightly longer time to recover some aspects of normal muscle function in comparison to mice treated with the nAChR antagonist MLA or deltaline. -- Highlights: ► Mice treated with nAChR agonists and antagonists have a loss in motor function. ► These deficits are temporary as near normal motor function returns within 10 min. ► There are compound-specific differences in the effects on motor function.

  3. Genetic deletion of the adenosine A(2A) receptor prevents nicotine-induced upregulation of α7, but not α4β2* nicotinic acetylcholine receptor binding in the brain.

    PubMed

    Metaxas, Athanasios; Al-Hasani, Ream; Farshim, Pamela; Tubby, Kristina; Berwick, Amy; Ledent, Catherine; Hourani, Susanna; Kitchen, Ian; Bailey, Alexis

    2013-08-01

    Considerable evidence indicates that adenosine A(2A) receptors (A(2A)Rs) modulate cholinergic neurotransmission, nicotinic acetylcholine receptor (nAChR) function, and nicotine-induced behavioural effects. To explore the interaction between A(2A) and nAChRs, we examined if the complete genetic deletion of adenosine A(2A)Rs in mice induces compensatory alterations in the binding of different nAChR subtypes, and whether the long-term effects of nicotine on nAChR regulation are altered in the absence of the A(2A)R gene. Quantitative autoradiography was used to measure cytisine-sensitive [¹²⁵I]epibatidine and [¹²⁵I]α-bungarotoxin binding to α4β2* and α7 nAChRs, respectively, in brain sections of drug-naïve (n = 6) or nicotine treated (n = 5-7), wild-type and adenosine A(2A)R knockout mice. Saline or nicotine (7.8 mg/kg/day; free-base weight) were administered to male CD1 mice via subcutaneous osmotic minipumps for a period of 14 days. Blood plasma levels of nicotine and cotinine were measured at the end of treatment. There were no compensatory developmental alterations in nAChR subtype distribution or density in drug-naïve A(2A)R knockout mice. In nicotine treated wild-type mice, both α4β2* and α7 nAChR binding sites were increased compared with saline treated controls. The genetic ablation of adenosine A(2A)Rs prevented nicotine-induced upregulation of α7 nAChRs, without affecting α4β2* receptor upregulation. This selective effect was observed at plasma levels of nicotine that were within the range reported for smokers (10-50 ng ml⁻¹). Our data highlight the involvement of adenosine A(2A)Rs in the mechanisms of nicotine-induced α7 nAChR upregulation, and identify A(2A)Rs as novel pharmacological targets for modulating the long-term effects of nicotine on α7 receptors. PMID:23583933

  4. Heterogeneity of Drosophila nicotinic acetylcholine receptors: SAD, a novel developmentally regulated alpha-subunit.

    PubMed Central

    Sawruk, E; Schloss, P; Betz, H; Schmitt, B

    1990-01-01

    Two genes, ard and als, are known to encode subunits of the nicotinic acetylcholine receptor (nAChR) in Drosophila. Here we describe the isolation of cDNA clones encoding a novel member (SAD, or alpha 2) of this receptor protein family. The deduced amino acid sequence displays high homology to the ALS protein and shares structural features with ligand binding nAChR alpha-subunits. Sad transcripts accumulate during major periods of neuronal differentiation and, in embryos, are localized in the central nervous system. Expression of SAD cRNA in Xenopus oocytes generates cation channels that are gated by nicotine. These data indicate heterogeneity of nAChRs in Drosophila. Images Fig. 3. Fig. 4. PMID:1697262

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

    PubMed

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

    2006-09-01

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

  6. Activation of α7 nicotinic acetylcholine receptors protects astrocytes against oxidative stress-induced apoptosis: implications for Parkinson's disease.

    PubMed

    Liu, Yuan; Zeng, Xiaoning; Hui, Yujian; Zhu, Chenlei; Wu, Jie; Taylor, Devin H; Ji, Juan; Fan, Weimin; Huang, Zuhu; Hu, Jun

    2015-04-01

    Astrocytes have been implicated in the immune responses associated with Parkinson's disease (PD). Inhibition of astrocyte apoptosis is a novel strategy for the treatment of PD. Recent studies suggest that α7 nicotinic acetylcholine receptors (α7-nAChRs) expressed in glial cells are critical links between inflammation and neurodegeneration in PD. However, little is known about their contribution to astrocyte apoptosis during the development of this disorder. In the present study, we showed that nicotine exerts a protective effect on H2O2-induced astrocyte apoptosis and glial cell-derived neurotrophic factor (GDNF) downregulation, and this effect was abolished by an α7-nAChR-selective antagonist. The underlying mechanisms might involve alleviation of mitochondrial membrane potential loss, stabilization of the Bax/Bcl-2 balance, and inhibition of cleaved caspase-9 activity through α7-nAChR activation. Systemic administration of nicotine dramatically alleviated MPTP-induced symptoms, protected dopaminergic neurons against degeneration, inhibited astrocytes and microglia activation in the substantia nigra pars compacta (SNpc) and blocked the decrease of GDNF in the striatum by activating α7-nAChRs. Taken together these findings demonstrate, for the first time, that nicotine suppresses H2O2-induced astrocyte apoptosis via the mitochondrial pathway through the stimulation of α7-nAChRs. Targeting α7-nAChRs expressed in astrocytes may be a novel therapeutic strategy for the treatment of neurodegenerative disorders. PMID:25486621

  7. N-Ethylmaleimide Dissociates α7 ACh Receptor from a Complex with NSF and Promotes Its Delivery to the Presynaptic Membrane.

    PubMed

    Nishizaki, Tomoyuki

    2016-08-01

    N-Ethylmaleimide (NEM)-sensitive factor (NSF) associates with soluble NSF attachment protein (SNAP), that binds to SNAP receptors (SNAREs) including syntaxin, SNAP25, and synaptobrevin. The complex of NSF/SNAP/SNAREs plays a critical role in the regulation of vesicular traffic. The present study investigated NEM-regulated α7 ACh receptor translocation. NSF associated with β-SNAP and the SNAREs syntaxin 1 and synaptobrevin 2 in the rat hippocampus. NSF also associated with the α7 ACh receptor subunit, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, and the γ-aminobutyric acid A (GABAA) receptor γ2 subunit. NEM, an inhibitor of NSF, significantly dissociated the α7 ACh receptor subunit from a complex with NSF and increased cell surface localization of the receptor subunit, but such effect was not obtained with the GluA1, GluA2 or γ2 subunits. NEM, alternatively, dissociated synaptobrevin 2 from an assembly of NSF/β-SNAP/syntaxin 1/synaptobrevin 2. NEM significantly increased the rate of nicotine-triggered AMPA receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, in rat hippocampal slices. The results of the present study indicate that NEM releases the α7 ACh receptor subunit and synaptobrevin 2 from an assembly of α7 ACh receptor subunit/NSF/β-SNAP/syntaxin 1/synaptobrevin 2, thereby promoting delivery of the α7 ACh receptor subunit to presynaptic membrane. PMID:27105867

  8. Insect Nicotinic Receptor Agonists as Flea Adulticides in Small Animals

    PubMed Central

    Vo, Dai Tan; Hsu, Walter H.; Martin, Richard J.

    2013-01-01

    Fleas are significant ectoparasites of small animals. They can be a severe irritant to animals and serve as a vector for a number of infectious diseases. In this article, we discuss the pharmacological characteristics of four insect nicotinic acetylcholine receptor (nAChR) agonists used as fleacides in dogs and cats, which include three neonicotinoids (imidacloprid, nitenpyram, and dinotefuran) and spinosad. Insect nAChR agonists are one of the most important new classes of insecticides, which are used to control sucking insects both on plants and on companion animals. These new compounds provide a new approach for practitioners to safely and effectively eliminate fleas. PMID:20646191

  9. Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study.

    PubMed

    Brown, L A; Jones, A K; Buckingham, S D; Mee, C J; Sattelle, D B

    2006-05-31

    Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation. PMID:16620825

  10. Role of β4* Nicotinic Acetylcholine Receptors in the Habenulo-Interpeduncular Pathway in Nicotine Reinforcement in Mice.

    PubMed

    Harrington, Lauriane; Viñals, Xavier; Herrera-Solís, Andrea; Flores, Africa; Morel, Carole; Tolu, Stefania; Faure, Philippe; Maldonado, Rafael; Maskos, Uwe; Robledo, Patricia

    2016-06-01

    Nicotine exerts its psychopharmacological effects by activating the nicotinic acetylcholine receptor (nAChR), composed of alpha and/or beta subunits, giving rise to a diverse population of receptors with a distinct pharmacology. β4-containing (β4*) nAChRs are located almost exclusively in the habenulo-interpeduncular pathway. We examined the role of β4* nAChRs in the medial habenula (MHb) and the interpeduncular nucleus (IPN) in nicotine reinforcement using behavioral, electrophysiological, and molecular techniques in transgenic mice. Nicotine intravenous self-administration (IVSA) was lower in constitutive β4 knockout (KO) mice at all doses tested (7.5, 15, 30, and 60 μg/kg/infusion) compared with wild-type (WT) mice. In vivo microdialysis showed that β4KO mice have higher extracellular dopamine (DA) levels in the nucleus accumbens than in WT mice, and exhibit a differential sensitivity to nicotine-induced DA outflow. Furthermore, electrophysiological recordings in the ventral tegmental area (VTA) demonstrated that DA neurons of β4KO mice are more sensitive to lower doses of nicotine than that of WT mice. Re-expression of β4* nAChRs in IPN neurons fully restored nicotine IVSA, and attenuated the increased sensitivity of VTA DA neurons to nicotine. These findings suggest that β4* nAChRs in the IPN have a role in maintaining nicotine IVSA. PMID:26585290

  11. Heterologous expression and nonsense suppression provide insights into agonist behavior at α6β2 nicotinic acetylcholine receptors.

    PubMed

    Post, Michael R; Limapichat, Walrati; Lester, Henry A; Dougherty, Dennis A

    2015-10-01

    The α6-containing subtypes of the nicotinic acetylcholine receptor (nAChR) are localized to presynaptic terminals of the dopaminergic pathways of the central nervous system. Selective ligands for these nAChRs are potentially useful in both Parkinson's disease and addiction. For these and other goals, it is important to distinguish the binding behavior of agonists at the α6-β2 binding site versus other subtypes. To study this problem, we apply nonsense suppression-based non-canonical amino acid mutagenesis. We report a combination of four mutations in α6β2 that yield high-level heterologous expression in Xenopus oocytes. By varying mRNA injection ratios, two populations were observed with unique characteristics, likely due to differing stoichiometries. Responses to nine known nAChR agonists were analyzed at the receptor, and their corresponding EC50 values and efficacies are reported. The system is compatible with nonsense suppression, allowing structure-function studies between Trp149 - a conserved residue on loop B found to make a cation-π interaction at several nAChR subtypes - and several agonists. These studies reveal that acetylcholine forms a strong cation-π interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the α6β2 nAChR. PMID:25908401

  12. Nicotine receptors mediating sensorimotor gating and its enhancement by systemic nicotine

    PubMed Central

    Pinnock, Farena; Bosch, Daniel; Brown, Tyler; Simons, Nadine; Yeomans, John R.; DeOliveira, Cleusa; Schmid, Susanne

    2015-01-01

    Prepulse inhibition (PPI) of startle occurs when intensity stimuli precede stronger startle-inducing stimuli by 10–1000 ms. PPI deficits are found in individuals with schizophrenia and other psychiatric disorders, and they correlate with other cognitive impairments. Animal research and clinical studies have demonstrated that both PPI and cognitive function can be enhanced by nicotine. PPI has been shown to be mediated, at least in part, by mesopontine cholinergic neurons that project to pontine startle neurons and activate muscarinic and potentially nicotine receptors (nAChRs). The subtypes and anatomical location of nAChRs involved in mediating and modulating PPI remain unresolved. We tested the hypothesis that nAChRs that are expressed by pontine startle neurons contribute to PPI. We also explored whether or not these pontine receptors are responsible for the nicotine enhancement of PPI. While systemic administration of nAChR antagonists had limited effects on PPI, PnC microinfusions of the non-α7nAChR preferring antagonist TMPH, but not of the α7nAChR antagonist MLA, into the PnC significantly reduced PPI. Electrophysiological recordings from startle-mediating PnC neurons confirmed that nicotine affects excitability of PnC neurons, which could be antagonized by TMPH, but not by MLA, indicating the expression of non-α7nAChR. In contrast, systemic nicotine enhancement of PPI was only reversed by systemic MLA and not by TMPH or local microinfusions of MLA into the PnC. In summary, our data indicate that non-α7nAChRs in the PnC contribute to PPI at stimulus intervals of 100 ms or less, whereas activation of α7nAChRs in other brain areas is responsible for the systemic nicotine enhancement of PPI. This is important knowledge for the correct interpretation of behavioral, preclinical, and clinical data as well as for developing drugs for the amelioration of PPI deficits and the enhancement of cognitive function. PMID:25717295

  13. Development of a solid-phase receptor-based assay for the detection of cyclic imines using a microsphere-flow cytometry system.

    PubMed

    Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Louzao, M Carmen; Taylor, Palmer; Talley, Todd; Botana, Luis M

    2013-02-19

    Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a "fast-acting" toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing a receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay, two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin, and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however, the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops, and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10-6000 μg/kg of shellfish meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive, and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins. PMID:23343192

  14. Development of a Solid-Phase Receptor-Based Assay for the Detection of Cyclic Imines Using a Microsphere-Flow Cytometry System

    PubMed Central

    Rodríguez, Laura P.; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Louzao, M. Carmen; Taylor, Palmer; Talley, Todd; Botana, Luis M.

    2013-01-01

    Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a “fast-acting” toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10–6000 μg/kg of shellfish meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins. PMID:23343192

  15. The stereotypy-inducing and OCD-like effects of chronic ‘binge’ cocaine are modulated by distinct subtypes of nicotinic acetylcholine receptors

    PubMed Central

    Metaxas, A; Keyworth, HL; Yoo, JH; Chen, Y; Kitchen, I; Bailey, A

    2012-01-01

    BACKGROUND AND PURPOSE High rates of cigarette smoking occur in cocaine-dependent individuals, reflecting an involvement of nicotinic acetylcholine receptors (nAChRs) in cocaine-elicited behaviour. This study was designed to assess the contribution of different nAChR subtypes to the behavioural and neurochemical effects of chronic cocaine treatment. EXPERIMENTAL APPROACH Cocaine (15 mg·kg−1, i.p.) was administered to male C57BL/6J mice in a chronic ‘binge’ paradigm, with and without the coadministration of the α7 preferring nAChR antagonist methyllycaconitine (MLA; 5 mg·kg−1, i.p.) or the β2* nAChR antagonist dihydro-β-erythroidine (DHβE; 2 mg·kg−1, i.p.). Quantitative autoradiography was used to examine the effect of cocaine exposure on α7 and α4β2* nAChRs, and on the high-affinity choline transporter. KEY RESULTS MLA+cocaine administration induced an intense self-grooming behaviour, indicating a likely role for α7 nAChRs in modulating this anxiogenic, compulsive-like effect of cocaine. In the major island of Calleja, a key area of action for neuroleptics, MLA+cocaine reduced choline transporter binding compared with cocaine (with or without DHβE) administration. DHβE treatment prevented the induction of stereotypy sensitisation to cocaine but prolonged locomotor sensitisation, implicating heteromeric β2* nAChRs in the neuroadaptations mediating cocaine-induced behavioural sensitisation. ‘Binge’ cocaine treatment region-specifically increased α4β2* nAChR binding in the midbrain dopaminergic regions: ventral tegmental area and substantia nigra pars compacta. CONCLUSIONS AND IMPLICATIONS We have shown a differential, subtype-selective, contribution of nAChRs to the behavioural and neurochemical sequelae of chronic cocaine administration. These data support the clinical utility of targeting specific nAChR subtypes for the alleviation of cocaine-abuse symptomatology. PMID:22568685

  16. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    PubMed Central

    Kudryavtsev, Denis; Bychkov, Maxim L.; Kulbatskii, Dmitrii S.; Kasheverov, Igor E.; Astapova, Maria V.; Feofanov, Alexey V.; Thomsen, Morten S.; Mikkelsen, Jens D.; Shenkarev, Zakhar O.; Tsetlin, Victor I.; Dolgikh, Dmitry A.; Kirpichnikov, Mikhail P.

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that

  17. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.

    PubMed

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kudryavtsev, Denis; Bychkov, Maxim L; Kulbatskii, Dmitrii S; Kasheverov, Igor E; Astapova, Maria V; Feofanov, Alexey V; Thomsen, Morten S; Mikkelsen, Jens D; Shenkarev, Zakhar O; Tsetlin, Victor I; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that

  18. Aporphine metho salts as neuronal nicotinic acetylcholine receptor blockers.

    PubMed

    Iturriaga-Vásquez, Patricio; Pérez, Edwin G; Slater, E Yvonne; Bermúdez, Isabel; Cassels, Bruce K

    2007-05-15

    (S)-Aporphine metho salts with the 1,2,9,10 oxygenation pattern displaced radioligands from recombinant human alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChR) at low micromolar concentrations. The affinity of the nonphenolic glaucine methiodide (4) (vs [(3)H]cytisine) was the lowest at alpha4beta2 nAChR (K(i)=10 microM), and predicentrine methiodide (2) and xanthoplanine iodide (3), with free hydroxyl groups at C-2 or C-9, respectively, had the highest affinity at these receptors (K(i) approximately 1 microM), while the affinity of the diphenolic boldine methiodide (1) was intermediate between these values. At homomeric alpha7 nAChR, xanthoplanine had the highest affinity (K(i)=10 microM) vs [(125)I]alpha-bungarotoxin while the other three compounds displaced the radioligand with K(i) values between 15 and 21 microM. At 100 microM, all four compounds inhibited the responses of these receptors to EC(50) concentrations of ACh. The effects of xanthoplanine iodide (3) were studied in more detail. Xanthoplanine fully inhibited the EC(50) ACh responses of both alpha7 and alpha4beta2 nACh receptors with estimated IC(50) values of 9+/-3 microM (alpha7) and 5+/-0.8 microM (alpha4beta2). PMID:17391965

  19. Nicotine ameliorates NMDA receptor antagonist-induced deficits in contextual fear conditioning through high-affinity nicotinic acetylcholine receptors in the hippocampus.

    PubMed

    André, Jessica M; Leach, Prescott T; Gould, Thomas J

    2011-03-01

    NMDA glutamate receptors (NMDARs) and nicotinic acetylcholine receptors (nAChRs) are both involved in learning and synaptic plasticity. Increasing evidence suggests processes mediated by these receptors may interact to modulate learning; however, little is known about the neural substrates involved in these interactive processes. The present studies investigated the effects of nicotine on MK-801 hydrogen maleate (MK-801) and DL-2-Amino-5-phosphonovaleric acid (APV)-induced disruption of contextual fear conditioning in male C57BL/6J mice, using direct drug infusion and selective nAChR antagonists to define the brain regions and the nAChR subtypes involved. Mice treated with MK-801 showed a deficit in contextual fear conditioning that was ameliorated by nicotine. Direct drug infusion demonstrated that the NMDAR antagonists disrupted hippocampal function and that nicotine acted in the dorsal hippocampus to ameliorate the deficit in learning. The high-affinity nAChR antagonist Dihydro-β-erythroidine hydrobromide (DhβE) blocked the effects of nicotine on MK-801-induced deficits while the α7 nAChR antagonist methyllycaconitine citrate salt hydrate (MLA) did not. These results suggest that NMDARs and nAChRs may mediate similar hippocampal processes involved in contextual fear conditioning. Furthermore, these results may have implications for developing effective therapeutics for the cognitive deficits associated with schizophrenia because a large subset of patients with schizophrenia exhibit cognitive deficits that may be related to NMDAR dysfunction and smoke at much higher rates than the healthy population, which may be an attempt to ameliorate cognitive deficits. PMID:21167848

  20. Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus.

    PubMed

    Endo, Toshiaki; Yanagawa, Yuchio; Obata, Kunihiko; Isa, Tadashi

    2005-12-01

    The superficial superior colliculus (sSC) is a key station in the sensory processing related to visual salience. The sSC receives cholinergic projections from the parabigeminal nucleus, and previous studies have revealed the presence of several different nicotinic acetylcholine receptor (nAChR) subunits in the sSC. In this study, to clarify the role of the cholinergic inputs to the sSC, we examined current responses induced by ACh in GABAergic and non-GABAergic sSC neurons using in vitro slice preparations obtained from glutamate decarboxylase 67-green fluorescent protein (GFP) knock-in mice in which GFP is specifically expressed in GABAergic neurons. Brief air pressure application of acetylcholine (ACh) elicited nicotinic inward current responses in both GABAergic and non-GABAergic neurons. The inward current responses in the GABAergic neurons were highly sensitive to a selective antagonist for alpha3beta2- and alpha6beta2-containing receptors, alpha-conotoxin MII (alphaCtxMII). A subset of these neurons exhibited a faster alpha-bungarotoxin-sensitive inward current component, indicating the expression of alpha7-containing nAChRs. We also found that the activation of presynaptic nAChRs induced release of GABA, which elicited a burst of miniature inhibitory postsynaptic currents mediated by GABA(A) receptors in non-GABAergic neurons. This ACh-induced GABA release was mediated mainly by alphaCtxMII-sensitive nAChRs and resulted from the activation of voltage-dependent calcium channels. Morphological analysis revealed that recorded GFP-positive neurons are interneurons and GFP-negative neurons include projection neurons. These findings suggest that nAChRs are involved in the regulation of GABAergic inhibition and modulate visual processing in the sSC. PMID:16107532

  1. Integration of inhibitory and excitatory effects of α7 nicotinic acetylcholine receptor activation in the prelimbic cortex regulates network activity and plasticity.

    PubMed

    Udakis, Matthew; Wright, Victoria Louise; Wonnacott, Susan; Bailey, Christopher Philip

    2016-06-01

    Cognitive and attentional processes governed by the prefrontal cortex (PFC) are influenced by cholinergic innervation. Here we have explored the role of α7 nicotinic acetylcholine receptors (nAChRs) as mediators of cholinergic signalling in the dorsomedial (prelimbic) PFC, using mouse brain slice electrophysiology. Activation of α7 nAChRs located on glutamatergic terminals and cell soma of GABAergic interneurons increased excitation and inhibition, respectively, in layer V of the prelimbic cortex. These actions were distinguished by their differential dependence on local acetylcholine (ACh): potentiation of endogenous cholinergic signalling with the positive allosteric modulator, PNU-120596, enhanced spontaneous excitatory events, an effect that was further increased by inhibition of acetylcholinesterase. In contrast, α7 nicotinic modulation of inhibitory signalling required addition of exogenous agonist (PNU-282987) as well as PNU-120596, and was unaffected by acetylcholinesterase inhibition. Thus α7 nAChRs can bi-directionally regulate network activity in the prelimbic cortex, depending on the magnitude and localisation of cholinergic signalling. This bidirectional influence is manifest in dual effects of α7 nAChRs on theta-burst-induced long-term potentiation (LTP) in layer V of the prelimbic cortex. Antagonism of α7 nAChRs significantly decreased LTP implicating a contribution from endogenous ACh, consistent with the ability of local ACh to enhance glutamatergic signalling. Exogenous agonist plus potentiator also decreased LTP, indicative of the influence of this drug combination on inhibitory signalling. Thus α7 nAChRs make a complex contribution to network activity and synaptic plasticity in the prelimbic cortex. PMID:26921769

  2. The impact of a parkinsonian lesion on dynamic striatal dopamine transmission depends on nicotinic receptor activation.

    PubMed

    Jennings, Katie A; Platt, Nicola J; Cragg, Stephanie J

    2015-10-01

    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

  3. 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

  4. Amyloid-β peptide increases cell surface localization of α7 ACh receptor to protect neurons from amyloid β-induced damage.

    PubMed

    Jin, Yu; Tsuchiya, Ayako; Kanno, Takeshi; Nishizaki, Tomoyuki

    Amyloid-β peptide 1-42 (Aβ1-42) reduced PC-12 cell viability in a concentration (1-10 μM)- and treatment time (48-72 h)-dependent manner. Nicotine prevented Aβ1-42-induced PC-12 cell death, but conversely, the α7 ACh receptor antagonist α-bungarotoxin enhanced Aβ1-42-induced cell toxicity. Extracellularly applied Aβ1-42 significantly increased cell surface localization of α7 ACh receptor in PC-12 cells as compared with that for non-treated control cells. Cell surface localization of α7 ACh receptor in the brain of 5xFAD mouse, an animal model of Alzheimer's disease (AD), apparently increased in an age (1-12 months)-dependent manner in association with increased accumulation of Aβ1-42 in the plasma membrane component. Taken together, these results indicate that Aβ1-42 promotes translocation of α7 ACh receptor towards the cell surface and that α7 ACh receptor rescues neuronal cells from Aβ1-42-induced damage. PMID:26522221

  5. Muscle aches

    MedlinePlus

    ... common cause of muscle aches and pain is fibromyalgia , a condition that causes tenderness in your muscles ... imbalance, such as too little potassium or calcium Fibromyalgia Infections, including the flu, Lyme disease , malaria , muscle ...

  6. Chemistry and Behavioral Studies Identify Chiral Cyclopropanes as Selective α4β2-Nicotinic Acetylcholine Receptor Partial Agonists Exhibiting an Antidepressant Profile

    PubMed Central

    Zhang, Hankun; Tückmantel, Werner; Eaton, J. Brek; Yuen, Po-wai; Yu, Li-Fang; Bajjuri, Krishna Mohan; Fedolak, Allison; Wang, Daguang; Ghavami, Afshin; Caldarone, Barbara; Paterson, Neil E.; Lowe, David A.; Brunner, Daniela; Lukas, Ronald J.; Kozikowski, Alan P.

    2012-01-01

    Despite their discovery in the early 20th century and intensive study over the last twenty years, nicotinic acetylcholine receptors (nAChRs) are still far from being well understood. Only a few chemical entities targeting nAChRs are currently undergoing clinical trials, and even fewer have reached the marketplace. In our efforts to discover novel and truly selective nAChR ligands, we designed and synthesized a series of chiral cyclopropane-containing α4β2-specific ligands that display low nanomolar binding affinities and excellent subtype selectivity, while acting as partial agonists at α4β2-nAChRs. Their favorable antidepressant-like properties were demonstrated in the classical mouse forced swim test. Preliminary ADMET studies and broad screening towards other common neurotransmitter receptors were also carried out to further evaluate their safety profile and eliminate their potential off-target activity. These highly potent cyclopropane ligands possess superior subtype selectivity compared to other α4β2-nAChR agonists reported to date, including the marketed drug varenicline, and therefore may fully satisfy the crucial prerequisite for avoiding adverse side effects. These novel chemical entities could potentially be advanced to the clinic as new drug candidates for treating depression. PMID:22171543

  7. Positive allosteric modulators as an approach to nicotinic acetylcholine receptor- targeted therapeutics: advantages and limitations

    PubMed Central

    Williams, Dustin K.; Wang, Jingyi; Papke, Roger L.

    2011-01-01

    Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues. PMID:21575610

  8. The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera

    PubMed Central

    Jones, Andrew K.; Raymond-Delpech, Valerie; Thany, Steeve H.; Gauthier, Monique; Sattelle, David B.

    2006-01-01

    Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission and play roles in many cognitive processes. They are under intense research as potential targets of drugs used to treat neurodegenerative diseases and neurological disorders such as Alzheimer's disease and schizophrenia. Invertebrate nAChRs are targets of anthelmintics as well as a major group of insecticides, the neonicotinoids. The honey bee, Apis mellifera, is one of the most beneficial insects worldwide, playing an important role in crop pollination, and is also a valuable model system for studies on social interaction, sensory processing, learning, and memory. We have used the A. mellifera genome information to characterize the complete honey bee nAChR gene family. Comparison with the fruit fly Drosophila melanogaster and the malaria mosquito Anopheles gambiae shows that the honey bee possesses the largest family of insect nAChR subunits to date (11 members). As with Drosophila and Anopheles, alternative splicing of conserved exons increases receptor diversity. Also, we show that in one honey bee nAChR subunit, six adenosine residues are targeted for RNA A-to-I editing, two of which are evolutionarily conserved in Drosophila melanogaster and Heliothis virescens orthologs, and that the extent of editing increases as the honey bee lifecycle progresses, serving to maximize receptor diversity at the adult stage. These findings on Apis mellifera enhance our understanding of nAChR functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species. PMID:17065616

  9. Airway-related vagal preganglionic neurons express multiple nicotinic acetylcholine receptor subunits

    PubMed Central

    Dehkordi, Ozra; Kc, Prabha; Balan, Kannan V.; Haxhiu, Musa A.

    2007-01-01

    Nicotine acting centrally increases bronchomotor tone and airway secretion, suggesting that airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) express nicotinic acetylcholine receptors (nAChRs). In the present study, we examined the three main functionally characterized subtypes of nAChRs in the CNS, the α7 homomeric and α4β2 heteromeric receptors. First, we characterized the expression of these subunits at the message (mRNA) and protein levels in brain tissues taken from the rNA region, the site where AVPNs are located. In addition, double labeling fluorescent immunohistochemistry and confocal laser microscopy were used to define the presence of α7, α4, and β2 nAChRs on AVPNs that were retrogradely labeled with cholera toxin h subunit (CTb), injected into the upper lung lobe (n = 4) or extrathoracic trachea (n = 4). Our results revealed expression of all three studied subunits at mRNA and protein levels within the rNA region. Furthermore, virtually all identified AVPNs innervating intrapulmonary airways express α7 and α4 nAChR subunits. Similarly, a majority of labeled AVPNs projecting to extrathoracic trachea contain α7 and β2 subunits, but less than half of them show detectable α4 nAChR traits. These results suggest that AVPNs express three major nAChR subunits (α7, α4, and β2) that could assemble into functional homologous or heterologous pentameric receptors, mediating fast and sustained nicotinic effects on cholinergic outflow to the airways. PMID:16616705

  10. Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

    PubMed

    Mineur, Y S; Brunzell, D H; Grady, S R; Lindstrom, J M; McIntosh, J M; Marks, M J; King, S L; Picciotto, M R

    2009-04-01

    High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation. PMID:19077117

  11. Nicotinic α4 Receptor-Mediated Cholinergic Influences on Food Intake and Activity Patterns in Hypothalamic Circuits

    PubMed Central

    Schaaf, Laura; Heeley, Nicholas; Heuschmid, Lena; Bai, Yunjing; Barrantes, Francisco J.; Apergis-Schoute, John

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine’s (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4β2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHβE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHβE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHβE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4β2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4β2 nAChR-mediated transmission. PMID:26247203

  12. Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors.

    PubMed Central

    Nef, P; Oneyser, C; Alliod, C; Couturier, S; Ballivet, M

    1988-01-01

    Four genes encode the related protein subunits that assemble to form the nicotinic acetylcholine receptor (nAChR) at the motor endplate of vertebrates. We have isolated from the chicken genome four additional members of the same gene family whose protein products, termed alpha 2, alpha 3, alpha 4 and n alpha (non-alpha) probably define three distinct neuronal nAChR subtypes. The neuronal nAChR genes have identical structures consisting of six protein-coding exons and specify proteins that are best aligned with the chicken endplate alpha subunit, whose gene we have also characterized. mRNA transcripts encoding alpha 4 and n alpha are abundant in embryonic and in adult avian brain, whereas alpha 2 and alpha 3 transcripts are much scarcer. The same set of neuronal genes probably exists in all vertebrates since their counterparts have also been identified in the rat genome. Images PMID:3267226

  13. Characterization of alpha-conotoxin interactions with the nicotinic acetylcholine receptor and monoclonal antibodies.

    PubMed Central

    Ashcom, J D; Stiles, B G

    1997-01-01

    The venoms of predatory marine cone snails, Conus species, contain numerous peptides and proteins with remarkably diverse pharmacological properties. One group of peptides are the alpha-conotoxins, which consist of 13-19 amino acids constrained by two disulphide bonds. A biologically active fluorescein derivative of Conus geographus alpha-conotoxin GI (FGI) was used in novel solution-phase-binding assays with purified Torpedo californica nicotinic acetylcholine receptor (nAchR) and monoclonal antibodies developed against the toxin. The binding of FGI to nAchR or antibody had apparent dissociation constants of 10-100 nM. Structure-function studies with alpha-conotoxin GI analogues composed of a single disulphide loop revealed that different conformational restraints are necessary for effective toxin interactions with nAchR or antibodies. PMID:9359860

  14. Chronic ethanol (EtOH) feeding increases muscarinic receptor (mAChR) density in esophagus without parallel change in dose response (D-R) to cholinergic agonists

    SciTech Connect

    Keshavarzian, A.; Gordon, J.H.; Urban, G.; Fields, J.Z. VA Hospital, Hines, IL )

    1991-03-11

    The mAChR/effector pathway for signal transduction is important in the physiology of esophagus and mAChR alterations are involved in EtOH induced changes in several organs. To see if EtOH-induced increases in lower esophageal sphincter pressure (LESP) are due to upregulation of mAChR, the authors evaluated mAChR binding and D-R curves for bethanechol (IV) induced increases in LESP, and compared these values to changes in LESP after acute and chronic EtOH. EtOH was given to cats acutely or chronically. The number of mAChR sites (Bmax) in esophagus was lowered by acute EtOH, withdrawal from chronic EtOH raised Bmax. Acute injection of EtOH to cats in withdrawal reversed this increase in mAChR density. These changes correlated with the earlier data on EtOH-induced changes in LESP. In contrast, the D-R curve for bethanechol shifted to the right. Thus, the withdrawal-associated increase in Bmax is more likely to be a compensatory response to deficits distal to the receptor recognition site than to proximal deficits and doesn't cause LESP hyperactivity. Also, receptor binding changes do not necessarily translate into physiological changes.

  15. Gastrointestinal hypomotility with loss of enteric nicotinic acetylcholine receptors: active immunization model in mice

    PubMed Central

    Meeusen, Jeffrey W.; Haselkorn, Keegan E.; Fryer, James P.; Kryzer, Thomas J.; Gibbons, Simon J.; Xiao, Yingxian; Lennon, Vanda A.

    2012-01-01

    Background Autoimmune gastrointestinal dysmotility (AGID) is a limited form of dysautonomia. The only proven effector to date is IgG specific for ganglionic nicotinic-acetylcholine receptors containing α3 subunits (α3*-nAChR). Rabbits immunized with recombinant α3-polypeptide produce α3*-nAChR autoantibodies, and profound AGID ensues. Human and rabbit α3*-nAChR-specific-IgGs induce transient hypomotility when injected into mice. Here we describe success and problems encountered inducing gastrointestinal hypomotility in mice by active immunization. Methods We repeatedly injected young adult mice of seven different strains susceptible to autoimmunity (spontaneous diabetes or neural antigen immunization-induced myasthenia gravis or encephalomyelitis) with: i) α3-polypeptide, intradermally, or ii) live α3*-nAChR-expressing xenogeneic cells, intraperitoneally. We measured serum α3*-nAChR-IgG twice monthly, and terminally assessed blue dye gastrointestinal transit, total small intestinal α3*-nAChR content (radiochemically) and myenteric plexus neuron numbers (immunohistochemically, ileal-jejunal whole-mount preparations). Key Results Standard cutaneous inoculation with α3-polypeptide was minimally immunogenic, regardless of dose. Intraperitoneally-injected live cells were potently immunogenic. Self-reactive α3*-nAChR-IgG was induced only by rodent immunogen; small intestinal transit slowing and enteric α3*-nAChR loss required high serum levels. Ganglionic neurons were not lost. Conclusions & Inferences AGID is inducible in mice by active immunization. Accompanying enteric α3*-nAChR reduction without neuronal death is consistent with an IgG-mediated rather than T cell-mediated pathogenesis, as is improvement of symptoms in patients receiving antibody-depleting therapies. PMID:23072523

  16. Mutation Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Reduces Low-Sensitivity α4β2, and Increases α5α4β2, Nicotinic Receptor Surface Expression

    PubMed Central

    Nichols, Weston A.; Henderson, Brandon J.; Marotta, Christopher B.; Yu, Caroline Y.; Richards, Chris; Dougherty, Dennis A.; Lester, Henry A.

    2016-01-01

    A number of mutations in α4β2-containing (α4β2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the β2 subunit called β2V287L. Two α4β2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (α4)2(β2)3 subunit stoichiometry and a low-sensitivity subtype with (α4)3(β2)2 stoichiometry. The α5 nicotinic subunit also co-assembles with α4β2 to form a high-sensitivity α5α4β2 nAChR. Previous studies suggest that the β2V287L mutation suppresses low-sensitivity α4β2* nAChR expression in a knock-in mouse model and also that α5 co-expression improves the surface expression of ADNFLE mutant nAChRs in a cell line. To test these hypotheses further, we expressed mutant and wild-type (WT) nAChRs in oocytes and mammalian cell lines, and measured the effects of the β2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The β2V287L mutation reduced the EC50 values of high- and low-sensitivity α4β2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity α4β2 expression. In contrast, it did not affect the EC50 of α5α4β2 nAChRs for ACh. Measurements of the ACh responses of WT and mutant nAChRs expressed in mammalian cell lines using a voltage-sensitive fluorescent dye and whole-cell patch-clamping confirm the oocyte data. They also show that, despite reducing the maximum response, β2V287L increased the α4β2 response to a sub-saturating ACh concentration (1 μM). Finally, imaging SEP-tagged α5, α4, β2, and β2V287L subunits showed that β2V287L reduced total α4β2 nAChR surface expression, increased the number of β2 subunits per α4β2 receptor, and increased surface α5α4β2 nAChR expression. Thus, the β2V287L mutation alters the subunit

  17. Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice.

    PubMed

    Cole, R N; Ghazanfari, N; Ngo, S T; Gervásio, O L; Reddel, S W; Phillips, W D

    2010-09-01

    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(1)i.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 beta-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

  18. Multiple binding sites in the nicotinic acetylcholine receptors: An opportunity for polypharmacolgy.

    PubMed

    Iturriaga-Vásquez, Patricio; Alzate-Morales, Jans; Bermudez, Isabel; Varas, Rodrigo; Reyes-Parada, Miguel

    2015-11-01

    For decades, the development of selective compounds has been the main goal for chemists and biologists involved in drug discovery. However, diverse lines of evidence indicate that polypharmacological agents, i.e. those that act simultaneously at various protein targets, might show better profiles than selective ligands, regarding both efficacy and side effects. On the other hand, the availability of the crystal structure of different receptors allows a detailed analysis of the main interactions between drugs and receptors in a specific binding site. Neuronal nicotinic acetylcholine receptors (nAChRs) constitute a large and diverse family of ligand-gated ion channels (LGICs) that, as a product of its modulation, regulate neurotransmitter release, which in turns produce a global neuromodulation of the central nervous system. nAChRs are pentameric protein complexes in such a way that expression of compatible subunits can lead to various receptor assemblies or subtypes. The agonist binding site, located at the extracellular region, exhibits different properties depending on the subunits that conform the receptor. In the last years, it has been recognized that nAChRs could also contain one or more allosteric sites which could bind non-classical nicotinic ligands including several therapeutically useful drugs. The presence of multiple binding sites in nAChRs offers an interesting possibility for the development of novel polypharmacological agents with a wide spectrum of actions. PMID:26318763

  19. Presynaptic GABAB Autoreceptor Regulation of Nicotinic Acetylcholine Receptor Mediated [3H]-GABA Release from Mouse Synaptosomes

    PubMed Central

    McClure-Begley, Tristan D.; Grady, Sharon R.; Marks, Michael J.; Collins, Allan C.; Stitzel, Jerry A.

    2014-01-01

    Activation of nicotinic acetylcholine receptors (nAChRs) can elicit neurotransmitter release from presynaptic nerve terminals. Mechanisms contributing to cell-and-terminal specific regulation of nAChR-mediated neurotransmitter exocytosis are not fully understood. The experiments discussed here examine how activation of GABAB auto- and hetero-receptors suppress nAChR-mediated release of [3H]-GABA and [3H]-dopamine (3H-DA) from mouse striatal synaptosomes. Activation of presynaptic GABAB receptors with (R)-baclofen decreased both [3H]-GABA and [3H]-DA release evoked by potassium depolarization. However, when nAChRs were activated with ACh to evoke neurotransmitter release, (R)-baclofen had no effect on [3H]-DA release, but potently inhibited ACh-evoked [3H]-GABA release. Inhibition of nAChR-evoked [3H]-GABA release by (R)-baclofen was time sensitive and the effect was lost after prolonged exposure to the GABAB agonist. The early inhibitory effect of GABA activation on ACh-evoked [3H]-GABA release was partially attenuated by antagonists of the phosphatase, calcineurin. Furthermore, antagonists of protein kinase C (PKC) prevented the time-dependent loss of the inhibitory (R)-baclofen effect on [3H]-GABA release. These results suggest that α4β2*-nAChRs present on GABAergic nerve terminals in the striatum are subject to functional regulation by GABAB autoreceptors that is apparently cell-type specific, since it is absent from DAergic striatal nerve terminals. In addition, the functional modulation of α4β2*-type nAChRs on striatal GABAergic nerve terminals by GABAB autoreceptor activation is time-sensitive and appears to involve opposing actions of calcineurin and PKC. PMID:24953818

  20. Anxiolytic-like and anxiogenic-like effects of nicotine are regulated via diverse action at β2*nicotinic acetylcholine receptors

    PubMed Central

    Anderson, S M; Brunzell, D H

    2015-01-01

    Background and Purpose Nicotine dose-dependently activates or preferentially desensitizes β2 subunit containing nicotinic ACh receptors (β2*nAChRs). Genetic and pharmacological manipulations assessed effects of stimulation versus inhibition of β2*nAChRs on nicotine-associated anxiety-like phenotype. Experimental Approach Using a range of doses of nicotine in β2*nAChR subunit null mutant mice (β2KO; backcrossed to C57BL/6J) and their wild-type (WT) littermates, administration of the selective β2*nAChR agonist, 5I-A85380, and the selective β2*nAChR antagonist dihydro-β-erythroidine (DHβE), we determined the behavioural effects of stimulation and inhibition of β2*nAChRs in the light–dark and elevated plus maze (EPM) assays. Key Results Low-dose i.p. nicotine (0.05 mg·kg−1) supported anxiolysis-like behaviour independent of genotype whereas the highest dose (0.5 mg·kg−1) promoted anxiogenic-like phenotype in WT mice, but was blunted in β2KO mice for the measure of latency. Administration of 5I-A85380 had similar dose-dependent effects in C57BL/6J WT mice; 0.001 mg·kg−1 5I-A85380 reduced anxiety on an EPM, whereas 0.032 mg·kg−1 5I-A85380 promoted anxiogenic-like behaviour in both the light–dark and EPM assays. DHβE pretreatment blocked anxiogenic-like effects of 0.5 mg·kg−1 nicotine. Similarly to DHβE, pretreatment with low-dose 0.05 mg·kg−1 nicotine did not accumulate with 0.5 mg·kg−1 nicotine, but rather blocked anxiogenic-like effects of high-dose nicotine in the light–dark and EPM assays. Conclusions and Implications These studies provide direct evidence that low-dose nicotine inhibits nAChRs and demonstrate that inhibition or stimulation of β2*nAChRs supports the corresponding anxiolytic-like or anxiogenic-like effects of nicotine. Inhibition of β2*nAChRs may relieve anxiety in smokers and non-smokers alike. PMID:25625469

  1. Nicotine increases GABAergic input on rat dorsal raphe serotonergic neurons through alpha7 nicotinic acetylcholine receptor.

    PubMed

    Hernández-Vázquez, F; Chavarría, K; Garduño, J; Hernández-López, S; Mihailescu, S P

    2014-12-15

    The dorsal raphe nucleus (DRN) contains large populations of serotonergic (5-HT) neurons. This nucleus receives GABAergic inhibitory afferents from many brain areas and from DRN interneurons. Both GABAergic and 5-HT DRN neurons express functional nicotinic acetylcholine receptors (nAChRs). Previous studies have demonstrated that nicotine increases 5-HT release and 5-HT DRN neuron discharge rate by stimulating postsynaptic nAChRs and by increasing glutamate and norepinephrine release inside DRN. However, the influence of nicotine on the GABAergic input to 5-HT DRN neurons was poorly investigated. Therefore, the aim of this work was to determine the effect of nicotine on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of 5-HT DRN neurons and the subtype of nAChR(s) involved in this response. Experiments were performed in coronal slices obtained from young Wistar rats. GABAergic sIPSCs were recorded from post hoc-identified 5-HT DRN neurons with the whole cell voltage patch-clamp technique. Administration of nicotine (1 μM) increased sIPSC frequency in 72% of identified 5-HT DRN neurons. This effect was not reproduced by the α4β2 nAChR agonist RJR-2403 and was not influenced by TTX (1 μM). It was mimicked by the selective agonist for α7 nAChR, PNU-282987, and exacerbated by the positive allosteric modulator of the same receptor, PNU-120596. The nicotine-induced increase in sIPSC frequency was independent on voltage-gated calcium channels and dependent on Ca(2+)-induced Ca(2+) release (CICR). These results demonstrate that nicotine increases the GABAergic input to most 5-HT DRN neurons, by activating α7 nAChRs and producing CICR in DRN GABAergic terminals. PMID:25231613

  2. Nicotinic Acetylcholine Receptors Containing the α7-Like Subunit Mediate Contractions of Muscles Responsible for Space Positioning of the Snail, Helix pomatia L. Tentacle

    PubMed Central

    Kiss, Tibor; Krajcs, Nóra; Pirger, Zsolt; Hernádi, László

    2014-01-01

    Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh) responses in muscles to determine the properties of the ACh receptor (AChR), the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium), methyllycaconitine, α-Bungarotoxin (αBgTx) and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na+ and Ca2+ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery. PMID:25303328

  3. Positive allosteric modulation of alpha-7 nicotinic receptors promotes cell death by inducing Ca(2+) release from the endoplasmic reticulum.

    PubMed

    Guerra-Álvarez, María; Moreno-Ortega, Ana J; Navarro, Elisa; Fernández-Morales, José Carlos; Egea, Javier; López, Manuela G; Cano-Abad, María F

    2015-05-01

    Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca(2+) signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca(2+) signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca(2+) with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca(2+) concentration; (iii) released Ca(2+) from the ER by a Ca(2+) -induced Ca(2+) release mechanism only in α7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7-nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of α7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca(2+) , overloading of intracellular Ca(2+) , and neuronal cell death. This study focuses on how the type II positive allosteric modulator PNU120596 (PAM II PNU12) affects intracellular Ca(2+) signaling and cell viability. Using SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH), we find that PAM of α7-nAChRs with PNU120596: (i) increases inward calcium current (ICa ) and cytosolic Ca(2+) concentration ([Ca(2+) ]cyt ); (ii) releases Ca(2+) from the ER ([Ca(2

  4. Discovery of Potent Positive Allosteric Modulators of the α3β2 Nicotinic Acetylcholine Receptor by a Chemical Space Walk in ChEMBL

    PubMed Central

    2014-01-01

    While a plethora of ligands are known for the well studied α7 and α4β2 nicotinic acetylcholine receptor (nAChR), only very few ligands address the related α3β2 nAChR expressed in the central nervous system and at the neuromuscular junction. Starting with the public database ChEMBL organized in the chemical space of Molecular Quantum Numbers (MQN, a series of 42 integer value descriptors of molecular structure), a visual survey of nearest neighbors of the α7 nAChR partial agonist N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide (PNU-282,987) pointed to N-(2-halobenzyl)-3-aminoquinuclidines as possible nAChR modulators. This simple “chemical space walk” was performed using a web-browser available at www.gdb.unibe.ch. Electrophysiological recordings revealed that these ligands represent a new and to date most potent class of positive allosteric modulators (PAMs) of the α3β2 nAChR, which also exert significant effects in vivo. The present discovery highlights the value of surveying chemical space neighbors of known drugs within public databases to uncover new pharmacology. PMID:24593915

  5. The use of α-conotoxin ImI to actualize the targeted delivery of paclitaxel micelles to α7 nAChR-overexpressing breast cancer.

    PubMed

    Mei, Dong; Lin, Zhiqiang; Fu, Jijun; He, Bing; Gao, Wei; Ma, Ling; Dai, Wenbing; Zhang, Hua; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Lu, Wanliang; Zhou, Demin; Zhang, Qiang

    2015-02-01

    Alpha7 nicotinic acetylcholine receptor (α7 nAChR), a ligand-gated ion channel, is increasingly emerging as a new tumor target owing to its expression specificity and significancy for cancer. In an attempt to increase the targeted drug delivery to the α7 nAChR-overexpressing tumors, herein, α-conotoxin ImI, a disulfide-rich toxin with highly affinity for α7 nAChR, was modified on the PEG-DSPE micelles (ImI-PMs) for the first time. The DLS, TEM and HPLC detections showed the spherical nanoparticle morphology about 20 nm with negative charge and high drug encapsulation. The ligand modification did not induce significant differences. The immunofluorescence assay confirmed the expression level of α7 nAChR in MCF-7 cells. In vitro and in vivo experiments demonstrated that the α7 nAChR-targeted nanomedicines could deliver more specifically and faster into α7 nAChR-overexpressing MCF-7 cells. Furthermore, fluo-3/AM fluorescence imaging technique indicated that the increased specificity was attributed to the ligand-receptor interaction, and the inducitivity for intracellular Ca(2+) transient by ImI was still remained after modification. Moreover, paclitaxel, a clinical frequently-used anti-tumor drug for breast cancer, was loaded in ImI-modified nanomedicines to evaluate the targeting efficacy. Besides of exhibiting greater cytotoxicity and inducing more cell apoptosis in vitro, paclitaxel-loaded ImI-PMs displayed stronger anti-tumor efficacy in MCF-7 tumor-bearing nu/nu mice. Finally, the active targeting system showed low systemic toxicity and myelosuppression evidenced by less changes in body weight, white blood cells, neutrophilic granulocyte and platelet counts. In conclusion, α7 nAChR is also a promising target for anti-tumor drug delivery and in this case, α-conotoxin ImI-modified nanocarrier is a potential delivery system for targeting α7 nAChR-overexpressing tumors. PMID:25542793

  6. Probing the Non-Canonical Interface for Agonist Interaction with an α5 Containing Nicotinic Acetylcholine Receptor*

    PubMed Central

    Marotta, Christopher B.; Dilworth, Crystal N.; Lester, Henry A.; Dougherty, Dennis A.

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit are of interest because genome-wide association studies and candidate gene studies have identified polymorphisms in the α5 gene that are linked to an increased risk for nicotine dependence, lung cancer, and/or alcohol addiction. To probe the functional impact of an α5 subunit on nAChRs, a method to prepare a homogeneous population of α5-containing receptors must be developed. Here we use a gain of function (9') mutation to isolate populations of α5-containing nAChRs for characterization by electrophysiology. We find that the α5 subunit modulates nAChR rectification when co-assembled with α4 and β2 subunits. We also probe the α5–α4 interface for possible ligand binding interactions. We find that mutations expected to ablate an agonist binding site involving the α5 subunit have no impact on receptor function. The most straightforward interpretation of this observation is that agonists do not bind at the α5–α4 interface, in contrast to what has recently been demonstrated for the α4–α4 interface in related receptors. In addition, our mutational results suggest that the α5 subunit does not replace the α4 or β2 subunits and is relegated to occupying only the auxiliary position of the pentameric receptor. PMID:24144909

  7. Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle-specific kinase autoantibody.

    PubMed

    Morsch, Marco; Reddel, Stephen W; Ghazanfari, Nazanin; Toyka, Klaus V; Phillips, William D

    2013-05-15

    In myasthenia gravis, the neuromuscular junction is impaired by the antibody-mediated loss of postsynaptic acetylcholine receptors (AChRs). Muscle weakness can be improved upon treatment with pyridostigmine, a cholinesterase inhibitor, or with 3,4-diaminopyridine, which increases the release of ACh quanta. The clinical efficacy of pyridostigmine is in doubt for certain forms of myasthenia. Here we formally examined the effects of these compounds in the antibody-induced mouse model of anti-muscle-specific kinase (MuSK) myasthenia gravis. Mice received 14 daily injections of IgG from patients with anti-MuSK myasthenia gravis. This caused reductions in postsynaptic AChR densities and in endplate potential amplitudes. Systemic delivery of pyridostigmine at therapeutically relevant levels from days 7 to 14 exacerbated the anti-MuSK-induced structural alterations and functional impairment at motor endplates in the diaphragm muscle. No such effect of pyridostigmine was found in mice receiving control human IgG. Mice receiving smaller amounts of MuSK autoantibodies did not display overt weakness, but 9 days of pyridostigmine treatment precipitated generalised muscle weakness. In contrast, one week of treatment with 3,4-diaminopyridine enhanced neuromuscular transmission in the diaphragm muscle. Both pyridostigmine and 3,4-diaminopyridine increase ACh in the synaptic cleft yet only pyridostigmine potentiated the anti-MuSK-induced decline in endplate ACh receptor density. These results thus suggest that ongoing pyridostigmine treatment potentiates anti-MuSK-induced AChR loss by prolonging the activity of ACh in the synaptic cleft. PMID:23440963

  8. Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle-specific kinase autoantibody

    PubMed Central

    Morsch, Marco; Reddel, Stephen W; Ghazanfari, Nazanin; Toyka, Klaus V; Phillips, William D

    2013-01-01

    In myasthenia gravis, the neuromuscular junction is impaired by the antibody-mediated loss of postsynaptic acetylcholine receptors (AChRs). Muscle weakness can be improved upon treatment with pyridostigmine, a cholinesterase inhibitor, or with 3,4-diaminopyridine, which increases the release of ACh quanta. The clinical efficacy of pyridostigmine is in doubt for certain forms of myasthenia. Here we formally examined the effects of these compounds in the antibody-induced mouse model of anti-muscle-specific kinase (MuSK) myasthenia gravis. Mice received 14 daily injections of IgG from patients with anti-MuSK myasthenia gravis. This caused reductions in postsynaptic AChR densities and in endplate potential amplitudes. Systemic delivery of pyridostigmine at therapeutically relevant levels from days 7 to 14 exacerbated the anti-MuSK-induced structural alterations and functional impairment at motor endplates in the diaphragm muscle. No such effect of pyridostigmine was found in mice receiving control human IgG. Mice receiving smaller amounts of MuSK autoantibodies did not display overt weakness, but 9 days of pyridostigmine treatment precipitated generalised muscle weakness. In contrast, one week of treatment with 3,4-diaminopyridine enhanced neuromuscular transmission in the diaphragm muscle. Both pyridostigmine and 3,4-diaminopyridine increase ACh in the synaptic cleft yet only pyridostigmine potentiated the anti-MuSK-induced decline in endplate ACh receptor density. These results thus suggest that ongoing pyridostigmine treatment potentiates anti-MuSK-induced AChR loss by prolonging the activity of ACh in the synaptic cleft. PMID:23440963

  9. Characterization of a Multiple Ligand-Gated Ion Channel Cellular Membrane Affinity Chromatography Column and Identification of Endogenously Expressed Receptors in Astrocytoma Cell Lines

    PubMed Central

    Kitabatake, T.; Moaddel, R.; Cole, R.; Gandhari, M.; Frazier, C.; Hartenstein, J.; Rosenberg, A.; Bernier, M.; Wainer, I. W.

    2008-01-01

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [3H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric α7 nicotinic acetylcholine receptors (α7 nAChR) and heteromeric nicotinic acetylcholine receptors (αxβy nAChRs), which was confirmed by the addition of subtype-specific inhibitors, κ-bungarotoxin (α7 nAChR) and K-bungarotoxin (αxβy nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), γ-aminobutyric acid (GABAA) and N-methyl-d-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABAA receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  10. Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines.

    PubMed

    Kitabatake, T; Moaddel, R; Cole, R; Gandhari, M; Frazier, C; Hartenstein, J; Rosenberg, A; Bernier, M; Wainer, I W

    2008-11-15

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  11. Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine

    PubMed Central

    Alberola-Die, Armando; Fernández-Ballester, Gregorio; González-Ros, José M.; Ivorra, Isabel; Morales, Andrés

    2016-01-01

    Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (IACh) in a dose-dependent manner (IC50 close to 70 μM), but unlike lidocaine, DEA did not affect IACh desensitization. IACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced IACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and

  12. Alpha9 nicotinic acetylcholine receptors and the treatment of pain.

    PubMed

    McIntosh, J Michael; Absalom, Nathan; Chebib, Mary; Elgoyhen, Ana Belén; Vincler, Michelle

    2009-10-01

    Chronic pain is a vexing worldwide problem that causes substantial disability and consumes significant medical resources. Although there are numerous analgesic medications, these work through a small set of molecular mechanisms. Even when these medications are used in combination, substantial amounts of pain often remain. It is therefore highly desirable to develop treatments that work through distinct mechanisms of action. While agonists of nicotinic acetylcholine receptors (nAChRs) have been intensively studied, new data suggest a role for selective antagonists of nAChRs. alpha-Conotoxins are small peptides used offensively by carnivorous marine snails known as Conus. A subset of these peptides known as alpha-conotoxins RgIA and Vc1.1 produces both acute and long lasting analgesia. In addition, these peptides appear to accelerate the recovery of function after nerve injury, possibly through immune mediated mechanisms. Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of alpha9alpha10 nAChRs. A recent study also reported that these alpha9alpha10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on alpha9* nAChRs.(1). PMID:19477168

  13. Nicotine-Mediated Regulation of Nicotinic Acetylcholine Receptors in Non-Small Cell Lung Adenocarcinoma by E2F1 and STAT1 Transcription Factors

    PubMed Central

    Schaal, Courtney; Chellappan, Srikumar

    2016-01-01

    Cigarette smoking is the major risk factor for non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. Nicotine, the addictive component of tobacco smoke, can induce proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, and survival in NSCLC cell lines, as well as growth and metastasis of NSCLC in mice. This nicotine-mediated tumor progression is facilitated through activation of nicotinic acetylcholine receptors (nAChRs), specifically the α7 subunit; however, how the α7 nAChR gene is regulated in lung adenocarcinoma is not fully clear. Here we demonstrate that the α7 nAChR gene promoter is differentially regulated by E2F and STAT transcription factors through a competitive interplay; E2F1 induces the promoter, while STAT transcription factors repress it by binding to an overlapping site at a region -294 through -463bp upstream of the transcription start site. Treatment of cells with nicotine induced the mRNA and protein levels of α7 nAChR; this could be abrogated by treatment with inhibitors targeting Src, PI3K, MEK, α7 nAChR, CDK4/6 or a disruptor of the Rb-Raf-1 interaction. Further, nicotine–mediated induction of α7 nAChR was reduced when E2F1 was depleted and in contrast elevated when STAT1 was depleted by siRNAs. Interestingly, extracts from e-cigarettes, which have recently emerged as healthier alternatives to traditional cigarette smoking, can also induce α7 nAChR expression in a manner similar to nicotine. These results suggest an autoregulatory feed-forward loop that induces the levels of α7 nAChR upon exposure to nicotine, which enhances the strength of the signal. It can be imagined that such an induction of α7 nAChR contributes to the tumor-promoting functions of nicotine. PMID:27228072

  14. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  15. Molecular-Dynamics Simulations of ELIC a Prokaryotic Homologue of the Nicotinic Acetylcholine Receptor

    SciTech Connect

    Cheng, Xiaolin; Ivanov, Ivaylo N; Wang, Hailong; McCammon, Jonathan

    2009-01-01

    The ligand-gated ion channel from Erwinia chrysanthemi (ELIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor (nAChR) that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. ELIC is similar to the nAChR in its primary sequence and overall subunit organization, but despite their structural similarity, it is not clear whether these two ligand-gated ion channels operate in a similar manner. Further, it is not known to what extent mechanistic insights gleaned from the ELIC structure translate to eukaryotic counterparts such as the nAChR. Here we use molecular-dynamics simulations to probe the conformational dynamics and hydration of the transmembrane pore of ELIC. The results are compared with those from our previous simulation of the human ?7 nAChR. Overall, ELIC displays increased stability compared to the nAChR, whereas the two proteins exhibit remarkable similarity in their global motion and flexibility patterns. The majority of the increased stability of ELIC does not stem from the deficiency of the models used in the simulations, and but rather seems to have a structural basis. Slightly altered dynamical correlation features are also observed among several loops within the membrane region. In sharp contrast to the nAChR, ELIC is completely dehydrated from the pore center to the extracellular end throughout the simulation. Finally, the simulation of an ELIC mutant substantiates the important role of F246 on the stability, hydration and possibly function of the ELIC channel.

  16. 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. PMID:27130855

  17. The positive allosteric modulator of α7 nicotinic acetylcholine receptors, 3-furan-2-yl-N-p-tolyl-acrylamide, enhances memory processes and stimulates ERK1/2 phosphorylation in mice.

    PubMed

    Targowska-Duda, Katarzyna M; Wnorowski, Artur; Budzynska, Barbara; Jozwiak, Krzysztof; Biala, Grazyna; Arias, Hugo R

    2016-04-01

    To determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs), improves memory processes, passive avoidance tests were conducted in male mice after acute and chronic treatments. To determine the neuronal mechanisms underlying the promnesic activity elicited by PAM-2, the effect of this ligand on α7 nAChR up-regulation and ERK1/2 phosphorylation was assessed in the hippocampus and prefrontal cortex. The results indicate that: (1) PAM-2 improves memory acquisition/consolidation after acute treatment (Day 2) and memory consolidation after chronic treatment (Day 22). Although no effect was observed on α7 nAChR up-regulation, the chronic, but not acute, PAM-2 treatment increases ERK1/2 kinase phosphorylation, (2) the promnesic activity of PAM-2 was inhibited by methyllycaconitine, a selective α7-antagonist, confirming the role of α7 nAChRs, (3) a synergistic (acute) effect was observed between inactive doses of PAM-2 (0.1mg/kg) and DMXBA (0.3mg/kg), a selective α7-agonist, and (4) PAM-2 reversed the memory impairment elicited by scopolamine, a muscarinic antagonist. The results demonstrate that PAM-2 presents promnesic activity mediated by α7 nAChRs, and is able to trigger ERK1/2 phosphorylation only after chronic treatment. PMID:26778787

  18. Cell-surface translational dynamics of nicotinic acetylcholine receptors

    PubMed Central

    Barrantes, Francisco J.

    2014-01-01

    Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor–receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional “trapping,” and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain α7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the α7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain. PMID

  19. Bladder outlet obstruction causes up-regulation of nicotinic acetylcholine receptors in bladder-projecting pelvic ganglion neurons.

    PubMed

    Chung, Hyun-Chul; Lee, Choong-Ku; Park, Kwang-Hwa; Jeong, Seong-Woo

    2015-03-30

    Pelvic ganglion (PG) neurons relay sympathetic and parasympathetic signals to the lower urinary tract, comprising the urinary bladder and bladder outlet, and are thus essential for both storage and voiding reflexes. Autonomic transmission is mediated by activation of the nicotinic acetylcholine receptor (nAChR) in PG neurons. Previously, bladder outlet obstruction (BOO), secondary to benign prostatic hyperplasia, was found to increase soma sizes of bladder-projecting PG neurons. To date, however, it remains unknown whether these morphological changes are accompanied by functional plasticity in PG neurons. In the present study, we investigated whether BOO alters acetylcholine receptor (nAChR) transcript expression and current density in bladder PG neurons. Partial ligation of the rat urethra for six weeks induced detrusor overactivity (DO), as observed during cystometrical measurement. In rats exhibiting DO, membrane capacitance of parasympathetic bladder PG neurons was selectively increased. Real-time PCR analysis revealed that BOO enhanced the expression of the transcripts encoding the nAChR α3 and β4 subunits in PG neurons. Notably, BOO significantly increased ACh-evoked current density in parasympathetic bladder PG neurons, whereas no changes were observed in sympathetic bladder and parasympathetic penile PG neurons. In addition, other ligand-gated ionic currents were immune to BOO in bladder PG neurons. Taken together, these data suggest that BOO causes upregulation of nAChR in parasympathetic bladder PG neurons, which in turn may potentiate ganglionic transmission and contribute to the development of DO. PMID:25625357

  20. Modulation of muscarinic and micotinic cholinergic receptor mediated catecholamine secretion in guinea pig chromaffin cells by phorbol esters

    SciTech Connect

    Figueiredo, J.C.; Fisher, S.K.; Horowitz, M.I.

    1986-05-01

    Isolated guinea pig chromaffin cells possess both nicotinic (nAChR) and muscarinic (mAChR) cholinergic receptors that are positively coupled to catecholamine (CA) release. Sixty to 70% of CA release is mediated by nAChRs and 30-40% by mAChRs. In the absence of added calcium, nAChR mediated CA release was reduced by 65% whereas the muscarinic response was unaffected. The addition of 100nM 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), also resulted in an increased CA release. Temporally and quantitatively, this response resembled that of mAChR activation. Addition of optimal concentrations of nicotine (50..mu..M) and TPA (100nM) induced a synergistic increase in CA release. Addition of muscarine (1mM) and TPA resulted in an additive response despite a 40-60% inhibition of mAChR mediated inositol phosphate release by TPA. Thus, in guinea pig chromaffin cells, it appears that PKC activation alone is a sufficient stimulus for CA release and that activation of both nicotinic and muscarinic receptors may further increase this enzyme's activity.

  1. The non-competitive blockade of GABAA receptors by an aqueous extract of water hemlock (Cicuta douglassi) tubers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement from the desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChR). In this study, we tested the hypothesis that the piperidine alkaloid anabaseine a 1,2-dehydropiperidine and anabasin...

  2. Activation and desensitization of peripheral muscle and neuronal nicotinic acetylcholine receptors by selected, naturally-occurring pyridine alkaloids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Teratogenic alkaloids can cause developmental defects due to inhibition of fetal movement that results from desensitization of fetal muscletype nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiper...

  3. The non-competitive blockade of GABAA receptors by an aqueous extract of water hemlock )Cicuta douglassi) tubers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement from the desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChR). In this study, we tested the hypothesis that the piperidine alkaloid anabaseine a 1,2-dehydropiperidine and anabasin...

  4. Neuronal nicotinic receptors as brain targets for pharmacotherapy of drug addiction.

    PubMed

    Rahman, Shafiqur; López-Hernández, Gretchen Y; Corrigall, William A; Papke, Roger L

    2008-11-01

    Nicotine addiction and other forms of drug addiction continue to be significant public health problems in the United States and the rest of the world. Accumulated evidence indicates that brain nicotinic acetylcholine receptors (nAChRs) are a heterogenous family of ion channels expressed in the various parts of the brain. A growing body of preclinical studies suggests that brain nAChRs are critical targets for the development of pharmacotherapies for nicotine and other drug addictions. In this review, we will discuss the nAChR subtypes, their function in response to endogenous brain transmitters, and how their functions are regulated in the presence of nicotine. Furthermore, we will discuss the role of nAChRs in mediating nicotine-induced addictive behavior in animal models. Additionally, we will provide an overview of the effects of nicotine and nicotinic compounds on the mesolimbic dopamine system, part of the reinforcement/reward circuitry of the brain, as an example of the neurochemical basis of nicotine addiction and other drug addictions. An appreciation of the complexity of nicotinic receptors and their regulation will be necessary for the development of nicotinic receptor modulators as potential pharmacotherapy for drug addiction. PMID:19128201

  5. Hippocampal GABAergic interneurons coexpressing alpha7-nicotinic receptors and connexin-36 are able to improve neuronal viability under oxygen-glucose deprivation.

    PubMed

    Voytenko, L P; Lushnikova, I V; Savotchenko, A V; Isaeva, E V; Skok, M V; Lykhmus, O Yu; Patseva, M A; Skibo, G G

    2015-08-01

    The hippocampal interneurons are very diverse by chemical profiles and rather inconsistent by sensitivity to CI. Some hippocampal GABAergic interneurons survive certain time after ischemia while ischemia-sensitive interneurons and pyramidal neurons are damaged. GABAergic signaling, nicotinic receptors expressing α7-subunit (α7nAChRs(+)) and connexin-36 (Cx36(+), electrotonic gapjunctions protein) contradictory modulate post-ischemic environment. We hypothesized that hippocampal ischemia-resistant GABAergic interneurons coexpressing glutamate decarboxylase-67 isoform (GAD67(+)), α7nAChRs(+), Cx36(+) are able to enhance neuronal viability. To check this hypothesis the histochemical and electrophysiological investigations have been performed using rat hippocampal organotypic culture in the condition of 30-min oxygen-glucose deprivation (OGD). Post-OGD reoxygenation (4h) revealed in CA1 pyramidal layer numerous damaged cells, decreased population spike amplitude and increased pair-pulse depression. In these conditions GAD67(+) interneurons displayed the OGD-resistance and significant increase of GABA synthesis/metabolism (GAD67-immunofluorescence, mitochondrial activity). The α7nAChRs(+) and Cx36(+) co-localizations were revealed in resistant GAD67(+) interneurons. Under OGD: GABAA-receptors (GABAARs) blockade increased cell damage and exacerbated the pair-pulse depression in CA1 pyramidal layer; α7nAChRs and Cx36-channels separate blockades sufficiently decreased cell damage while interneuronal GAD67-immunofluorescence and mitochondrial activity were similar to the control. Thus, hippocampal GABAergic interneurons co-expressing α7nAChRs and Cx36 remained resistant certain time after OGD and were able to modulate CA1 neuron survival through GABAARs, α7nAChRs and Cx36-channels activity. The enhancements of the neuronal viability together with GABA synthesis/metabolism normalization suggest cooperative neuroprotective mechanism that could be used for increase in

  6. A comparative study of the effects of the intravenous self-administration or subcutaneous minipump infusion of nicotine on the expression of brain neuronal nicotinic receptor subtypes.

    PubMed

    Moretti, Milena; Mugnaini, Manolo; Tessari, Michela; Zoli, Michele; Gaimarri, Annalisa; Manfredi, Irene; Pistillo, Francesco; Clementi, Francesco; Gotti, Cecilia

    2010-08-01

    Long-term nicotine exposure changes neuronal acetylcholine nicotinic receptor (nAChR) subtype expression in the brains of smokers and experimental animals. The aim of this study was to investigate nicotine-induced changes in nAChR expression in two models commonly used to describe the effects of nicotine in animals: operant (two-lever presses) intravenous self-administration (SA) and passive subcutaneous nicotine administration via an osmotic minipump (MP). In the MP group, alpha4beta2 nAChRs were up-regulated in all brain regions, alpha6beta2* nAChRs were down-regulated in the nucleus accumbens (NAc) and caudate-putamen, and alpha7 nAChRs were up-regulated in the caudal cerebral cortex (CCx); the up-regulation of alpha4beta2alpha5 nAChRs in the CCx was also suggested. In the SA group, alpha4beta2 up-regulation was lower and limited to the CCx and NAc; there were no detectable changes in alpha6beta2* or alpha7 nACRs. In the CCx of the MP rats, there was a close correlation between the increase in alpha4beta2 binding and alpha4 and beta2 subunit levels measured by means of Western blotting, demonstrating that the up-regulation was due to an increase in alpha4beta2 proteins. Western blotting also showed that the increase in the beta2 subunit exceeded that of the alpha4 subunit, suggesting that a change in alpha4beta2 stoichiometry may occur in vivo as has been shown in vitro. These results show that nicotine has an area-specific effect on receptor subtypes, regardless of its administration route, but the effect is quantitatively greater in the case of MP administration. PMID:20439469

  7. Functional nicotinic acetylcholine receptor reconstitution in Au(111)-supported thiolipid monolayers

    NASA Astrophysics Data System (ADS)

    Pissinis, Diego E.; Diaz, Carolina; Maza, Eliana; Bonini, Ida C.; Barrantes, Francisco J.; Salvarezza, Roberto C.; Schilardi, Patricia L.

    2015-09-01

    The insertion and function of the muscle-type nicotinic acetylcholine receptor (nAChR) in Au(111)-supported thiolipid self-assembled monolayers have been studied by atomic force microscopy (AFM), surface plasmon resonance (SPR), and electrochemical techniques. It was possible for the first time to resolve the supramolecular arrangement of the protein spontaneously inserted in a thiolipid monolayer in an aqueous solution. Geometric supramolecular arrays of nAChRs were observed, most commonly in a triangular form compatible with three nAChR dimers of ~20 nm each. Addition of the full agonist carbamoylcholine activated and opened the nAChR ion channel, as revealed by the increase in capacitance relative to that of the nAChR-thiolipid system under basal conditions. Thus, the self-assembled system appears to be a viable biomimetic model to measure ionic conductance mediated by ion-gated ion channels under different experimental conditions, with potential applications in biotechnology and pharmacology.

  8. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    SciTech Connect

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  9. Functional expression of human α9* nicotinic acetylcholine receptors in X. laevis oocytes is dependent on the α9 subunit 5' UTR.

    PubMed

    Filchakova, Olena; McIntosh, J Michael

    2013-01-01

    Nicotinic acetylcholine receptors (nAChRs) containing the α9 subunit are expressed in a wide variety of non-neuronal tissues ranging from immune cells to breast carcinomas. The α9 subunit is able to assemble into a functional homomeric nAChR and also co-assemble with the α10 subunit into functional heteromeric nAChRs. Despite the increasing awareness of the important roles of this subunit in vertebrates, the study of human α9-containing nAChRs has been severely limited by difficulties in its expression in heterologous systems. In Xenopus laevis oocytes, functional expression of human α9α10 nAChRs is very low compared to that of rat α9α10 nAChRs. When oocytes were co-injected with cRNA of α9 and α10 subunits of human versus those of rat, oocytes with the rat α9 human α10 combination had an ∼-fold higher level of acetylcholine-gated currents (I(ACh)) than those with the human α9 rat α10 combination, suggesting difficulties with human α9 expression. When the ratio of injected human α9 cRNA to human α10 cRNA was increased from 1∶1 to 5∶1, I(ACh) increased 36-fold (from 142±23 nA to 5171±748 nA). Functional expression of human α9-containing receptors in oocytes was markedly improved by appending the 5'-untranslated region of alfalfa mosaic virus RNA4 to the 5'-leader sequence of the α9 subunit cRNA. This increased the functional expression of homomeric human α9 receptors by 70-fold (from 7±1 nA to 475±158 nA) and of human α9α10 heteromeric receptors by 80-fold (from 113±62 nA to 9192±1137 nA). These findings indicate the importance of the composition of the 5' untranslated leader sequence for expression of α9-containing nAChRs. PMID:23717646

  10. Agonist and antagonist effects of tobacco-related nitrosamines on human α4β2 nicotinic acetylcholine receptors

    PubMed Central

    Brusco, Simone; Ambrosi, Paola; Meneghini, Simone; Becchetti, Andrea

    2015-01-01

    Regulation of the “neuronal” nicotinic acetylcholine receptors (nAChRs) is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) on human α4β2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 μM. At 100 μM, it activated 16% of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4β2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 μM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo. PMID:26441658

  11. The Role of Nicotinic Acetylcholine Receptors in the Medial Prefrontal Cortex and Hippocampus in Trace Fear Conditioning

    PubMed Central

    Raybuck, J. D.; Gould, T. J.

    2010-01-01

    Acute nicotine enhances multiple types of learning including trace fear conditioning but the underlying neural substrates of these effects are not well understood. Trace fear conditioning critically involves the medial prefrontal cortex and hippocampus, which both express nicotinic acetylcholine receptors (nAChRs). Therefore, nicotine could act in either or both areas to enhance trace fear conditioning. To identify the underlying neural areas and nAChR subtypes, we examined the effects of infusion of nicotine, or nicotinic antagonists dihydro-beta-erythroidine (DHβE: high-affinity nAChRs) or methyllycaconitine (MLA: low-affinity nAChRs) into the dorsal hippocampus, ventral hippocampus, and medial prefrontal cortex (mPFC) on trace and contextual fear conditioning. We found that the effects of nicotine on trace and contextual fear conditioning vary by brain region and nAChR subtype. The dorsal hippocampus was involved in the effects of nicotine on both trace and contextual fear conditioning but each task was sensitive to different doses of nicotine. Additionally, dorsal hippocampal infusion of the antagonist DHβE produced deficits in trace but not contextual fear conditioning. Nicotine infusion into the ventral hippocampus produced deficits in both trace and contextual fear conditioning. In the mPFC, nicotine enhanced trace but not contextual fear conditioning. Interestingly, infusion of the antagonists MLA or DHβE in the mPFC also enhanced trace fear conditioning. These findings suggest that nicotine acts on different substrates to enhance trace versus contextual fear conditioning, and that nicotine-induced desensitization of nAChRs in the mPFC may contribute to the effects of nicotine on trace fear conditioning. PMID:20727979

  12. Functional Impact of 14 Single Nucleotide Polymorphisms Causing Missense Mutations of Human α7 Nicotinic Receptor

    PubMed Central

    Zhang, Qinhui; Du, Yingjie; Zhang, Jianliang; Xu, Xiaojun; Xue, Fenqin; Guo, Cong; Huang, Yao; Lukas, Ronald J.; Chang, Yongchang

    2015-01-01

    The α7nicotinic receptor (nAChR) is a major subtype of the nAChRs in the central nervous system, and the receptor plays an important role in brain function. In the dbSNP database, there are 55 single nucleotide polymorphisms (SNPs) that cause missense mutations of the human α7nAChR in the coding region. In this study, we tested the impact of 14 SNPs that cause missense mutations in the agonist binding site or the coupling region between binding site and channel gate on the receptor function. The wild type or mutant receptors were expressed or co-expressed in Xenopus oocytes, and the agonist-induced currents were tested using two-electrode voltage clamp. Our results demonstrated that 6 mutants were nonfunctional, 4 mutants had reduced current expression, and 1 mutants altered ACh and nicotine efficacy in the opposite direction, and one additional mutant had slightly reduced agonist sensitivity. Interestingly, the function of most of these nonfunctional mutants could be rescued by α7nAChR positive allosteric modulator PNU-120596 and agonist-PAM 4BP-TQS. Finally, when coexpressed with the wild type, the nonfunctional mutants could also influence the receptor function. These changes of the receptor properties by the mutations could potentially have an impact on the physiological function of the α7nAChR-mediated cholinergic synaptic transmission and anti-inflammatory effects in the human SNP carriers. Rescuing the nonfunctional mutants could provide a novel way to treat the related disorders. PMID:26340537

  13. Functional Impact of 14 Single Nucleotide Polymorphisms Causing Missense Mutations of Human α7 Nicotinic Receptor.

    PubMed

    Zhang, Qinhui; Du, Yingjie; Zhang, Jianliang; Xu, Xiaojun; Xue, Fenqin; Guo, Cong; Huang, Yao; Lukas, Ronald J; Chang, Yongchang

    2015-01-01

    The α7nicotinic receptor (nAChR) is a major subtype of the nAChRs in the central nervous system, and the receptor plays an important role in brain function. In the dbSNP database, there are 55 single nucleotide polymorphisms (SNPs) that cause missense mutations of the human α7nAChR in the coding region. In this study, we tested the impact of 14 SNPs that cause missense mutations in the agonist binding site or the coupling region between binding site and channel gate on the receptor function. The wild type or mutant receptors were expressed or co-expressed in Xenopus oocytes, and the agonist-induced currents were tested using two-electrode voltage clamp. Our results demonstrated that 6 mutants were nonfunctional, 4 mutants had reduced current expression, and 1 mutants altered ACh and nicotine efficacy in the opposite direction, and one additional mutant had slightly reduced agonist sensitivity. Interestingly, the function of most of these nonfunctional mutants could be rescued by α7nAChR positive allosteric modulator PNU-120596 and agonist-PAM 4BP-TQS. Finally, when coexpressed with the wild type, the nonfunctional mutants could also influence the receptor function. These changes of the receptor properties by the mutations could potentially have an impact on the physiological function of the α7nAChR-mediated cholinergic synaptic transmission and anti-inflammatory effects in the human SNP carriers. Rescuing the nonfunctional mutants could provide a novel way to treat the related disorders. PMID:26340537

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

    PubMed

    Jensen, Majbrit M; Arvaniti, Maria; Mikkelsen, Jens D; Michalski, Dominik; Pinborg, Lars H; Härtig, Wolfgang; Thomsen, Morten S

    2015-04-01

    Alzheimer's disease (AD) is a neurodegenerative disorder involving impaired cholinergic neurotransmission and dysregulation of nicotinic acetylcholine receptors (nAChRs). Ly-6/neurotoxin (Lynx) proteins have been shown to modulate cognition and neural plasticity by binding to nAChR subtypes and modulating their function. Hence, changes in nAChR regulatory proteins such as Lynx proteins could underlie the dysregulation of nAChRs in AD. Using Western blotting, we detected bands corresponding to the Lynx proteins prostate stem cell antigen (PSCA) and Lypd6 in human cortex indicating that both proteins are present in the human brain. We further showed that PSCA forms stable complexes with the α4 nAChR subunit and decreases nicotine-induced extracellular-signal regulated kinase phosphorylation in PC12 cells. In addition, we analyzed protein levels of PSCA and Lypd6 in postmortem tissue of medial frontal gyrus from AD patients and found significantly increased PSCA levels (approximately 70%). In contrast, no changes in Lypd6 levels were detected. In concordance with our findings in AD patients, PSCA levels were increased in the frontal cortex of triple transgenic mice with an AD-like pathology harboring human transgenes that cause both age-dependent β-amyloidosis and tauopathy, whereas Tg2576 mice, which display β-amyloidosis only, had unchanged PSCA levels compared to wild-type animals. These findings identify PSCA as a nAChR-binding protein in the human brain that is affected in AD, suggesting that PSCA-nAChR interactions may be involved in the cognitive dysfunction observed in AD. PMID:25680266

  15. The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity.

    PubMed

    Kharrat, Riadh; Servent, Denis; Girard, Emmanuelle; Ouanounou, Gilles; Amar, Muriel; Marrouchi, Riadh; Benoit, Evelyne; Molgó, Jordi

    2008-11-01

    Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples. PMID:18990115

  16. Neuronal Nicotinic Acetylcholine Receptor Modulators Reduce Sugar Intake

    PubMed Central

    Shariff, Masroor; Quik, Maryka; Holgate, Joan; Morgan, Michael; Patkar, Omkar L.; Tam, Vincent; Belmer, Arnauld; Bartlett, Selena E.

    2016-01-01

    Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases α4β2 * and decreases α6β2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption. PMID:27028298

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

    PubMed

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

    2014-12-01

    Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts. PMID:25260255

  18. UBXN2A regulates nicotinic receptor degradation by modulating the E3 ligase activity of CHIP.

    PubMed

    Teng, Yanfen; Rezvani, Khosrow; De Biasi, Mariella

    2015-10-15

    Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α3 subunit are known for their prominent role in normal ganglionic transmission while their involvement in the mechanisms underlying nicotine addiction and smoking-related disease has been emerging only in recent years. The amount of information available on the maturation and trafficking of α3-containing nAChRs is limited. We previously showed that UBXN2A is a p97 adaptor protein that facilitates the maturation and trafficking of α3-containing nAChRs. Further investigation of the mechanisms of UBXN2A actions revealed that the protein interacts with CHIP (carboxyl terminus of Hsc70 interacting protein), whose ubiquitin E3 ligase activity regulates the degradation of several disease-related proteins. We show that CHIP displays E3 ligase activity toward the α3 nAChR subunit and contributes to its ubiquitination and subsequent degradation. UBXN2A interferes with CHIP-mediated ubiquitination of α3 and protects the nicotinic receptor subunit from endoplasmic reticulum associated degradation (ERAD). UBXN2A also cross-talks with VCP/p97 and HSC70/HSP70 proteins in a complex where α3 is likely to be targeted by CHIP. Overall,we identify CHIP as an E3 ligase for α3 and UBXN2A as a protein that may efficiently regulate the stability of CHIP's client substrates. PMID:26265139

  19. Marine Natural Products Acting on the Acetylcholine-Binding Protein and Nicotinic Receptors: From Computer Modeling to Binding Studies and Electrophysiology

    PubMed Central

    Kudryavtsev, Denis; Makarieva, Tatyana; Utkina, Natalia; Santalova, Elena; Kryukova, Elena; Methfessel, Christoph; Tsetlin, Victor; Stonik, Valentin; Kasheverov, Igor

    2014-01-01

    For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([125I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (Ki 0.5–1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH4C1 cell line, no competition with [125I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (Ki ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources. PMID:24686559

  20. Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors.

    PubMed

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

    2010-09-01

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

  1. The α7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to taxanes.

    PubMed

    Tu, Chao-Chiang; Huang, Chien-Yu; Cheng, Wan-Li; Hung, Chin-Sheng; Uyanga, Batzorig; Wei, Po-Li; Chang, Yu-Jia

    2016-04-01

    Gastric cancer is difficult to cure because most patients are diagnosed at an advanced disease stage. Systemic chemotherapy remains an important therapy for gastric cancer, but both progression-free survival and disease-free survival associated with various combination regimens are limited because of refractoriness and chemoresistance. Accumulating evidence has revealed that the homomeric α7-nicotinic acetylcholine receptor (A7-nAChR) promotes human gastric cancer by driving cancer cell proliferation, migration, and metastasis. Therefore, A7-nAChR may serve as a potential therapeutic target for gastric cancer. However, the role of A7-nAChR in taxane therapy for gastric cancer was unclear. Cells were subjected to A7-nAChR knockdown (A7-nAChR KD) using short interfering RNA (siRNA). The anti-proliferative effects of taxane were assessed via 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), and cell cycle distribution assays. A7-nAChR-KD cells exhibited low resistance to docetaxel and paclitaxel treatment, as measured by the MTT assay. Following paclitaxel treatment, the proportion of apoptotic cells was higher among A7-nAChR-KD cells than among scrambled control cells, as measured by cell cycle distribution and TUNEL assays. Further molecular analyses showed a reduction in the pAKT levels and a dramatic increase in the Bad levels in paclitaxel-treated A7-nAChR-KD cells but not in scrambled control cells. Following paclitaxel treatment, the level of Bax was slightly increased in both cell populations, whereas Poly (ADP-ribose) polymerase (PARP) cleavage was increased only in A7-nAChR-KD cells. These findings indicate that A7-nAChR-KD cells are more sensitive to paclitaxel treatment. We conclude that A7-nAChR may be a key biomarker for assessing the chemosensitivity of gastric cancer cells to taxane. PMID:26499946

  2. Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells

    PubMed Central

    Elgueta, Claudio; Vielma, Alex H.; Palacios, Adrian G.; Schmachtenberg, Oliver

    2015-01-01

    Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine >> RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca2+ accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca2+ stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing. PMID:25709566

  3. Characterization of [(3)H]CHIBA-1001 binding to alpha7 nicotinic acetylcholine receptors in the brain from rat, monkey, and human.

    PubMed

    Tanibuchi, Yuko; Wu, Jin; Toyohara, Jun; Fujita, Yuko; Iyo, Masaomi; Hashimoto, Kenji

    2010-08-12

    Accumulating evidence suggests that the alpha7 subtype of nicotinic acetylcholine receptors (nAChRs) plays a role in the pathophysiology of neuropsychiatric diseases, including schizophrenia and Alzheimer's disease. Currently, there are no suitable small molecule radioligands for alpha7 nAChRs in the brain, although [(125)I]alpha-bungarotoxin has been widely used as a radioligand for alpha7 nAChRs. In the present study, we characterized a new radioligand, 4-[(3)H]methylphenyl 2,5-diazabicyclo[3.2.2]nonane-2-carboxylate ([(3)H]CHIBA-1001), a derivative of the selective alpha7 nAChR agonist SSR180711, in brain membranes from rat, monkey, and human. Scatchard analysis revealed an apparent equilibrium dissociation constant (Kd) of 193.4nM in rat brain membranes at 4 degrees C, and the maximal number of binding sites (Bmax) was 346.2fmol/mg protein. The order of drugs for the inhibition of [(3)H]CHIBA-1001 binding to rat brain membranes is SSR180711>A-844606>MG624>epibatidine>DMAB>A-582941, suggesting a similarity of alpha7 nAChR pharmacological profiles. In contrast, alpha-bungarotoxin, MLA, and nicotine were found to be very weak. The distribution of [(3)H]CHIBA-1001 binding to crude membranes from dissected regions of rat, monkey, and human brain was different from that of [(125)I]alpha-bungarotoxin binding, suggesting that [(3)H]CHIBA-1001 binding sites may not be identical to [(125)I]alpha-bungarotoxin binding in the brain. In summary, [(3)H]CHIBA-1001 would be a useful radioligand for alpha7 nAChRs in the brains of rodents, non-human primates, and humans. PMID:20537987

  4. Pharmacological identification of cholinergic receptor subtypes on Drosophila melanogaster larval heart.

    PubMed

    Malloy, Cole A; Ritter, Kyle; Robinson, Jonathan; English, Connor; Cooper, Robin L

    2016-01-01

    The Drosophila melanogaster heart is a popular model in which to study cardiac physiology and development. Progress has been made in understanding the role of endogenous compounds in regulating cardiac function in this model. It is well characterized that common neurotransmitters act on many peripheral and non-neuronal tissues as they flow through the hemolymph of insects. Many of these neuromodulators, including acetylcholine (ACh), have been shown to act directly on the D. melanogaster larval heart. ACh is a primary neurotransmitter in the central nervous system (CNS) of vertebrates and at the neuromuscular junctions on skeletal and cardiac tissue. In insects, ACh is the primary excitatory neurotransmitter of sensory neurons and is also prominent in the CNS. A full understanding regarding the regulation of the Drosophila cardiac physiology by the cholinergic system remains poorly understood. Here we use semi-intact D. melanogaster larvae to study the pharmacological profile of cholinergic receptor subtypes, nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), in modulating heart rate (HR). Cholinergic receptor agonists, nicotine and muscarine both increase HR, while nAChR agonist clothianidin exhibits no significant effect when exposed to an open preparation at concentrations as low as 100 nM. In addition, both nAChR and mAChR antagonists increase HR as well but also display capabilities of blocking agonist actions. These results provide evidence that both of these receptor subtypes display functional significance in regulating the larval heart's pacemaker activity. PMID:26438517

  5. Differential Effects of Quercetin and Quercetin Glycosides on Human α7 Nicotinic Acetylcholine Receptor-Mediated Ion Currents.

    PubMed

    Lee, Byung-Hwan; Choi, Sun-Hye; Kim, Hyeon-Joong; Jung, Seok-Won; Hwang, Sung-Hee; Pyo, Mi-Kyung; Rhim, Hyewhon; Kim, Hyoung-Chun; Kim, Ho-Kyoung; Lee, Sang-Mok; Nah, Seung-Yeol

    2016-07-01

    Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin- 3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance α7 nicotinic acetylcholine receptor (α7 nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of α7 nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current (IACh) in Xenopus oocytes expressing the α7 nAChR. IACh was measured with a two-electrode voltage clamp technique. In oocytes injected with α7 nAChR copy RNA, quercetin enhanced IACh, whereas quercetin glycosides inhibited IACh. Quercetin glycosides mediated an inhibition of IACh, which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of IACh inhibition by quercetin glycosides was Rutin≥Rham1>Rham2. Quercetin glycosides-mediated IACh enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated IACh inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated α7 nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the α7 nAChR in a differential manner. PMID:27098860

  6. Differential Effects of Quercetin and Quercetin Glycosides on Human α7 Nicotinic Acetylcholine Receptor-Mediated Ion Currents

    PubMed Central

    Lee, Byung-Hwan; Choi, Sun-Hye; Kim, Hyeon-Joong; Jung, Seok-Won; Hwang, Sung-Hee; Pyo, Mi-Kyung; Rhim, Hyewhon; Kim, Hyoung-Chun; Kim, Ho-Kyoung; Lee, Sang-Mok; Nah, Seung-Yeol

    2016-01-01

    Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin-3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance α7 nicotinic acetylcholine receptor (α7 nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of α7 nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current (IACh) in Xenopus oocytes expressing the α7 nAChR. IACh was measured with a two-electrode voltage clamp technique. In oocytes injected with α7 nAChR copy RNA, quercetin enhanced IACh, whereas quercetin glycosides inhibited IACh. Quercetin glycosides mediated an inhibition of IACh, which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of IACh inhibition by quercetin glycosides was Rutin≥Rham1>Rham2. Quercetin glycosides-mediated IACh enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated IACh inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated α7 nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the α7 nAChR in a differential manner. PMID:27098860

  7. Nicotinic acetylcholine receptors contribute to learning-induced metaplasticity in the hippocampus.

    PubMed

    Becker, Benjamin; Klein, Eva M; Striepens, Nadine; Mihov, Yoan; Schlaepfer, Thomas E; Reul, Juergen; Goossens, Liesbet; Schruers, Koen; Kendrick, Keith M; Hurlemann, René

    2013-07-01

    Hippocampal learning is thought to induce metaplasticity, which can facilitate subsequent learning. Administered at single low doses, the N-methyl-d-aspartate-type glutamate receptor antagonist memantine predominantly blocks α7 nicotinic acetylcholine receptors (α7 nAChRs). Placebo-controlled administration of a single low dose of memantine in a pharmaco-fMRI experiment may thus help characterize the role of α7 nAChRs in hippocampal metaplasticity. We hypothesized that if α7 nAChRs contribute to learning-induced metaplasticity in the hippocampus, blockade of these receptors with low-dose memantine would selectively interfere with a facilitation of subsequent learning without impairing hippocampal learning per se. To specifically test this hypothesis, we devised a randomized controlled trial in which healthy volunteers were administered a 20-mg single oral dose of memantine or placebo and scanned on three subsequent runs of a hippocampal learning task. Our results indicate no discrepancies in behavioral learning between low-dose memantine- and placebo-treated participants in the first and second run of this task. In the third run, however, only the placebo-treated group showed facilitated behavioral learning, an effect paralleled by decreased neural responses in the hippocampal cornu ammonis region. Our findings suggest that blockade of α7 nAChRs selectively interfered with a learning-induced facilitation of subsequent learning while leaving unimpaired hippocampal learning per se. Taken together, our results provide support for a relevant contribution of α7 nAChRs to learning-associated metaplasticity in the hippocampus. PMID:23469888

  8. Selectivity optimization of substituted 1,2,3-triazoles as α7 nicotinic acetylcholine receptor agonists.

    PubMed

    Arunrungvichian, Kuntarat; Fokin, Valery V; Vajragupta, Opa; Taylor, Palmer

    2015-08-19

    Three series of substituted anti-1,2,3-triazoles (IND, PPRD, and QND), synthesized by cycloaddition from azide and alkyne building blocks, were designed to enhance selectivity and potency profiles of a lead α7 nicotinic acetylcholine receptor (α7-nAChR) agonist, TTIn-1. Designed compounds were synthesized and screened for affinity by a radioligand binding assay. Their functional characterization as agonists and antagonists was performed by fluorescence resonance energy transfer assay using cell lines expressing transfected cDNAs, α7-nAChRs, α4β2-nAChRs, and 5HT3A receptors, and a fluorescence cell reporter. In the IND series, a tropane ring of TTIn-1, substituted at N1, was replaced by mono- and bicyclic amines to vary length and conformational flexibility of a carbon linker between nitrogen atom and N1 of the triazole. Compounds with a two-carbon atom linker optimized binding with Kd's at the submicromolar level. Further modification at the hydrophobic indole of TTIn-1 was made in PPRD and QND series by fixing the amine center with the highest affinity building blocks in the IND series. Compounds from IND and PPRD series are selective as agonists for the α7-nAChRs over α4β2-nAChRs and 5HT3A receptors. Lead compounds in the three series have EC50's between 28 and 260 nM. Based on the EC50, affinity, and selectivity determined from the binding and cellular responses, two of the leads have been advanced to behavioral studies described in the companion article (DOI: 10.1021/acschemneuro.5b00059). PMID:25932897

  9. Absence of nicotinic acetylcholine receptor α7 subunit amplifies inflammation and accelerates onset of fibrosis: an inflammatory kidney model

    PubMed Central

    Truong, Luan D.; Trostel, Jessica; Garcia, Gabriela E.

    2015-01-01

    Inflammation is regulated by endogenous mechanisms, including anti-inflammatory cytokines, adenosine, and the nicotinic acetylcholine receptor α7 subunit (α7nAChR). We investigated the role of α7nAChR in protection against the progression of tissue injury in a model of severe, macrophage-mediated, cytokine-dependent anti-glomerular basement membrane (GBM) glomerulonephritis (GN), in α7nAChR-deficient (α7−/−) mice . At d 7 after the injection of anti-GBM antibody, kidneys from α7−/− mice displayed severe glomeruli (P < 0.0001) and tubulointerstitial lesions (P < 0.001) compared to kidneys from WT mice. An important finding was the presence of severe glomerulosclerosis in α7−/− mice in this early phase of the disease. Kidneys of α7−/− mice showed greater accumulation of inflammatory cells and higher expression of chemokines and cytokines than did those of WT mice. In addition, in α7−/− fibrotic kidneys, the expression of fibrin, collagen, TGF-β, and tissue inhibitor of metalloproteinase (TIMP)-2 increased, and the expression of TIMP3 declined. The increase in counterregulatory responses to inflammation in α7−/− nephritic kidneys did not compensate for the lack of α7nAChR. These findings indicate that α7nAChR plays a key role in regulating the inflammatory response in anti-GBM GN and that disruption of the endogenous protective α7nAChR amplifies inflammation to accelerate kidney damage and fibrosis.—Truong, L. D., Trostel. J., Garcia, G. E. Absence of nicotinic acetylcholine receptor α7 subunit amplifies inflammation and accelerates onset of fibrosis: an inflammatory kidney model. PMID:25985801

  10. Acetylcholinesterase inhibitors used in treatment of Alzheimer's disease prevent glutamate neurotoxicity via nicotinic acetylcholine receptors and phosphatidylinositol 3-kinase cascade.

    PubMed

    Takada-Takatori, Yuki; Kume, Toshiaki; Sugimoto, Mitsuhiro; Katsuki, Hiroshi; Sugimoto, Hachiro; Akaike, Akinori

    2006-09-01

    We show here that donepezil, galanathamine and tacrine, therapeutic acetylcholinesterase inhibitors currently being used for treatment of Alzheimer's disease, protect neuronal cells in a time- and concentration-dependent manner from glutamate neurotoxicity that involves apoptosis. The neuroprotective effects were antagonized by mecamylamine, an inhibitor of nicotinic acetylcholine receptors (nAChRs). Dihydro-beta-erythroidine and methyllycaconitine, antagonists for alpha4-nAChR and alpha7-nAChR, respectively, antagonized the protective effect of donepezil and galanthamine, but not that of tacrine. Previous reports suggest the involvement of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway in the nicotine-induced neuroprotection. Inhibitors for a non-receptor type tyrosine kinase, Fyn, and janus-activated kinase 2, suppressed the neuroprotective effect of donepezil and galanthamine, but not that of tacrine. Furthermore, LY294002, a PI3K inhibitor, also suppressed the neuroprotective effect of donepezil and galanthamine, but not that of tacrine. The phosphorylation of Akt, an effector of PI3K, and the expression level of Bcl-2, an anti-apoptotic protein, increased with donepezil and galanthamine treatment, but not with tacrine treatment. These results suggest that donepezil and galanthamine prevent glutamate neurotoxicity through alpha4- and alpha7-nAChRs, followed by the PI3K-Akt pathway, and that tacrine protects neuronal cells through a different pathway. PMID:16762377

  11. Lynx1 Shifts α4β2 Nicotinic Receptor Subunit Stoichiometry by Affecting Assembly in the Endoplasmic Reticulum*

    PubMed Central

    Nichols, Weston A.; Henderson, Brandon J.; Yu, Caroline; Parker, Rell L.; Richards, Christopher I.; Lester, Henry A.; Miwa, Julie M.

    2014-01-01

    Glycosylphosphatidylinositol-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx1 affects assembly of nascent α4 and β2 subunits and alters the stoichiometry of the receptor population that reaches the plasma membrane. Additionally, these data suggest that lynx1 shifts nAChR stoichiometry to low sensitivity (α4)3(β2)2 pentamers primarily through this interaction in the endoplasmic reticulum, rather than solely via direct modulation of activity on the plasma membrane. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any glycosylphosphatidylinositol-anchored protein, could act within the cell to alter assembly of a multisubunit protein. PMID:25193667

  12. Lynx1 shifts α4β2 nicotinic receptor subunit stoichiometry by affecting assembly in the endoplasmic reticulum.

    PubMed

    Nichols, Weston A; Henderson, Brandon J; Yu, Caroline; Parker, Rell L; Richards, Christopher I; Lester, Henry A; Miwa, Julie M

    2014-11-01

    Glycosylphosphatidylinositol-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx1 affects assembly of nascent α4 and β2 subunits and alters the stoichiometry of the receptor population that reaches the plasma membrane. Additionally, these data suggest that lynx1 shifts nAChR stoichiometry to low sensitivity (α4)3(β2)2 pentamers primarily through this interaction in the endoplasmic reticulum, rather than solely via direct modulation of activity on the plasma membrane. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any glycosylphosphatidylinositol-anchored protein, could act within the cell to alter assembly of a multisubunit protein. PMID:25193667

  13. Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor α4 via activating calpain-2

    PubMed Central

    Yin, Yaling; Wang, Yali; Gao, Di; Ye, Jinwang; Wang, Xin; Fang, Lin; Wu, Dongqin; Pi, Guilin; Lu, Chengbiao; Zhou, Xin-Wen; Yang, Ying; Wang, Jian-Zhi

    2016-01-01

    Cholinergic impairments and tau accumulation are hallmark pathologies in sporadic Alzheimer’s disease (AD), however, the intrinsic link between tau accumulation and cholinergic deficits is missing. Here, we found that overexpression of human wild-type full-length tau (termed hTau) induced a significant reduction of α4 subunit of nicotinic acetylcholine receptors (nAChRs) with an increased cleavage of the receptor producing a ~55kDa fragment in primary hippocampal neurons and in the rat brains, meanwhile, the α4 nAChR currents decreased. Further studies demonstrated that calpains, including calpain-1 and calpain-2, were remarkably activated with no change of caspase-3, while simultaneous suppression of calpain-2 by selective calpain-2 inhibitor but not calpain-1 attenuated the hTau-induced degradation of α4 nAChR. Finally, we demonstrated that hTau accumulation increased the basal intracellular calcium level in primary hippocampal neurons. We conclude that the hTau accumulation inhibits nAChRs α4 by activating calpain-2. To our best knowledge, this is the first evidence showing that the intracellular accumulation of tau causes cholinergic impairments. PMID:27277673

  14. Solution structure of {alpha}-conotoxin PIA, a novel antagonist of {alpha}6 subunit containing nicotinic acetylcholine receptors

    SciTech Connect

    Chi, Seung-Wook; Lee, Si-Hyung; Kim, Do-Hyoung; Kim, Jae-Sung; Olivera, Baldomero M.; McIntosh, J. Michael; Han, Kyou-Hoon . E-mail: khhan600@kribb.re.kr

    2005-12-30

    {alpha}-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing {alpha}6 and {alpha}3 subunits. {alpha}-conotoxin PIA displays 75-fold higher affinity for rat {alpha}6/{alpha}3{beta}2{beta}3 nAChRs than for rat {alpha}3{beta}2 nAChRs. We have determined the three-dimensional structure of {alpha}-conotoxin PIA by nuclear magnetic resonance spectroscopy. The {alpha}-conotoxin PIA has an '{omega}-shaped' overall topology as other {alpha}4/7 subfamily conotoxins. Yet, unlike other neuronally targeted {alpha}4/7-conotoxins, its N-terminal tail Arg{sup 1}-Asp{sup 2}-Pro{sup 3} protrudes out of its main molecular body because Asp{sup 2}-Pro{sup 3}-Cys{sup 4}-Cys{sup 5} forms a stable type I {beta}-turn. In addition, a kink introduced by Pro{sup 15} in the second loop of this toxin provides a distinct steric and electrostatic environment from those in {alpha}-conotoxins MII and GIC. By comparing the structure of {alpha}-conotoxin PIA with other functionally related {alpha}-conotoxins we suggest structural features in {alpha}-conotoxin PIA that may be associated with its unique receptor recognition profile.

  15. Five subunit genes of the human muscle nicotinic acetylcholine receptor are mapped to two linkage groups on chromosome 2 and 17

    SciTech Connect

    Lobos, E.A. )

    1993-09-01

    RFLPs were detected in the five subunit genes of the human muscle nicotinic acetylcholine receptor (nAChR) using genomic DNA or cDNA probes from the homologous mouse loci. The RFLPs at the alpha-, beta-, gamma-, delta-, and epsilon-subunit gene loci were analyzed for genetic linkage in 16 families (n = 188). Significant evidence was obtained for close linkage of the [beta]- and [epsilon]-nAChR genes and much greater genetic distance between the [alpha]-nAChR gene and the [gamma]/[delta]-nAChR gene complex. The linkage analysis program CRI-MAP was used to map the positions of the [beta]- and [epsilon]-nAChR genes relative to seven markers on chromosome 17. The results indicate the [beta]- and [epsilon]-nAChR probes to a panel of human-hamster somatic cell hybrids. The [alpha]-, [gamma]-, and [delta]-nAChR genes were placed on a map of 13 chromosome 2 markers. The linkage analysis placed the nAChR genes at two sites on chromosome 2q about equidistant from the marker CRYGP1, with the [alpha]-nAChR gene about 27 cM proximal and the [gamma]/[delta]-nAChR gene complex about 31 cM distal to CRYGP1. 46 refs., 2 figs., 5 tabs.

  16. A positive relationship between harm avoidance and brain nicotinic acetylcholine receptor availability.

    PubMed

    Storage, Steven; Mandelkern, Mark A; Phuong, Jonathan; Kozman, Maggie; Neary, Meaghan K; Brody, Arthur L

    2013-12-30

    Prior research indicates that disturbance of cholinergic neurotransmission reduces anxiety, leading to the hypothesis that people with heightened cholinergic function have a greater tendency toward anxiety-like and/or harm-avoidant behavior. We sought to determine if people with elevated levels of harm avoidance (HA), a dimension of temperament from the Temperament and Character Inventory (TCI), have high α4β2* nicotinic acetylcholine receptor (nAChR) availability. Healthy adults (n=105; 47 non-smokers and 58 smokers) underwent bolus-plus-continuous infusion positron emission tomography (PET) scanning using the radiotracer 2-[18F]fluoro-3-(2(S)azetidinylmethoxy) pyridine (abbreviated as 2-FA). During the uptake period of 2-FA, participants completed the TCI. The central study analysis revealed a significant association between total HA and mean nAChR availability, with higher total HA scores being linked with greater nAChR availability. In examining HA subscales, both 'Fear of Uncertainty' and 'Fatigability' were significant, based on higher levels of these characteristics being associated with greater nAChR availabilities. This study adds to a growing body of knowledge concerning the biological basis of personality and may prove useful in understanding the pathophysiology of psychiatric disorders (such as anxiety disorders) that have similar characteristics to HA. Study findings may indicate that heightened cholinergic neurotransmission is associated with increased anxiety-like traits. PMID:24148908

  17. Neuronal Acetylcholine Nicotinic Receptors as New Targets for Lung Cancer Treatment.

    PubMed

    Mucchietto, Vanessa; Crespi, Arianna; Fasoli, Francesca; Clementi, Francesco; Gotti, Cecilia

    2016-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Smoking accounts for approximately 70% of the cases of non- small cell lung cancer (NSCLC) and 90% of the cases of small-cell lung cancer (SCLC), although some patients develop lung cancer without a history of smoking. Nicotine is the most active addictive component of tobacco smoke. It does not initiate tumorigenesis in humans and rodents, but it alters the pathophysiology of lung cells by inducing the secretion of growth factors, neurotransmitters and cytokines, and promotes tumour growth and metastases by inducing cell cycle progression, migration, invasion, angiogenesis and the evasion of apoptosis. Most of these effects are a result of nicotine binding and activation of cell-surface neuronal nicotinic acetylcholine receptors (nAChRs) and downstream intracellular signalling cascades, and many are blocked by nAChR subtype-selective antagonists. Recent genome-wide association studies have revealed single nucleotide polymorphisms of nAChR subunits that influence nicotine dependence and lung cancer. This review describes the molecular basis of nAChR structural and functional diversity in normal and cancer lung cells, and the genetic alterations facilitating smoking-induced lung cancers. It also summarises current knowledge concerning the intracellular pathways activated by nicotine and other compounds present in tobacco smoke. PMID:26845123

  18. Alpha9 alpha10 nicotinic acetylcholine receptors as target for the treatment of chronic pain.

    PubMed

    Del Bufalo, Alessandra; Cesario, Alfredo; Salinaro, Gianluca; Fini, Massimo; Russo, Patrizia

    2014-01-01

    Chronic pain is a widespread healthcare problem affecting not only the patient but in many ways all the society. Chronic pain is a disease itself that endures for a long period of time and it is resistant to the majority of medical treatments that provide modest improvements in pain and minimum improvements in physical and emotional functioning. More co-existing chronic pain conditions may be present in the same individual (patient). The α9α10 nicotinic acetylcholine receptor (nAChR) may be a potential target in the pathophysiology of chronic pain, as well in the development of breast and lung cancers. α-conotoxins (α-CNT) are small peptides used offensively by carnivorous marine snails known as Conus that target nAChR. Among α-CNT there are potent and selective antagonists of α9α10 nAChR such as RgIA and Vc1.1 that produces both acute and long lasting analgesia. Moreover, these peptides accelerate the recovery of nerve function after injury, likely through immune/inflammatory-mediated mechanisms. We review the background, findings, implications and problems in using compounds that act on α9α10 nAChR. PMID:24641230

  19. Recent Developments in Novel Antidepressants Targeting α4β2-Nicotinic Acetylcholine Receptors

    PubMed Central

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) have been investigated for developing drugs that can potentially treat various central nervous system disorders. Considerable evidence supports the hypothesis that modulation of the cholinergic system through activation and/or desensitization/inactivation of nAChR holds promise for the development of new antidepressants. The introductory portion of this Miniperspective discusses the basic pharmacology that underpins the involvement of α4β2-nAChRs in depression, along with the structural features that are essential to ligand recognition by the α4β2-nAChRs. The remainder of this Miniperspective analyzes reported nicotinic ligands in terms of drug design considerations and their potency and selectivity, with a particular focus on compounds exhibiting antidepressant-like effects in preclinical or clinical studies. This Miniperspective aims to provide an in-depth analysis of the potential for using nicotinic ligands in the treatment of depression, which may hold some promise in addressing an unmet clinical need by providing relief from depressive symptoms in refractory patients. PMID:24901260

  20. Varenicline, a Partial Agonist at Neuronal Nicotinic Receptors, Reduces Nicotine-Induced Increases in 20% Ethanol Operant Self-Administration in Sprague-Dawley Rats

    PubMed Central

    Bito-Onon, Jade J.; Simms, Jeffrey A.; Chatterjee, Susmita; Holgate, Joan; Bartlett, Selena E.

    2010-01-01

    Alcohol and nicotine use disorders are often treated as separate diseases, despite evidence that approximately 80–90% of alcohol dependent individuals are also heavy smokers. Both nicotine and ethanol have been shown to interact with neuronal nicotinic acetylcholine receptors (nAChRs), suggesting these receptors are a common biological target for the effects of nicotine and ethanol in the brain. There are few studies that have examined the effects of co-administered nicotine and ethanol on the activity of nAChRs in rodents. In the present study, we show that Sprague-Dawley rats, a strain often used for nicotine studies but not as often for voluntary ethanol intake studies, will consume 20% ethanol using both the intermittent-access two-bottle-choice and operant self-administration models without the need for sucrose fading. We show that nicotine (0.2mg/kg and 0.8mg/kg, s.c.) significantly increases operant 20% ethanol self-administration and varenicline (2mg/kg, s.c), a partial agonist at nAChRs, significantly decreases operant ethanol self-administration and nicotine-induced increases in ethanol self-administration. This suggests that nAChRs play an important role in increasing ethanol self-administration and that varenicline may be an efficacious treatment for alcohol and nicotine co-dependencies. PMID:21392178

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  2. α7 Nicotinic Receptor-Mediated Astrocytic Gliotransmitter Release: Aβ Effects in a Preclinical Alzheimer’s Mouse Model

    PubMed Central

    Pirttimaki, Tiina Maria; Codadu, Neela Krushna; Awni, Alia; Pratik, Pandey; Nagel, David Andrew; Hill, Eric James; Dineley, Kelly Tennyson; Parri, H. Rheinallt

    2013-01-01

    It is now recognized that astrocytes participate in synaptic communication through intimate interactions with neurons. A principal mechanism is through the release of gliotransmitters (GTs) such as ATP, D-serine and most notably, glutamate, in response to astrocytic calcium elevations. We and others have shown that amyloid-β (Aβ), the toxic trigger for Alzheimer’s disease (AD), interacts with hippocampal α7 nicotinic acetylcholine receptors (nAChRs). Since α7nAChRs are highly permeable to calcium and are expressed on hippocampal astrocytes, we investigated whether Aβ could activate astrocytic α7nAChRs in hippocampal slices and induce GT glutamate release. We found that biologically-relevant concentrations of Aβ1-42 elicited α7nAChR-dependent calcium elevations in hippocampal CA1 astrocytes and induced NMDAR-mediated slow inward currents (SICs) in CA1 neurons. In the Tg2576 AD mouse model for Aβ over-production and accumulation, we found that spontaneous astrocytic calcium elevations were of higher frequency compared to wildtype (WT). The frequency and kinetic parameters of AD mice SICs indicated enhanced gliotransmission, possibly due to increased endogenous Aβ observed in this model. Activation of α7nAChRs on WT astrocytes increased spontaneous inward currents on pyramidal neurons while α7nAChRs on astrocytes of AD mice were abrogated. These findings suggest that, at an age that far precedes the emergence of cognitive deficits and plaque deposition, this mouse model for AD-like amyloidosis exhibits augmented astrocytic activity and glutamate GT release suggesting possible repercussions for preclinical AD hippocampal neural networks that contribute to subsequent cognitive decline. PMID:24312364

  3. The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors.

    PubMed

    Couesnon, Aurélie; Aráoz, Rómulo; Iorga, Bogdan I; Benoit, Evelyne; Reynaud, Morgane; Servent, Denis; Molgó, Jordi

    2016-01-01

    The cyclic imine toxin 20-methyl spirolide G (20-meSPX-G), produced by the toxigenic dinoflagellate Alexandrium ostenfeldii/Alexandrium peruvianum, has been previously reported to contaminate shellfish in various European coastal locations, as revealed by mouse toxicity bioassay. The aim of the present study was to determine its toxicological profile and its molecular target selectivity. 20-meSPX-G blocked nerve-evoked isometric contractions in isolated mouse neuromuscular preparations, while it had no action on contractions elicited by direct electrical stimulation, and reduced reversibly nerve-evoked compound muscle action potential amplitudes in anesthetized mice. Voltage-clamp recordings in Xenopus oocytes revealed that 20-meSPX-G potently inhibited currents evoked by ACh on Torpedo muscle-type and human α7 nicotinic acetylcholine receptors (nAChR), whereas lower potency was observed in human α4β2 nAChR. Competition-binding assays showed that 20-meSPX-G fully displaced [³H]epibatidine binding to HEK-293 cells expressing the human α3β2 (Ki = 0.040 nM), whereas a 90-fold lower affinity was detected in human α4β2 nAChR. The spirolide displaced [(125)I]α-bungarotoxin binding to Torpedo membranes (Ki = 0.028 nM) and in HEK-293 cells expressing chick chimeric α7-5HT₃ nAChR (Ki = 0.11 nM). In conclusion, this is the first study to demonstrate that 20-meSPX-G is a potent antagonist of nAChRs, and its subtype selectivity is discussed on the basis of molecular docking models. PMID:27563924

  4. Modulation of nicotinic ACh-, GABAA- and 5-HT3-receptor functions by external H-7, a protein kinase inhibitor, in rat sensory neurones

    PubMed Central

    Hu, Hong-Zhen; Li, Zhi-Wang

    1997-01-01

    The effects of external H-7, a potent protein kinase inhibitor, on the responses mediated by γ-aminobutyric acid A type (GABAA)-, nicotinic acetylcholine (nicotinic ACh)-, ionotropic 5-hydroxytryptamine (5-HT3)-, adenosine 5′-triphosphate (ATP)-, N-methyl-D-aspartate (NMDA)- and kainate (KA)-receptors were studied in freshly dissociated rat dorsal root ganglion neurone by use of whole cell patch-clamp technique. External H-7 (1–1000 μM) produced a reversible, dose-dependent inhibition of whole cell currents activated by GABA, ACh and 5-HT. Whole-cell currents evoked by ATP, 2-methylthio-ATP, NMDA and KA were insensitive to external H-7. External H-7 shifted the dose-response curve of GABA-activated currents downward without changing the EC50 significantly (from 15.0±4.0 μM to 18.0±5.0 μM). The maximum response to GABA was depressed by 34.0±5.3%. This inhibitory action of H-7 was voltage-independent. Intracellular application of H-7 (20 μM), cyclic AMP (1 mM) and BAPTA (10 mM) could not reverse the H-7 inhibition of GABA-activated currents. The results suggest that external H-7 selectively and allosterically modulates the functions of GABAA-, nicotine ACh- and 5-HT3 receptors via a common conserved site in the external domain of these receptors. PMID:9401786

  5. Virtual screening studies of Chinese medicine Coptidis Rhizoma as alpha7 nicotinic acetylcholine receptor agonists for treatment of Alzheimer's disease

    NASA Astrophysics Data System (ADS)

    Xiang, Li; Xu, Youdong; Zhang, Yan; Meng, Xianli; Wang, Ping

    2015-04-01

    Alzheimer's disease (AD) is an age-related neurodegenerative disease. Extensive in vitro and in vivo experiments have proved that the decreased activity of the cholinergic neuron is responsible for the memory and cognition deterioration. The alpha7 nicotinic acetylcholine receptor (α7-nAChR) is proposed to a drug target of AD, and compounds which acting as α7-nAChR agonists are considered as candidates in AD treatment. Chinese medicine CoptidisRhizoma and its compounds are reported in various anti-AD effects. In this study, virtual screening, docking approaches and hydrogen bond analyses were applied to screen potential α7-nAChR agonists from CoptidisRhizome. The 3D structure of the protein was obtained from PDB database. 87 reported compounds were included in this research and their structures were accessed by NCBI Pubchem. Docking analysis of the compounds was performed using AutoDock 4.2 and AutoDock Vina. The images of the binding modes hydrogen bonds and the hydrophobic interaction were rendered with PyMOL1.5.0.4. and LigPlot+ respectively. Finally, N-tran-feruloyltyramine, isolariciresinol, flavanone, secoisolariciresinol, (+)-lariciresinol and dihydrochalcone, exhibited the lowest docking energy of protein-ligand complex. The results indicate these 6 compounds are potential α7 nAChR agonists, and expected to be effective in AD treatment.

  6. At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor.

    PubMed

    Otvos, Reka A; Mladic, Marija; Arias-Alpizar, Gabriela; Niessen, Wilfried M A; Somsen, Govert W; Smit, August B; Kool, Jeroen

    2016-06-01

    The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel expressed in different regions of the central nervous system (CNS). The α7-nAChR has been associated with Alzheimer's disease, epilepsy, and schizophrenia, and therefore is extensively studied as a drug target for the treatment of these diseases. Important sources for new compounds in drug discovery are natural extracts. Since natural extracts are complex mixtures, identification of the bioactives demands the use of analytical techniques to separate a bioactive from inactive compounds. This study describes screening methodology for identifying bioactive compounds in mixtures acting on the α7-nAChR. The methodology developed combines liquid chromatography (LC) coupled via a split with both an at-line calcium (Ca(2+))-flux assay and high-resolution mass spectrometry (MS). This allows evaluation of α7-nAChR responses after LC separation, while parallel MS enables compound identification. The methodology was optimized for analysis of agonists and positive allosteric modulators, and was successfully applied to screening of the hallucinogen mushroom Psilocybe Mckennaii The crude mushroom extract was analyzed using both reversed-phase and hydrophilic interaction liquid chromatography. Matching retention times and peak shapes of bioactives found with data from the parallel MS measurements allowed rapid pinpointing of accurate masses corresponding to the bioactives. PMID:26738519

  7. PRENATAL NICOTINE EXPOSURE SELECTIVELY AFFECTS NICOTINIC RECEPTOR EXPRESSION IN PRIMARY AND ASSOCIATIVE VISUAL CORTICES OF THE FETAL BABOON

    PubMed Central

    Duncan, Jhodie R.; Garland, Marianne; Stark, Raymond I.; Myers, Michael M.; Fifer, William P.; Mokler, David J.; Kinney, Hannah C.

    2014-01-01

    Exposure to nicotine during pregnancy via maternal cigarette smoking is associated with visual deficits in children. This is possibly due to activation of nicotinic acetylcholine receptors (nAChRs) in the occipital cortex which are important in the development of visual mapping. Using a baboon model we explored the effects of prenatal nicotine on parameters in the primary and associated visual cortices. Pregnant baboons were infused with nicotine (0.5 mg/hr, i.v.) or saline from 86 days gestation. At 161 days gestation fetal brains were collected (n=5/group) and the occipital lobe assessed for nAChRs and markers of the serotonergic and catecholaminergic systems using tissue autoradiography and/or high performance liquid chromatography. Neuronal nAChRs and serotonergic markers were expressed in a region and subunit dependent manner. Prenatal nicotine exposure was associated with increased binding for 3H-epibatidine sensitive nAChRs in the primary visual cortex (BA 17) and BA 18, but not BA 19, of the associative visual cortex (p<0.05). Markers of the serotonergic or catecholaminergic systems were not significantly altered. Thus, prenatal nicotine exposure is associated with alterations in the cholinergic system in the occipital lobe which may aid in the explanation of the appearance of visual deficits in children from mothers who smoke during pregnancy. PMID:24903536

  8. Conotoxins Targeting Nicotinic Acetylcholine Receptors: An Overview

    PubMed Central

    Lebbe, Eline K. M.; Peigneur, Steve; Wijesekara, Isuru; Tytgat, Jan

    2014-01-01

    Marine snails of the genus Conus are a large family of predatory gastropods with an unparalleled molecular diversity of pharmacologically active compounds in their venom. Cone snail venom comprises of a rich and diverse cocktail of peptide toxins which act on a wide variety of ion channels such as voltage-gated sodium- (NaV), potassium- (KV), and calcium- (CaV) channels as well as nicotinic acetylcholine receptors (nAChRs) which are classified as ligand-gated ion channels. The mode of action of several conotoxins has been the subject of investigation, while for many others this remains unknown. This review aims to give an overview of the knowledge we have today on the molecular pharmacology of conotoxins specifically interacting with nAChRs along with the structure–function relationship data. PMID:24857959

  9. Behavioral phenotyping of heterozygous acetylcholinesterase knockout (AChE+/-) mice showed no memory enhancement but hyposensitivity to amnesic drugs.

    PubMed

    Espallergues, Julie; Galvan, Laurie; Sabatier, Florence; Rana-Poussine, Vanessa; Maurice, Tangui; Chatonnet, Arnaud

    2010-01-20

    Decrease in the expression or activity of acetylcholinesterase (AChE) enzymatic activity results in increased cholinergic tonus in the brain and periphery, with concomitant regulations of nicotinic and muscarinic receptors expression. We generated AChE knockout mice and characterized the behavioral phenotype of heterozygous animals, focusing on learning and memory functions. Male and female, AChE+/- and AChE+/+ littermate controls (129 sv strain) were tested at 5-9 weeks of age. AChE activity was significantly decreased in the hippocampus and cortex of AChE+/- mice, but butyrylcholinesterase activity was preserved. AChE+/- mice failed to show any difference in terms of locomotion, exploration and anxiety parameters in the open-field test. Animals were then tested for place learning in the water-maze. They were trained using a 'sustained acquisition' protocol (3 swim trials per day) or a 'mild acquisition' protocol (2 swim trials per day) to locate an invisible platform in fixed position (reference memory procedure). Then, during 3 days, they were trained to locate the platform in a variable position (working memory procedure). Learning profiles and probe test performances were similar for AChE+/- and AChE+/+ mice. Mice were then treated with the muscarinic receptor antagonist scopolamine (0.5, 5 mg/kg) 20 min before each training session. Scopolamine impaired learning at both doses in AChE+/+ mice, but only at the highest dose in AChE+/- mice. Moreover, the intracerebroventricular injection of amyloid-beta25-35 peptide, 9 nmol, 7 days before water-maze acquisition, failed to induce learning deficits in AChE+/- mice, but impaired learning in AChE+/+ controls. The peptide failed to be toxic in forebrain structures of AChE+/- mice, since an increase in lipid peroxidation levels was measured in the hippocampus of AChE+/+ but not AChE+/- mice. We conclude that the increase in cholinergic tonus observed in AChE+/- mice did not result in increased memory functions but

  10. Subunit composition of α5-containing nicotinic receptors in the rodent habenula

    PubMed Central

    Scholze, Petra; Koth, Gabriele; Orr-Urtreger, Avi; Huck, Sigismund

    2012-01-01

    Gene association studies in humans have linked the α5 subunit gene CHRNA5 to an increased risk for nicotine dependence. In the CNS, nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit are expressed at relatively high levels in the habenulo-interpeduncular system. Recent experimental evidence furthermore suggests that α5-containing receptors in the habenula play a key role in controlling the intake of nicotine in rodents. We have now analysed the subunit composition of hetero-oligomeric nAChRs in the habenula of postnatal day 18 (P18) C57Bl/6J control mice and of mice with deletions of the α5, the β2, or the β4 subunit genes. Receptors consisting of α3β4*1 clearly outnumbered α4β2*-containing receptors not only in P18 but also in adult mice. We found low levels of α5-containing receptors in both mice (6%) and rats (2.5% of overall nAChRs). Observations in β2 and β4 null mice indicate that although α5 requires the presence of the β4 subunit for assembling (but not of β2), α5 in wild-type mice assembles into receptors that also contain the subunits α3, β2, and β4. PMID:22380605

  11. Vulnerability to nicotine self-administration in adolescent mice correlates with age-specific expression of α4* nicotinic receptors.

    PubMed

    Renda, Anthony; Penty, Nora; Komal, Pragya; Nashmi, Raad

    2016-09-01

    The majority of smokers begin during adolescence, a developmental period with a high susceptibility to substance abuse. Adolescents are affected differently by nicotine compared to adults, with adolescents being more vulnerable to nicotine's rewarding properties. It is unknown if the age-dependent molecular composition of a younger brain contributes to a heightened susceptibility to nicotine addiction. Nicotine, the principle pharmacological component of tobacco, binds and activates nicotinic acetylcholine receptors (nAChRs) in the brain. The most prevalent is the widely expressed α4-containing (α4*) subtype which mediates reward and is strongly implicated in nicotine dependence. Exposing different age groups of mice, postnatal day (P) 44-86 days old, to a two bottle-choice oral nicotine self-administration paradigm for five days yielded age-specific consumption levels. Nicotine self-administration was elevated in the P44 group, peaked at P54-60 and was drastically lower in the P66 through P86 groups. We also quantified α4* nAChR expression via spectral confocal imaging of brain slices from α4YFP knock-in mice, in which the α4 nAChR subunit is tagged with a yellow fluorescent protein. Quantitative fluorescence revealed age-specific α4* nAChR expression in dopaminergic and GABAergic neurons of the ventral tegmental area. Receptor expression showed a strong positive correlation with daily nicotine dose, suggesting that α4* nAChR expression levels are age-specific and may contribute to the propensity to self-administer nicotine. PMID:27102349

  12. Non-competitive Inhibition of Nicotinic Acetylcholine Receptors by Ladybird Beetle Alkaloids.

    PubMed

    Leong, Ron L; Xing, Hong; Braekman, Jean-Claude; Kem, William R

    2015-10-01

    Ladybird beetles (Family Coccinellidae) secrete an alkaloid rich venom from their leg joints that protects them from predators. Coccinellines, the major venom constituents, are alkaloids composed of three fused piperidine rings that share a common nitrogen atom. Although many coccinellines have been isolated and chemically characterized, their pharmacological properties are essentially unknown. Using radioligand binding and functional assays we investigated the actions of several coccinellines on skeletal muscle and α7 nicotinic acetylcholine receptors (nAChRs). The alkaloids were shown to displace the specific binding of tritiated piperidyl-N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([(3)H]-TCP), which has been shown to bind deep within the ion channel of the electric fish (Torpedo) muscle nAChR. The stereoisomers precoccinelline and hippodamine (whose nitrogens are predicted to be ionized at physiological pH) and their respective analogs N-methyl-precoccinelline and N-methyl-hippodamine (whose quaternary nitrogens are permanently charged) displayed similar IC50s for inhibition of [(3)H]-TCP binding. However, the corresponding precoccinelline and hippodamine N-oxides, coccinelline and convergine (which have an electronegative oxygen bonded to an electropositive nitrogen) displayed significantly higher binding IC50s. Finally, exochomine, a dimeric coccinelline containing the hippodamine structure, displayed the highest IC50 (lowest affinity) for displacing specific [(3)H]-TCP binding. The presence of a desensitizing concentration (10(-3) M) of carbachol (CCh) had little or no effect on the affinity of the Torpedo nAChR for the three coccinellines tested. High concentrations of the coccinellid alkaloids did not affect binding of [(3)H]-cytisine to Torpedo receptor ACh binding sites. Inhibition of the alpha7 nAChR with pre-equilibrated precoccinelline was insurmountable with respect to ACh concentration. We conclude that the coccinellines bind to one or more

  13. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal ventral horn neurons.

    PubMed

    Mine, N; Taniguchi, W; Nishio, N; Izumi, N; Miyazaki, N; Yamada, H; Nakatsuka, T; Yoshida, M

    2015-04-01

    Nicotinic acetylcholine receptors (nAChRs) are distributed widely in the central nervous system and play important roles in higher brain functions, including learning, memory, and recognition. However, functions of the cholinergic system in spinal motoneurons remain poorly understood. In this study, we investigated the actions of presynaptic and postsynaptic nAChRs in spinal ventral horn neurons by performing whole-cell patch-clamp recordings on lumbar slices from male rats. The application of nicotine or acetylcholine generated slow inward currents and increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Slow inward currents by acetylcholine or nicotine were not inhibited by tetrodotoxin (TTX) or glutamate receptor antagonists. In the presence of TTX, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were also increased by acetylcholine or nicotine. A selective α4β2 nicotinic receptor antagonist, dihydro-β-erythroidine hydrobromide (DhβE), significantly decreased nicotine-induced inward currents without affecting the enhancement of sEPSCs and mEPSCs. In addition, a selective α7 nicotinic receptor antagonist, methyllycaconitine, did not affect either nicotine-induced inward currents or the enhancement of sEPSCs and mEPSCs. These results suggest that α4β2 AChRs are localized at postsynaptic sites in the spinal ventral horn, non-α4β2 and non-α7 nAChRs are located presynaptically, and nAChRs enhance excitatory synaptic transmission in the spinal ventral horn. PMID:25613686

  14. The immunomodulation of nicotinic acetylcholine receptor subunits in Zhikong scallop Chlamys farreri.

    PubMed

    Shi, Xiaowei; Zhou, Zhi; Wang, Lingling; Wang, Mengqiang; Shi, Shaoying; Wang, Zhen; Song, Linsheng

    2015-11-01

    Nicotinic acetylcholine receptor (nAChR), the best-studied ionotropic neuron receptor protein, is a key player in neuronal communication, and it has been reported to play an important role in immunomodulation of vertebrates. Although nAChRs have also been identified in most invertebrates, the knowledge about their immunomodulation is still limited. In the present study, two scallop nAChR genes were identified from Chlamys farreri (designed as CfnAChR1 and CfnAChR2), which encoded 384 and 443 amino acids, respectively. The conserved disulfide-linked cystines, ion selectivity residues and the hydrophobic gating residues (L251, V255 and V259) were identified in CfnAChR1 and CfnAChR2. The immunoreactivities of CfnAChR1 and CfnAChR2 were observed in all the tested scallop tissues, including adductor muscle, mantle, gill, hepatopancreas, kidney and gonad. After LPS (0.5 mg mL(-1)) stimulation, the expression of CfnAChR1 mRNA in haemocytes increased significantly by 9.83-fold (P < 0.05) and 12.93-fold (P < 0.05) at 3 h and 24 h, respectively. While the expression level of CfnAChR2 mRNA increased 43.94% at 12 h after LPS stimulation (P < 0.05). After TNF-α (50 ng mL(-1)) stimulation, the expression levels of CfnAChR1 and CfnAChR2 both increased significantly at 1 h, which were 21.33-fold (P < 0.05) and 2.44-fold (P < 0.05) of that in the PBS group, respectively. The results collectively indicated that the cholinergic nervous system in scallops could be activated by immune stimulations through CfnAChR1 and CfnAChR2, which function as the links between the cholinergic nervous system and immune system. PMID:26455648

  15. Effects of adjunct galantamine to risperidone, or haloperidol, in animal models of antipsychotic activity and extrapyramidal side-effect liability: involvement of the cholinergic muscarinic receptor.

    PubMed

    Wadenberg, Marie-Louise G; Fjällström, Ann-Kristin; Federley, Malin; Persson, Pernilla; Stenqvist, Pia

    2011-06-01

    The acetylcholine esterase inhibitor/cholinergic nicotinic receptor (nAChR) allosteric modulator galantamine (Gal) is used against cognitive impairment in Alzheimer's disease. Negative/cognitive and psychotic symptom improvement in schizophrenia by adjunct Gal to antipsychotic drugs (APDs) has been reported. Cognitive symptoms in schizophrenia may involve brain prefrontal hypo-dopaminergia. Experimental data by others indicate nAChR involvement in animal pro-cognitive effects of Gal. The role of nAChRs in antipsychotic effects by Gal has, however, not been elucidated. Using the conditioned avoidance response (CAR) and the catalepsy tests for antipsychotic activity and extrapyramidal side-effect (EPS) liability, respectively, we here investigated the effects of adjunct Gal (1.25 mg/kg) to the typical APD haloperidol (Hal) (0.05 mg/kg), or the atypical APD risperidone (Ris) (0.2 mg/kg), in rats. Adjunct Gal significantly enhanced APD-like effects by low doses of Hal or Ris, but showed a safe EPS liability profile only in combination with Ris. Pretreatment with the muscarinic receptor (mAChR) antagonist scopolamine, but not the nAChR antagonist mecamylamine, completely reversed the enhancing effects of adjunct Gal to Hal treatment, in the CAR test. While the nAChR-modulating properties of Gal probably contribute to pro-cognitive activity, as shown by others, the present data suggest that any contribution to antipsychotic activity by Gal is mediated primarily via mAChRs. This property combination of Gal may offer a unique, favourable therapeutic profile for schizophrenia treatment. PMID:20701827

  16. Spontaneous opening of the acetylcholine receptor channel in developing muscle cells from normal and dystrophic mice

    SciTech Connect

    Franco-Obregon, A.; Lansman, J.B.

    1995-12-31

    Single-channel activity was recorded from cell-attached patches on skeletal muscle cells isolated from wild-type mice and from mice carrying the dy or mdx mutations. Spontaneous openings of the nicotinic acetylcholine receptor channel (nAChR) were detected in virtually all recordings from either 4v/dy or dyl + myotubes. but only infrequently from wild-type or mdx myotubes. Spontaneous openings were also present in most recordings from undifferentiated myoblasts from all of the mouse strains studied. The biophysical properties of the spontaneous activity were similar to those of the embryonic form of the nAChR in the presence of acetylcholine (ACh). Examination of the single-channel currents evoked by low concentrations of ACh showed a reduced sensitivity to the agonist in the dystrophic dy and mdx myotubes. but not in wild- type myotubes. The results suggest that alterations in nAChR function are associated with the pathogenesis of muscular dystrophy in the dy mouse.

  17. Infiltration of CCR2+Ly6Chigh Proinflammatory Monocytes and Neutrophils into the Central Nervous System Is Modulated by Nicotinic Acetylcholine Receptors in a Model of Multiple Sclerosis.

    PubMed

    Jiang, Wei; St-Pierre, Stéphanie; Roy, Patrick; Morley, Barbara J; Hao, Junwei; Simard, Alain R

    2016-03-01

    Myeloid cells, including proinflammatory monocytes and neutrophils, have important roles in the pathology of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). These cells infiltrate the CNS in the early stages of disease development and contribute to the inflammatory response that is associated with symptom severity. It is thus crucial to identify and understand new mechanisms that can regulate the CNS infiltration of proinflammatory myeloid cells. Nicotinic acetylcholine receptors (nAChRs) have been increasingly studied for their immune-regulatory properties. In this study, we assessed the ability of nicotine, an nAChR ligand, to modulate proinflammatory myeloid cell numbers within the bone marrow, spleen, blood, and CNS of EAE mice. We found that nicotine significantly inhibits the infiltration of proinflammatory monocytes and neutrophils into the CNS at time points where these cells are known to play critical roles in disease pathology. In contrast, nicotine does not affect the expansion of other monocytes. We also show that nicotine exerts these effects by acting on α7 and α9 nAChR subtypes. Finally, mRNA transcript levels for CCL2 and CXCL2, chemokines involved in the chemotaxis of proinflammatory monocytes and neutrophils, respectively, are reduced in the brain of nicotine-treated EAE mice before the massive infiltration of these cells. Taken together, our data provide evidence that nAChRs can regulate proinflammatory cell infiltration into the CNS, which could be of significant value for the treatment of neuroinflammatory disorders. PMID:26810225

  18. Infiltration of CCR2+Ly6Chigh Proinflammatory Monocytes and Neutrophils into the Central Nervous System Is Modulated by Nicotinic Acetylcholine Receptors in a Model of Multiple Sclerosis

    PubMed Central

    Jiang, Wei; St-Pierre, Stéphanie; Roy, Patrick; Morley, Barbara J.

    2016-01-01

    Myeloid cells, including proinflammatory monocytes and neutrophils, have important roles in the pathology of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). These cells infiltrate the CNS in the early stages of disease development and contribute to the inflammatory response that is associated with symptom severity. It is thus crucial to identify and understand new mechanisms that can regulate the CNS infiltration of proinflammatory myeloid cells. Nicotinic acetylcholine receptors (nAChRs) have been increasingly studied for their immune-regulatory properties. In this study, we assessed the ability of nicotine, an nAChR ligand, to modulate proinflammatory myeloid cell numbers within the bone marrow, spleen, blood, and CNS of EAE mice. We found that nicotine significantly inhibits the infiltration of proinflammatory monocytes and neutrophils into the CNS at time points where these cells are known to play critical roles in disease pathology. In contrast, nicotine does not affect the expansion of other monocytes. We also show that nicotine exerts these effects by acting on α7 and α9 nAChR subtypes. Finally, mRNA transcript levels for CCL2 and CXCL2, chemokines involved in the chemotaxis of proinflammatory monocytes and neutrophils, respectively, are reduced in the brain of nicotine-treated EAE mice before the massive infiltration of these cells. Taken together, our data provide evidence that nAChRs can regulate proinflammatory cell infiltration into the CNS, which could be of significant value for the treatment of neuroinflammatory disorders. PMID:26810225

  19. Structure-activity studies and analgesic efficacy of N-(3-pyridinyl)-bridged bicyclic diamines, exceptionally potent agonists at nicotinic acetylcholine receptors.

    PubMed

    Bunnelle, William H; Daanen, Jerome F; Ryther, Keith B; Schrimpf, Michael R; Dart, Michael J; Gelain, Arianna; Meyer, Michael D; Frost, Jennifer M; Anderson, David J; Buckley, Michael; Curzon, Peter; Cao, Ying-Jun; Puttfarcken, Pamela; Searle, Xenia; Ji, Jianguo; Putman, C Brent; Surowy, Carol; Toma, Lucio; Barlocco, Daniela

    2007-07-26

    A series of exceptionally potent agonists at neuronal nicotinic acetylcholine receptors (nAChRs) has been investigated. Several N-(3-pyridinyl) derivatives of bridged bicyclic diamines exhibit double-digit-picomolar binding affinities for the alpha 4 beta 2 subtype, placing them with epibatidine among the most potent nAChR ligands described to date. Structure-activity studies have revealed that substitutions, particularly hydrophilic groups in the pyridine 5-position, differentially modulate the agonist activity at ganglionic vs central nAChR subtypes, so that improved subtype selectivity can be demonstrated in vitro. Analgesic efficacy has been achieved across a broad range of pain states, including rodent models of acute thermal nociception, persistent pain, and neuropathic allodynia. Unfortunately, the hydrophilic pyridine substituents that were shown to enhance agonist selectivity for central nAChRs in vitro tend to limit CNS penetration in vivo, so that analgesic efficacy with an improved therapeutic window was not realized with those compounds. PMID:17585748

  20. [Influence of stimulation and blockade of α4β2 nicotinic acetylcholine receptors on learning of female rats in basic phases of ovary cycle].

    PubMed

    Fedotova, Iu O

    2014-03-01

    The present work was devoted to the comparative analysis of α4β2 nicotinic acetylcholine receptors (nAChRs) in learning/memory processes during ovary cycle in the adult female rats. RJR-2403 (1.0 mg/kg, i. p.), α4β2 nAChRs agonist and mecamylamine (1.0 mg/kg, i. p.), α4β2 nAChRs antagonist were injected chronically during 14 days. The processes of learning/memory were assessed in different models of learning: passive avoidance performance and Morris water maze. Chronic RJR-2403 administration to females improved the passive avoidance performance in proestrous and estrous as compared to the control animals. Also, RJR-2403 restored spatial learning of rats during proestrous phases in Morris water maze, and stimulated the dynamics of spatial learning during estrous phases. On the contrary, the chronic mecamylamine administration impaired non-spatial, and especially, spatial learning in females during key phases of ovary cycle. The results of the study suggest positive effect of α4β2 nAChRs stimulation in learning/memory processes during ovary cycle in the adult female rats. PMID:25464732

  1. Nicotine promotes cell proliferation via {alpha}7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells

    SciTech Connect

    Wong, Helen Pui Shan; Yu Le; Lam, Emily Kai Yee; Tai, Emily Kin Ki; Wu, William Ka Kei; Cho, Chi Hin . E-mail: chcho@cuhk.edu.hk

    2007-06-15

    Cigarette smoking has been implicated in colon cancer. Nicotine is a major alkaloid in cigarette smoke. In the present study, we showed that nicotine stimulated HT-29 cell proliferation and adrenaline production in a dose-dependent manner. The stimulatory action of nicotine was reversed by atenolol and ICI 118,551, a {beta}{sub 1}- and {beta}{sub 2}-selective antagonist, respectively, suggesting the role of {beta}-adrenoceptors in mediating the action. Nicotine also significantly upregulated the expression of the catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-{beta}-hydroxylase (D{beta}H) and phenylethanolamine N-methyltransferase]. Inhibitor of TH, a rate-limiting enzyme in the catecholamine-biosynthesis pathway, reduced the actions of nicotine on cell proliferation and adrenaline production. Expression of {alpha}7-nicotinic acetylcholine receptor ({alpha}7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an {alpha}7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and D{beta}H expression as well as adrenaline production. Taken together, through the action on {alpha}7-nAChR nicotine stimulates HT-29 cell proliferation via the upregulation of the catecholamine-synthesis pathway and ultimately adrenaline production and {beta}-adrenergic activation. These data reveal the contributory role {alpha}7-nAChR and {beta}-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer.

  2. Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid.

    PubMed

    Tomizawa, M; Wen, Z; Chin, H L; Morimoto, H; Kayser, H; Casida, J E

    2001-09-01

    The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs. PMID:11579144

  3. Biomolecular recognition of antagonists by α7 nicotinic acetylcholine receptor: Antagonistic mechanism and structure-activity relationships studies.

    PubMed

    Peng, Wei; Ding, Fei

    2015-08-30

    As the key constituent of ligand-gated ion channels in the central nervous system, nicotinic acetylcholine receptors (nAChRs) and neurodegenerative diseases are strongly coupled in the human species. In recently years the developments of selective agonists by using nAChRs as the drug target have made a large progress, but the studies of selective antagonists are severely lacked. Currently these antagonists rest mainly on the extraction of partly natural products from some animals and plants; however, the production of these crude substances is quite restricted, and artificial synthesis of nAChR antagonists is still one of the completely new research fields. In the context of this manuscript, our primary objective was to comprehensively analyze the recognition patterns and the critical interaction descriptors between target α7 nAChR and a series of the novel compounds with potentially antagonistic activity by means of virtual screening, molecular docking and molecular dynamics simulation, and meanwhile these recognition reactions were also compared with the biointeraction of α7 nAChR with a commercially natural antagonist - methyllycaconitine. The results suggested clearly that there are relatively obvious differences of molecular structures between synthetic antagonists and methyllycaconitine, while the two systems have similar recognition modes on the whole. The interaction energy and the crucially noncovalent forces of the α7 nAChR-antagonists are ascertained according to the method of Molecular Mechanics/Generalized Born Surface Area. Several amino acid residues, such as B/Tyr-93, B/Lys-143, B/Trp-147, B/Tyr-188, B/Tyr-195, A/Trp-55 and A/Leu-118 played a major role in the α7 nAChR-antagonist recognition processes, in particular, residues B/Tyr-93, B/Trp-147 and B/Tyr-188 are the most important. These outcomes tally satisfactorily with the discussions of amino acid mutations. Based on the explorations of three-dimensional quantitative structure

  4. Differential antagonism and tolerance/cross-tolerance among nicotinic acetylcholine receptor agonists: scheduled-controlled responding and hypothermia in C57BL/6J mice.

    PubMed

    de Moura, Fernando B; McMahon, Lance R

    2016-04-01

    The tobacco-dependence pharmacotherapies varenicline and cytisine act as partial α4β2 nAChR agonists. However, the extent to which α4β2 nicotinic acetylcholine receptors (nAChRs) mediate their in-vivo effects remains unclear. Nicotine, varenicline, cytisine, and epibatidine were studied in male C57BL/6J mice for their effects on rates of fixed ratio responding and rectal temperature alone and in combination with the nonselective nAChR antagonist mecamylamine and the α4β2 nAChR antagonist dihydro-β-erythroidine. The effects of nicotine, varenicline, cytisine, epibatidine, and cocaine were assessed before and during chronic nicotine treatment. The rate-decreasing and hypothermic effects of nicotine, varenicline, cytisine, and epibatidine were antagonized by mecamylamine (1 mg/kg), but only the effects of nicotine and epibatidine were antagonized by dihydro-β-erythroidine (3.2 mg/kg). Chronic nicotine produced 4.7 and 5.1-fold rightward shifts in the nicotine dose-effect functions to decrease response rate and rectal temperature, respectively. Nicotine treatment decreased the potency of epibatidine to decrease response rate and rectal temperature 2.2 and 2.9-fold, respectively, and shifted the varenicline dose-effect functions 2.0 and 1.7-fold rightward, respectively. Cross-tolerance did not develop from nicotine to cytisine. These results suggest that the in-vivo pharmacology of tobacco cessation aids cannot be attributed to a single nAChR subtype; instead, multiple receptor subtypes differentially mediate their effects. PMID:26910582

  5. Nicotinic modulation of glutamate receptor function at nerve terminal level: a fine-tuning of synaptic signals.

    PubMed

    Marchi, Mario; Grilli, Massimo; Pittaluga, Anna M

    2015-01-01

    This review focuses on a specific interaction occurring between the nicotinic cholinergic receptors (nAChRs) and the glutamatergic receptors (GluRs) at the nerve endings level. We have employed synaptosomes in superfusion and supplemented and integrated our findings with data obtained using techniques from molecular biology and immuno-cytochemistry, and the assessment of receptor trafficking. In particular, we characterize the following: (1) the direct and unequivocal localization of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) glutamatergic receptors on specific nerve terminals, (2) their pharmacological characterization and functional co-localization with nAChRs on the same nerve endings, and (3) the existence of synergistic or antagonistic interactions among them. Indeed, in the rat nucleus accumbens (NAc), the function of some AMPA and NMDA receptors present on the dopaminergic and glutamatergic nerve terminals can be regulated negatively or positively in response to a brief activation of nAChRs. This effect occurs rapidly and involves the trafficking of AMPA and NMDA receptors. The event takes place also at very low concentrations of nicotine and involves the activation of several nAChRs subtypes. This dynamic control by cholinergic nicotinic system of glutamatergic NMDA and AMPA receptors might therefore represent an important neuronal presynaptic adaptation associated with nicotine administration. The understanding of the role of these nicotine-induced functional changes might open new and interesting perspectives both in terms of explaining the mechanisms that underlie some of the effects of nicotine addiction and in the development of new drugs for smoking cessation. PMID:25972809

  6. Presynaptic α4β2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus.

    PubMed

    Garduño, Julieta; Galindo-Charles, Luis; Jiménez-Rodríguez, Javier; Galarraga, Elvira; Tapia, Dagoberto; Mihailescu, Stefan; Hernandez-Lopez, Salvador

    2012-10-24

    Several behavioral effects of nicotine are mediated by changes in serotonin (5-HT) release in brain areas that receive serotonergic afferents from the dorsal raphe nucleus (DRN). In vitro experiments have demonstrated that nicotine increases the firing activity in the majority of DRN 5-HT neurons and that DRN contains nicotinic acetylcholine receptors (nAChRs) located at both somata and presynaptic elements. One of the most common presynaptic effects of nicotine is to increase glutamate release. Although DRN receives profuse glutamatergic afferents, the effect of nicotine on glutamate release in the DRN has not been studied in detail. Using whole-cell recording techniques, we investigated the effects of nicotine on the glutamatergic input to 5-HT DRN neurons in rat midbrain slices. Low nicotine concentrations, in the presence of bicuculline and tetrodotoxin (TTX), increased the frequency but did not change the amplitude of glutamate-induced EPSCs, recorded from identified 5-HT neurons. Nicotine-induced increase of glutamatergic EPSC frequency persisted 10-20 min after drug withdrawal. This nicotinic effect was mimicked by exogenous administration of acetylcholine (ACh) or inhibition of ACh metabolism. In addition, the nicotine-induced increase in EPSC frequency was abolished by blockade of α4β2 nAChRs, voltage-gated calcium channels, or intracellular calcium signaling but not by α7 nAChR antagonists. These data suggest that both nicotine and endogenous ACh can increase glutamate release through activation of presynaptic α4β2 but not α7 nAChRs in the DRN. The effect involves long-term changes in synaptic function, and it is dependent on voltage-gated calcium channels and presynaptic calcium stores. PMID:23100436

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

    USGS Publications Warehouse

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

    2014-01-01

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

  8. Antagonizing amyloid-β/calcium-sensing receptor signaling in human astrocytes and neurons: a key to halt Alzheimer's disease progression?

    PubMed Central

    Dal Prà, Ilaria; Chiarini, Anna; Armato, Ubaldo

    2015-01-01

    Astrocytes’ roles in late-onset Alzheimer's disease (LOAD) promotion are important, since they survive soluble or fibrillar amyloid-β peptides (Aβs) neurotoxic effects, undergo alterations of intracellular and intercellular Ca2+ signaling and gliotransmitters release via the Aβ/α7-nAChR (α7-nicotinic acetylcholine receptor) signaling, and overproduce/oversecrete newly synthesized Aβ42 oligomers, NO, and VEGF-A via the Aβ/CaSR (calcium-sensing receptor) signaling. Recently, it was suggested that the NMDAR (N-methyl-D-aspartate receptor) inhibitor nitromemantine would block the synapse-destroying effects of Aβ/α7-nAChR signaling. Yet, this and the progressive extracellular accrual and spreading of Aβ42 oligomers would be stopped well upstream by NPS 2143, an allosteric CaSR antagonist (calcilytic). PMID:25883618

  9. Targeting the α7 nicotinic acetylcholine receptor to prevent progressive dementia and improve cognition in adults with Down's syndrome.

    PubMed

    Deutsch, Stephen I; Burket, Jessica A; Benson, Andrew D

    2014-10-01

    As persons with Down's syndrome (DS) age into the third decade and beyond, they develop Alzheimer's disease (AD)-like histopathological changes in brain and may manifest progressive worsening of adaptive functions. Increasingly, persons with DS have near-normal to normal life spans; thus, it has become a therapeutic imperative to preserve adaptive functions and ability to live as independently as possible in the least restrictive environment throughout adulthood. Data suggest that these histopathological changes and worsening adaptive functions result, at least in part, from the binding of the amyloidogenic Aβ1-42 peptide to α7 nicotinic acetylcholine receptors (α7nAChRs) on the surface of neurons, which can lead to the internalization of the tightly-bound complex and cell lysis. Pharmacotherapeutic targeting of the α7nAChR may inhibit the creation of the Aβ1-42-α7nAChR complex, which has been observed both intraneuronally and as a component of the amyloid plaque seen in AD. Additionally, selective α7nAChR agonists may improve memory and cognition independently of their potential ability to attenuate the cytotoxicity of Aβ1-42 and retard the deposition of amyloid plaques in adults with DS. However, there are conflicting data supporting an antagonist strategy to improve cognition in the presence of elevated levels of Aβ amyloidogenic peptides, as well as to prevent emergence of pyramidal neuron hyperexcitability. A major challenge to the implementation of clinical trials of targeted α7nAChR interventions in adults with DS will be the ability to detect medication-induced changes in cognition in the context of intellectual disability. The Review will consider some of the current evidence supporting both the role of the Aβ1-42-α7nAChR complex in the pathogenesis of the AD-like histopathology in adult persons with DS, and pharmacotherapeutic interventions with α7nAChR agonists. PMID:24865150

  10. Serotonin 5-HT3 receptors in rat CA1 hippocampal interneurons: functional and molecular characterization

    PubMed Central

    Sudweeks, Sterling N; van Hooft, Johannes A; Yakel, Jerrel L

    2002-01-01

    The molecular makeup of the serotonin 5-HT3 receptor (5-HT3R) channel was investigated in rat hippocampal CA1 interneurons in slices using single-cell RT-PCR and patch-clamp recording techniques. We tested for the expression of the 5-HT3A (both short and long splice variants) and 5-HT3B subunits, as well as the expression of the α4 subunit of the neuronal nicotinic ACh receptors (nAChRs), the latter of which has been shown to co-assemble with the 5-HT3A subunit in heterologous expression systems. Both the 5-HT3A-short and α4-nAChR subunits were expressed in these interneurons, but we could not detect any expression of either the 5-HT3B or the 5-HT3A-long subunits. Furthermore, there was a strong tendency for the 5-HT3A-short and α4-nAChR subunits to be co-expressed in individual interneurons. To assess whether there was any functional evidence for co-assembly between the 5-HT3A-short and α4-nAChR subunits, we used the sulphydryl agent 2-aminoethyl methanethiosulphonate (MTSEA), which has previously been shown to modulate expressed 5-HT3Rs that contain the α4-nAChR subunit. In half of the interneurons examined, MTSEA significantly enhanced the amplitude of the 5-HT3R-mediated responses, which is consistent with the notion that the α4-nAChR subunit co-assembles with the 5-HT3A subunit to form a native heteromeric 5-HT3R channel in rat CA1 hippocampal interneurons in vivo. In addition, the single-channel properties of the 5-HT3R were investigated in outside-out patches. No resolvable single-channel currents were observed. Using non-stationary fluctuation analysis, we obtained an estimate of the single-channel conductance of 4 pS, which is well below that expected for channels containing both the 5-HT3A and 5-HT3B subunits. PMID:12411518

  11. Desensitizing and non-desensitizing subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in cockroach neurons.

    PubMed

    Salgado, Vincent L; Saar, Raimund

    2004-10-01

    Two alpha-bungarotoxin-sensitive nicotinic receptor subtypes in cockroach neurons are identified as desensitizing (nAChD), selectively inhibitable with 100 nM imidacloprid, and non-desensitizing (nAChN), selectively inhibitable with 100 pM methyllycaconitine. Although the desensitization rate of nAChD receptors is highly variable, pharmacology is largely independent of desensitization rate. Because desensitized states tightly bind agonists, nAChD receptors are potently inhibited by neonicotinoids and specifically measured in radiolabeled imidacloprid binding assays. However, they are not usually detected in binding assays with radiolabeled alpha-bungarotoxin, which has a Kd for the resting state of 21 nM, but binds poorly to desensitized states often present in binding assays. In contrast, nAChN receptors are specifically measured in binding assays with radiolabeled alpha-bungarotoxin, which binds them with a Kd of 1.3 nM. nAChN receptors are activated by neonicotinoids at micromolar concentrations, and allosterically by spinosyn A, with an EC50 of 27 nM. Spinosyn A weakly antagonizes nAChD receptors -23% at 10 microM. The roles of the two nAChR subtypes in insecticide poisoning are discussed. PMID:15518655

  12. Neuregulin 1 as an endogenous regulator of nicotinic acetylcholine receptors in adult major pelvic ganglion neurons.

    PubMed

    Kim, Han-Gyu; Cho, Sung-Min; Lee, Choong-Ku; Jeong, Seong-Woo

    2015-08-01

    We investigated whether endogenous neuregulin 1 (NRG1) is released in a soluble form (called sNRG1) and upregulates expression of nicotinic acetylcholine receptor (nAChR) in autonomic major pelvic ganglion (MPG) neurons of adult rats. To elicit the release of sNRG1, either the hypogastric nerve or the pelvic nerve was electrically stimulated. Then, the MPG-conditioned medium (CM) was subjected to western blotting using an antibody directed against the N-terminal ectodomain of NRG1. Both sympathetic and parasympathetic nerve activation elicited the release of sNRG1 from MPG neurons in a frequency-dependent manner. The sNRG1 release was also induced by treatment of MPG neurons with either high KCl or neurotrophic factors. The biological activity of the released sNRG1 was detected by tyrosine phosphorylation (p185) of the ErbB2 receptors in MPG neurons. When MPG neurons were incubated for 6 h in the CM, the protein level of the nAChR α3 subunit and ACh-induced current (IACh) density were significantly increased. The CM-induced changes in IACh was abolished by a selective ErbB2 tyrosine kinase inhibitor. Taken together, these data suggest that NRG1 functions as an endogenous regulator of nAChR expression in adult MPG neurons. PMID:26043693

  13. Cognitive improvements in a mouse model with substituted 1,2,3-triazole agonists for nicotinic acetylcholine receptors.

    PubMed

    Arunrungvichian, Kuntarat; Boonyarat, Chantana; Fokin, Valery V; Taylor, Palmer; Vajragupta, Opa

    2015-08-19

    The α7 nicotinic acetylcholine receptor (nAChR) is a recognized drug target for dementias of aging and certain developmental disorders. Two selective and potent α7-nAChR agonists, winnowed from a list of 43 compounds characterized in a companion article (DOI: 10.1021/acschemneuro.5b00058), 5-((quinuclid-3-yl)-1H-1,2,3-triazol-4-yl)-1H-indole (IND8) and 3-(4-hydroxyphenyl-1,2,3-triazol-1-yl) quinuclidine (QND8), were evaluated for cognitive improvement in both short- and long-term memory. Tacrine, a centrally active acetylcholinesterase inhibitor, and PNU-282987, a congeneric α7 nAChR agonist, were employed as reference standards. Three behavioral tests, modified Y-maze, object recognition test (ORT), and water maze, were performed in scopolamine-induced amnesic mice. Intraperitoneal injection of these two compounds significantly improved the cognitive impairment in a modified Y-maze test (5 μmol/kg for IND8 and 10 μmol/kg for QND8), ORT (10 μmol/kg), and water maze test (25 μmol/kg). For delay induced memory deficit or natural memory loss in mice, IND8 and QND8 at 10 μmol/kg were able to enhance memory comparable to PNU-282987 when evaluated using ORT time delay model. Cognitive enhancement of IND8 and QND8 was mediated through α7-nAChRs as evidenced by its complete abolition after pretreatment with a selective α7-nAChR antagonist, methyllycaconitine. These data demonstrate that IND8 and QND8 and their congeners are potential candidates for treatment of cognitive disorders, and the substituted triazole series formed by cycloaddition of alkynes and azides warrant further preclinical optimization. PMID:25978789

  14. Mapping the agonist binding site of the nicotinic acetylcholine receptor. Orientation requirements for activation by covalent agonist.

    PubMed

    Sullivan, D A; Cohen, J B

    2000-04-28

    To characterize the structural requirements for ligand orientation compatible with activation of the Torpedo nicotinic acetylcholine receptor (nAChR), we used Cys mutagenesis in conjunction with sulfhydryl-reactive reagents to tether primary or quaternary amines at defined positions within the agonist binding site of nAChRs containing mutant alpha- or gamma-subunits expressed in Xenopus oocytes. 4-(N-Maleimido)benzyltrimethylammonium and 2-aminoethylmethanethiosulfonate acted as irreversible antagonists when tethered at alphaY93C, alphaY198C, or gammaE57C, as well as at alphaN94C (2-aminoethylmethanethiosulfonate only). [2-(Trimethylammonium)-ethyl]-methanethiosulfonate (MTSET), which attaches thiocholine to binding site Cys, also acted as an irreversible antagonist when tethered at alphaY93C, alphaN94C, or gammaE57C. However, MTSET modification of alphaY198C resulted in prolonged activation of the nAChR not reversible by washing but inhibitable by subsequent exposure to non-competitive antagonists. Modification of alphaY198C (or any of the other positions tested) by [(trimethylammonium)methyl]methanethiosulfonate resulted only in irreversible inhibition, while modification of alphaY198C by [3-(trimethylammonium)propyl]methanethiosulfonate resulted in irreversible activation of nAChR, but at lower efficacy than by MTSET. Thus changing the length of the tethering arm by less than 1 A in either direction markedly effects the ability of the covalent trimethylammonium to activate the nAChR, and agonist activation depends on a very selective orientation of the quaternary ammonium within the agonist binding site. PMID:10777557

  15. A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper).

    PubMed

    Liu, Zewen; Williamson, Martin S; Lansdell, Stuart J; Denholm, Ian; Han, Zhaojun; Millar, Neil S

    2005-06-14

    Neonicotinoids, such as imidacloprid, are nicotinic acetylcholine receptor (nAChR) agonists with potent insecticidal activity. Since its introduction in the early 1990s, imidacloprid has become one of the most extensively used insecticides for both crop protection and animal health applications. As with other classes of insecticides, resistance to neonicotinoids is a significant threat and has been identified in several pest species, including the brown planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. In this study, radioligand binding experiments have been conducted with whole-body membranes prepared from imidacloprid-susceptible and imidacloprid-resistant strains of N. lugens. The results reveal a much higher level of [3H]imidacloprid-specific binding to the susceptible strain than to the resistant strain (16.7 +/- 1.0 and 0.34 +/- 0.21 fmol/mg of protein, respectively). With the aim of understanding the molecular basis of imidacloprid resistance, five nAChR subunits (Nlalpha1-Nlalpha4 and Nlbeta1) have been cloned from N. lugens.A comparison of nAChR subunit genes from imidacloprid-sensitive and imidacloprid-resistant populations has identified a single point mutation at a conserved position (Y151S) in two nAChR subunits, Nlalpha1 and Nlalpha3. A strong correlation between the frequency of the Y151S point mutation and the level of resistance to imidacloprid has been demonstrated by allele-specific PCR. By expression of hybrid nAChRs containing N. lugens alpha and rat beta2 subunits, evidence was obtained that demonstrates that mutation Y151S is responsible for a substantial reduction in specific [3H]imidacloprid binding. This study provides direct evidence for the occurrence of target-site resistance to a neonicotinoid insecticide. PMID:15937112

  16. Prior nicotine self-administration attenuates subsequent dopaminergic deficits of methamphetamine in rats: role of nicotinic acetylcholine receptors.

    PubMed

    Baladi, Michelle G; Nielsen, Shannon M; McIntosh, J Michael; Hanson, Glen R; Fleckenstein, Annette E

    2016-08-01

    Preclinical studies have demonstrated that oral nicotine exposure attenuates long-term dopaminergic damage induced by toxins, including repeated, high doses of methamphetamine. It is suggested that alterations in nicotinic acetylcholine receptor (nAChR) expression, including α4β2* and α6β2* subtypes, likely contribute to this protection. The current study extended these findings by investigating whether nicotine self-administration in male, Sprague-Dawley rats (a) attenuates short-term dopaminergic damage induced by methamphetamine and (b) causes alterations in levels of α4β2* and α6β2* nAChR subtypes. The findings indicate that nicotine self-administration (0.032 mg/kg/infusion for 14 days) per se did not alter α4β2* and α6β2* nAChR expression or dopamine transporter (DAT) expression and function. Interestingly, prior nicotine self-administration attenuated methamphetamine-induced decreases in DAT function when assessed 24 h, but not 1 h, after methamphetamine treatment (4×7.5 mg/kg/injection). The ability of nicotine to attenuate the effects of methamphetamine on DAT function corresponded with increases in α4β2*, but not α6β2*, nAChR binding density. Understanding the role of nAChRs in methamphetamine-induced damage has the potential to elucidate mechanisms underlying the etiology of disorders involving dopaminergic dysfunction, as well as to highlight potential new therapeutic strategies for prevention or reduction of dopaminergic neurodegeneration. PMID:26871405

  17. In vivo chronic nicotine exposure differentially and reversibly affects upregulation and stoichiometry of α4β2 nicotinic receptors in cortex and thalamus.

    PubMed

    Fasoli, F; Moretti, M; Zoli, M; Pistillo, F; Crespi, A; Clementi, F; Mc Clure-Begley, T; Marks, M J; Gotti, C

    2016-09-01

    Studies with heterologous expression systems have shown that the α4β2 nicotinic acetylcholine receptor (nAChR) subtype can exist in two stoichiometries (with two [(α4)2(β2)3] or three [(α4)3(β2)2] copies of the α subunit in the receptor pentamer) which have different pharmacological and functional properties and are differently regulated by chronic nicotine treatment. However, the effects of nicotine treatment in vivo on native α4β2 nAChR stoichiometry are not well known. We investigated in C57BL/6 mice the in vivo effect of 14-day chronic nicotine treatment and subsequent withdrawal, on the subunit expression and β2/α4 subunit ratio of (3)H-epibatidine labeled α4β2*-nAChR in total homogenates of cortex and thalamus. We found that in basal conditions the ratio of the β2/α4 subunit in the cortex and thalamus is different indicating a higher proportion in receptors with (α4)2(β2)3 subunit stoichiometry in the thalamus. For cortex exposure to chronic nicotine elicited an increase in receptor density measured by (3)H-epibatidine binding, an increase in the α4 and β2 protein levels, and an increase in β2/α4 subunit ratio, that indicates an increased proportion of receptors with the (α4)2(β2)3 stoichiometry. For thalamus we did not find a significant increase in receptor density, α4 and β2 protein levels, or changes in β2/α4 subunit ratio. All the changes elicited by chronic nicotine in cortex were transient and returned to basal levels with an average half-life of 2.8 days following nicotine withdrawal. These data suggest that chronic nicotine exposure in vivo favors increased assembly of α4β2 nAChR containing three β2 subunits. A greater change in stoichiometry was observed for cortex (which has relatively low basal expression of (α4)2(β2)3 nAChR) than in thalamus (which has a relatively high basal expression of (α4)2(β2)3 nAChR). PMID:27157710

  18. Recent developments in the synthesis of nicotinic acetylcholine receptor ligands.

    PubMed

    Breining, Scott R

    2004-01-01

    The extraordinary pharmacology of nicotine and epibatidine have indicated the potential for nicotinic acetylcholine receptor (nAChR) ligands to serve as a new therapeutic class for a host of CNS disorders. Many such ligands are natural products, or analogs thereof, which represent a significant challenge to the synthetic chemist. Synthesis of such molecules often serves as a showcase to demonstrate the potential of newly developed methodology. This synthetic challenge coupled with the promise of pharmacological activity in compounds possessing the nicotinic pharmacophore has stimulated a great deal of synthetic activity over the last five years. The present report provides an overview of novel synthetic methodology occurring during this period directed toward the synthesis of compounds with presumed affinity for the neuronal nAChR. Syntheses chosen for review here represent the major efforts toward molecules such as epibatidine analogs, anatoxin-a, nicotine and related alkaloids, conformationally constrained nicotine derivatives, cytisine and methyllycaconitine (MLA). PMID:14965298

  19. Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends*

    PubMed Central

    Basu, Sreya; Sladecek, Stefan; Pemble, Hayley; Wittmann, Torsten; Slotman, Johan A.; van Cappellen, Wiggert; Brenner, Hans-Rudolf; Galjart, Niels

    2014-01-01

    The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2–9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2–8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends. PMID:25231989

  20. Light microscopic distribution of some cholinergic markers in the rat and rabbit locus coeruleus and the nucleus angularis grisea periventricularis of the domestic pig (Sus scrofa): a correlative electron microscopic investigation of cholinergic receptor proteins in the rabbit.

    PubMed

    Caffé, A R

    1994-10-15

    Cholinergic modulation of locus coeruleus (LC) neurons evokes a variety of neuronal and behavioural effects. In an attempt to understand the LC cholinergic circuit, several markers has been investigated and compared. (Immuno)-histochemical and autoradiographic methods have been used on rat, rabbit, and pig tissue. To identify the boundaries of the LC in each of these species, sections through the entire brainstem have been stained for tyrosine hydroxylase. The results indicate that the pig does not possess a LC proper that conforms to the accepted features of this cell group. However, in this location fusiform cells reminiscent of LC interneurons are still present. This group of fusiform neurons has been named the nucleus angularis grisea periventricularis (NAGP). LC cells of the rat and rabbit show strong acetylcholinesterase (AChE) activity. In the pig the NAGP is markedly free from AChE staining. Muscarinic binding sites are densely distributed over the rabbit LC and adjacent region. The rat and rabbit LC neurons synthesise both muscarinic (mAChR) and nicotinic receptor protein (nAChR). In the pig NAGP region mAChR and nAChR positive cell bodies are almost absent, while some nAChR immunoreactive dendrites are present. The light microscopic data in the rabbit have been confirmed by electron microscopic analysis. It is concluded that the general concept of a noradrenergic LC that is present throughout mammals is questionable. At present, choline acetyltransferase immunoreactive terminals that closely correspond to the other cholinergic components in the rat or rabbit LC have not been observed. However, in these species the cholinergic sensitivity of LC cells is mediated via both muscarinic and nicotinic receptors on somata and dendrites. PMID:7849322

  1. Synthesis and structure-activity relationships of 3,8-diazabicyclo[4.2.0]octane ligands, potent nicotinic acetylcholine receptor agonists.

    PubMed

    Frost, Jennifer M; Bunnelle, William H; Tietje, Karin R; Anderson, David J; Rueter, Lynne E; Curzon, Peter; Surowy, Carol S; Ji, Jianquo; Daanen, Jerome F; Kohlhaas, Kathy L; Buckley, Michael J; Henry, Rodger F; Dyhring, Tino; Ahring, Philip K; Meyer, Michael D

    2006-12-28

    A series of potent neuronal nicotinic acetylcholine receptor (nAChR) ligands based on a 3,8-diazabicyclo[4.2.0]octane core have been synthesized and evaluated for affinity and agonist efficacy at the human high affinity nicotine recognition site (halpha4beta2) and in a rat model of persistent nociceptive pain (formalin model). Numerous analogs in this series exhibit picomolar affinity in radioligand binding assays and nanomolar agonist potency in functional assays, placing them among the most potent nAChR ligands known for the halpha4beta2 receptor. Several of the compounds reported in this study (i.e., 24, 25, 28, 30, 32, and 47) exhibit equivalent or greater affinity for the halpha4beta2 receptor relative to epibatidine, and like epibatidine, many exhibit robust analgesic efficacy in the rat formalin model of persistent pain. PMID:17181167

  2. Low Dose Nicotine and Antagonism of β2 Subunit Containing Nicotinic Acetylcholine Receptors Have Similar Effects on Affective Behavior in Mice

    PubMed Central

    Anderson, Shawn M.; Brunzell, Darlene H.

    2012-01-01

    Nicotine leads to both activation and desensitization (inactivation) of nicotinic acetylcholine receptors (nAChRs). This study tested the hypothesis that nicotine and a selective antagonist of β2*nAChRs would have similar effects on affective behavior. Adult C57BL/6J male mice were tested in a conditioned emotional response (CER) assay which evaluates the ability of an aversive stimulus to inhibit goal-directed behavior. Mice lever-pressed for a saccharin reinforcer according to a variable schedule of reinforcement during sessions in which two presentations of a compound light/tone conditioned stimulus (CS) co-terminated with a 0.1 or 0.3 mA, 0.5 s footshock unconditioned stimulus (US). During testing in the absence of the US, mice received doses of i.p. nicotine (0, 0.0032, 0.01, 0.032, 0.1 mg/kg) or a selective β2 subunit containing nAChR (β2*nAChR) antagonist dihydro-beta-erythroidine (0, 0.1, 0.3, 1.0, 3.0 mg/kg DHβE). There was a dose-dependent effect of nicotine revealing that only low doses (0.01, 0.032 mg/kg) increased CER suppression ratios (SR) in these mice. DHβE also dose-dependently increased SR at the 3 mg/kg dose. In ethological measures of fear−/anxiety-like behavior, these doses of nicotine and DHβE significantly reduced digging behavior in a marble burying task and 0.3 mg/kg DHβE promoted open-arm activity in the elevated plus maze. Doses of nicotine and DHβE that altered affective behavior had no effect on locomotor activity. Similar to previous reports with anxiolytic drugs, low dose nicotine and DHβE reversed SR in a CER assay, decreased digging in a marble burying assay and increased open arm activity in the elevated plus maze. This study provides evidence that inactivation of β2*nAChRs reduces fear-like and anxiety-like behavior in rodents and suggests that smokers may be motivated to smoke in part to desensitize their β2*nAChRs. These data further identify β2*nAChR antagonism as a potential therapeutic strategy for relief of

  3. New potential AChE inhibitor candidates.

    PubMed

    de Paula, A A N; Martins, J B L; dos Santos, M L; Nascente, L de C; Romeiro, L A S; Areas, T F M A; Vieira, K S T; Gambôa, N F; Castro, N G; Gargano, R

    2009-09-01

    We have theoretically studied new potential candidates of acetylcholinesterase (AChE) inhibitors designed from cardanol, a non-isoprenoid phenolic lipid of cashew Anacardium occidentale nut-shell liquid. The electronic structure calculations of fifteen molecule derivatives from cardanol were performed using B3LYP level with 6-31G, 6-31G(d), and 6-311+G(2d,p) basis functions. For this study we used the following groups: methyl, acetyl, N,N-dimethylcarbamoyl, N,N-dimethylamine, N,N-diethylamine, piperidine, pyrrolidine, and N,N-methylbenzylamine. Among the proposed compounds we identified that the structures with substitution by N,N-dimethycarbamoyl, N,N-dimethylamine, and pyrrolidine groups were better correlated to rivastigmine, and represent possible AChE inhibitors against Alzheimer disease. PMID:19446931

  4. Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

    PubMed Central

    Engle, Staci E.; Broderick, Hilary J.; Drenan, Ryan M.

    2012-01-01

    Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority. Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9′A mice 1 and α6 L9′S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices. In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of

  5. A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella

    PubMed Central

    Wang, Jing; Wang, Xingliang; Lansdell, Stuart J.; Zhang, Jianheng; Millar, Neil S.; Wu, Yidong

    2016-01-01

    Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. PMID:26855198

  6. Activation of α-7 nicotinic acetylcholine receptor reduces ischemic stroke injury through reduction of pro-inflammatory macrophages and oxidative stress.

    PubMed

    Han, Zhenying; Shen, Fanxia; He, Yue; Degos, Vincent; Camus, Marine; Maze, Mervyn; Young, William L; Su, Hua

    2014-01-01

    Activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) has a neuro-protective effect on ischemic and hemorrhagic stroke. However, the underlying mechanism is not completely understood. We hypothesized that α-7 nAchR agonist protects brain injury after ischemic stroke through reduction of pro-inflammatory macrophages (M1) and oxidative stress. C57BL/6 mice were treated with PHA568487 (PHA, α-7 nAchR agonist), methyllycaconitine (MLA, nAchR antagonist), or saline immediately and 24 hours after permanent occlusion of the distal middle cerebral artery (pMCAO). Behavior test, lesion volume, CD68(+), M1 (CD11b(+)/Iba1(+)) and M2 (CD206/Iba1+) microglia/macrophages, and phosphorylated p65 component of NF-kB in microglia/macrophages were quantified using histological stained sections. The expression of M1 and M2 marker genes, anti-oxidant genes and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were quantified using real-time RT-PCR. Compared to the saline-treated mice, PHA mice had fewer behavior deficits 3 and 7 days after pMCAO, and smaller lesion volume, fewer CD68(+) and M1 macrophages, and more M2 macrophages 3 and 14 days after pMCAO, whereas MLA's effects were mostly the opposite in several analyses. PHA increased anti-oxidant genes and NADPH oxidase expression associated with decreased phosphorylation of NF-kB p65 in microglia/macrophages. Thus, reduction of inflammatory response and oxidative stress play roles in α-7 nAchR neuro-protective effect. PMID:25157794

  7. In vivo pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107: preclinical considerations in Alzheimer's disease.

    PubMed

    Bitner, R Scott; Bunnelle, William H; Decker, Michael W; Drescher, Karla U; Kohlhaas, Kathy L; Markosyan, Stella; Marsh, Kennan C; Nikkel, Arthur L; Browman, Kaitlin; Radek, Rich; Anderson, David J; Buccafusco, Jerry; Gopalakrishnan, Murali

    2010-09-01

    We previously reported that alpha7 nicotinic acetylcholine receptor (nAChR) agonism produces efficacy in preclinical cognition models correlating with activation of cognitive and neuroprotective signaling pathways associated with Alzheimer's disease (AD) pathology. In the present studies, the selective and potent alpha7 nAChR agonist 5-(6-[(3R)-1-azabicyclo[2.2.2]oct-3-yloxy] pyridazin-3-yl)-1H-indole (ABT-107) was evaluated in behavioral assays representing distinct cognitive domains. Studies were also conducted to address potential issues that may be associated with the clinical development of an alpha7 nAChR agonist. Specifically, ABT-107 improved cognition in monkey delayed matching to sample, rat social recognition, and mouse two-trial inhibitory avoidance, and continued to improve cognitive performance at injection times when exposure levels continued to decline. Rats concurrently infused with ABT-107 and donepezil at steady-state levels consistent with clinical exposure showed improved short-term recognition memory. Compared with nicotine, ABT-107 did not produce behavioral sensitization in rats or exhibit psychomotor stimulant activity in mice. Repeated (3 days) daily dosing of ABT-107 increased extracellular cortical acetylcholine in rats, whereas acute administration increased cortical extracellular signal-regulated kinase and cAMP response element-binding protein phosphorylation in mice, neurochemical and biochemical events germane to cognitive function. ABT-107 increased cortical phosphorylation of the inhibitory residue (Ser9) of glycogen synthase kinase-3, a primary tau kinase associated with AD pathology. In addition, continuous infusion of ABT-107 in tau/amyloid precursor protein transgenic AD mice reduced spinal tau hyperphosphorylation. These findings show that targeting alpha7 nAChRs may have potential utility for symptomatic alleviation and slowing of disease progression in the treatment AD, and expand the understanding of the potential

  8. Encenicline, an α7 Nicotinic Acetylcholine Receptor Partial Agonist, Reduces Immune Cell Infiltration in the Colon and Improves Experimental Colitis in Mice.

    PubMed

    Salaga, M; Blomster, L V; Piechota-Polańczyk, A; Zielińska, M; Jacenik, D; Cygankiewicz, A I; Krajewska, W M; Mikkelsen, J D; Fichna, Jakub

    2016-01-01

    The α7 pentamer nicotinic acetylcholine receptors (nAChRs) are a target in transduction of anti-inflammatory signals from the central nervous system to the gastrointestinal (GI) tract. The aim of this study was to investigate the anti-inflammatory action of the novel α7 nAChR partial agonist encenicline and to determine the mechanism underlying its activity. Anti-inflammatory activity of encenicline was evaluated using trinitrobenzenesulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced models of colitis. Macroscopic score, ulcer score, colon length and thickness, as well as myeloperoxidase (MPO) activity were recorded. Immunohistochemistry (IHC) was used to measure the infiltration of immune cells in the colon. Furthermore, we employed flow cytometry to determine the effect of encenicline on frequencies of FoxP3(+) and interleukin (IL)-17A(+) T cells in the mouse colon. Encenicline attenuated TNBS- and DSS-induced colitis in mice via α7 nAChRs, as indicated by significantly reduced macroscopic parameters and MPO activity. Treatment with encenicline significantly reduced the infiltration of macrophages, neutrophils, and B cells in the colon of TNBS-treated animals, as indicated by IHC. In the TNBS model encenicline reduced the frequency of FoxP3(+) IL-17A(+) T cells in the colon. In the DSS-model treatment encenicline increased the frequency of FoxP3(+) T cells and reduced IL-17A(+) T cells. Stimulation of α7 nAChR with partial agonist encenicline alleviates colitis via alteration of the number and/or activation status of the immune cells in the gut, emphasizing a potential role of α7 nAChRs as a target for anticolitic drugs. PMID:26462538

  9. A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella.

    PubMed

    Wang, Jing; Wang, Xingliang; Lansdell, Stuart J; Zhang, Jianheng; Millar, Neil S; Wu, Yidong

    2016-04-01

    Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. PMID:26855198

  10. Activation of muscarinic receptors by ACh release in hippocampal CA1 depolarizes VIP but has varying effects on parvalbumin-expressing basket cells

    PubMed Central

    Bell, L Andrew; Bell, Karen A; McQuiston, A Rory

    2015-01-01

    We investigated the effect of acetylcholine release on mouse hippocampal CA1 perisomatically projecting interneurons. Acetylcholine was optogenetically released in hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated virally mediated transfection. The effect of optogenetically released acetylcholine was assessed on interneurons expressing Cre recombinase in vasoactive intestinal peptide (VIP) or parvalbumin (PV) interneurons using whole cell patch clamp methods. Acetylcholine released onto VIP interneurons that innervate pyramidal neuron perisomatic regions (basket cells, BCs) were depolarized by muscarinic receptors. Although PV BCs were also excited by muscarinic receptor activation, they more frequently responded with hyperpolarizing or biphasic responses. Muscarinic receptor activation resulting from ACh release increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in downstream hippocampal CA1 pyramidal neurons with peak instantaneous frequencies occurring in both the gamma and theta bandwidths. Both PV and VIP BCs contributed to the increased sIPSC frequency in pyramidal neurons and optogenetic suppression of PV or VIP BCs inhibited sIPSCs occurring in the gamma range. Therefore, we propose acetylcholine release in CA1 has a complex effect on CA1 pyramidal neuron output through varying effects on perisomatically projecting interneurons. PMID:25556796

  11. Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking

    PubMed Central

    Watty, Anke; Methfessel, Christoph; Hucho, Ferdinand

    1997-01-01

    Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an “unconventional allosteric protein” which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3′-dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly “freezes” the nAChR in a high agonist affinity/closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a high-affinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states. PMID:9223339

  12. Role of α5-containing nicotinic receptors in neuropathic pain and response to nicotine.

    PubMed

    Xanthos, Dimitris N; Beiersdorf, Johannes W; Thrun, Ariane; Ianosi, Bogdan; Orr-Urtreger, Avi; Huck, Sigismund; Scholze, Petra

    2015-08-01

    Nicotinic receptors in the central nervous system (nAChRs) are known to play important roles in pain processing and modulate behavioral responses to analgesic drugs, including nicotine. The presence of the α5-neuronal nicotinic accessory subunit in the nicotinic receptor complex is increasingly understood to modulate reward and aversive states, addiction, and possibly pathological pain. In the current study, using α5-knockout (KO) mice and subunit-specific antibodies, we assess the role of α5-containing neuronal nicotinic receptors in neuropathic pain and in the analgesic response to nicotine. After chronic constriction injury (CCI) or partial sciatic nerve ligation (PSNL), no differences in mechanical, heat, or cold hyperalgesia were found in wild-type (WT) versus α5-KO littermate mice. The number of α5-containing nAChRs was decreased (rather than increased) after CCI in the spinal cord and in the thalamus. Nevertheless, thermal analgesic response to nicotine was marginally reduced in CCI α5-KO mice at 4 days after CCI, but not at later timepoints or after PSNL. Interestingly, upon daily intermittent nicotine injections in unoperated mice, WT animals developed tolerance to nicotine-induced analgesia to a larger extent than α5-KO mice. Our results suggest that α5-containing nAChRs mediate analgesic tolerance to nicotine but do not play a major role in neuropathic pain. PMID:25725336

  13. Neuronal nicotinic acetylcholine receptors: neuroplastic changes underlying alcohol and nicotine addictions

    PubMed Central

    Feduccia, Allison A.; Chatterjee, Susmita; Bartlett, Selena E.

    2012-01-01

    Addictive drugs can activate systems involved in normal reward-related learning, creating long-lasting memories of the drug's reinforcing effects and the environmental cues surrounding the experience. These memories significantly contribute to the maintenance of compulsive drug use as well as cue-induced relapse which can occur even after long periods of abstinence. Synaptic plasticity is thought to be a prominent molecular mechanism underlying drug-induced learning and memories. Ethanol and nicotine are both widely abused drugs that share a common molecular target in the brain, the neuronal nicotinic acetylcholine receptors (nAChRs). The nAChRs are ligand-gated ion channels that are vastly distributed throughout the brain and play a key role in synaptic neurotransmission. In this review, we will delineate the role of nAChRs in the development of ethanol and nicotine addiction. We will characterize both ethanol and nicotine's effects on nAChR-mediated synaptic transmission and plasticity in several key brain areas that are important for addiction. Finally, we will discuss some of the behavioral outcomes of drug-induced synaptic plasticity in animal models. An understanding of the molecular and cellular changes that occur following administration of ethanol and nicotine will lead to better therapeutic strategies. PMID:22876217

  14. Delayed procedural learning in α7-nicotinic acetylcholine receptor knockout mice

    PubMed Central

    Young, J. W.; Meves, J. M.; Tarantino, I. S.; Caldwell, S.; Geyer, M. A.

    2014-01-01

    The α7-nicotinic acetylcholine receptor (nAChR) has long been a procognitive therapeutic target to treat schizophrenia. Evidence on the role of this receptor in cognition has been lacking, however, in part due to the limited availability of suitable ligands. The behavior of α7-nAChR knockout (KO) mice has been examined previously, but cognitive assessments using tests with cross-species translatability have been limited to date. Here, we assessed the cognitive performance of α7-nAChR KO and wild-type (WT) littermate mice in the attentional set-shifting task of executive functioning, the radial arm maze test of spatial working memory span capacity and the novel object recognition test of short-term memory. The reward motivation of these mutants was assessed using the progressive ratio breakpoint test. In addition, we assessed the exploratory behavior and sensorimotor gating using the behavioral pattern monitor and prepulse inhibition, respectively. α7-nAChR KO mice exhibited normal set-shifting, but impaired procedural learning (rule acquisition) in multiple paradigms. Spatial span capacity, short-term memory, motivation for food, exploration and sensorimotor gating were all comparable to WT littermates. The data presented here support the notion that this receptor is important for such procedural learning, when patterns in the environment become clear and a rule is learned. In combination with the impaired attention observed previously in these mice, this finding suggests that agonist treatments should be examined in clinical studies of attention and procedural learning, perhaps in combination with cognitive behavioral therapy. PMID:21679297

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

    SciTech Connect

    Garcia-Rates, Sara; Camarasa, Jordi

    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 inhibited 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 the

  16. Structural and Functional Studies of the Modulator NS9283 Reveal Agonist-like Mechanism of Action at α4β2 Nicotinic Acetylcholine Receptors*

    PubMed Central

    Olsen, Jeppe A.; Ahring, Philip K.; Kastrup, Jette S.; Gajhede, Michael; Balle, Thomas

    2014-01-01

    Modulation of Cys loop receptor ion channels is a proven drug discovery strategy, but many underlying mechanisms of the mode of action are poorly understood. We report the x-ray structure of the acetylcholine-binding protein from Lymnaea stagnalis with NS9283, a stoichiometry selective positive modulator that targets the α4-α4 interface of α4β2 nicotinic acetylcholine receptors (nAChRs). Together with homology modeling, mutational data, quantum mechanical calculations, and pharmacological studies on α4β2 nAChRs, the structure reveals a modulator binding mode that overlaps the α4-α4 interface agonist (acetylcholine)-binding site. Analysis of contacts to residues known to govern agonist binding and function suggests that modulation occurs by an agonist-like mechanism. Selectivity for α4-α4 over α4-β2 interfaces is determined mainly by steric restrictions from Val-136 on the β2-subunit and favorable interactions between NS9283 and His-142 at the complementary side of α4. In the concentration ranges where modulation is observed, its selectivity prevents NS9283 from directly activating nAChRs because activation requires coordinated action from more than one interface. However, we demonstrate that in a mutant receptor with one natural and two engineered α4-α4 interfaces, NS9283 is an agonist. Modulation via extracellular binding sites is well known for benzodiazepines acting at γ-aminobutyric acid type A receptors. Like NS9283, benzodiazepines increase the apparent agonist potency with a minimal effect on efficacy. The shared modulatory profile along with a binding site located in an extracellular subunit interface suggest that modulation via an agonist-like mechanism may be a common mechanism of action that potentially could apply to Cys loop receptors beyond the α4β2 nAChRs. PMID:24982426

  17. Loop 2 of Ophiophagus hannah toxin b binds with neuronal nicotinic acetylcholine receptors and enhances intracranial drug delivery.

    PubMed

    Zhan, Changyou; Yan, Zhiqiang; Xie, Cao; Lu, Weiyue

    2010-12-01

    Three-finger snake neurotoxins have been widely investigated for their high binding affinities with nicotinic acetylcholine receptors (nAChRs), which are widely expressed in the central nervous system including the blood-brain barrier and thus mediate intracranial drug delivery. The loop 2 segments of three-finger snake neurotoxins are considered as the binding domain with nAChRs, and thus, they may have the potential to enhance drug or drug delivery system intracranial transport. In the present work, binding of the synthetic peptides to the neuronal nAChRs was assessed by measuring their ability to inhibit the binding of (125)I-α-bungarotoxin to the receptor. The loop 2 segment of Ophiophagus hannah toxin b (KC2S) showed high binding affinity, and the competitive binding IC(50) value was 32.51 nM. Furthermore, the brain targeting efficiency of KC2S had been investigated in vitro and in vivo. The specific uptake by brain capillary endothelial cells (BCECs) demonstrated that KC2S could be endocytosized after binding with nAChRs. In vivo, the qualitative and quantitative biodistribution results of fluorescent dyes (DiR or coumarin-6) indicated that KC2S modified poly(ethylene glycol)-poly(lactic acid) micelles (KC2S-PEG-PLA micelles) could enhance intracranial drug delivery. Furthermore, intravenous treatment with paclitaxel-encapsulated KC2S-PEG-PLA micelles (KC2S-PEG-PLA-PTX micelles) afforded robust inhibition of intracranial glioblastoma. The median survival time of KC2S-PEG-PLA-PTX-micelle-treated mice (47.5 days) was significantly longer than that of mice treated by mPEG-PLA-PTX micelles (41.5 days), Taxol (38.5 days), or saline (34 days). Compared with the short peptide derived from rabies virus glycoprotein (RVG29) that has been previously reported as an excellent brain targeting ligand, KC2S has a similar binding affinity with neuronal nAChRs but fewer amino acid residues. Thus, we concluded that the loop 2 segment of Ophiophagus hannah toxin b could bind

  18. 6,6-Spiroimine analogs of (-)-gymnodimine A: synthesis and biological evaluation on nicotinic acetylcholine receptors.

    PubMed

    Duroure, Leslie; Jousseaume, Thierry; Aráoz, Rómulo; Barré, Elvina; Retailleau, Pascal; Chabaud, Laurent; Molgó, Jordi; Guillou, Catherine

    2011-12-01

    Simple models of the spiroimine core of (-)-gymnodimine A have been synthesized in racemic and optically active forms. The quaternary carbon of the racemic spiroimines was created by Michael addition of a β-ketoester to acrolein, whereas the asymmetric allylic alkylation of the same β-ketoester was used to access the spiroimines in an enantioselective fashion. Both racemic and enantio-enriched mixtures were tested for their biological activities on Xenopus oocytes either expressing (human α4β2) or having incorporated (Torpedoα1(2)βγδ) nicotinic acetylcholine receptors (nAChRs). These spiroimine analogs of (-)-gymnodimine A inhibited acetylcholine-evoked nicotinic currents, but were less active than the phycotoxin. Our results reveal that the 6,6-spiroimine moiety is important for the blockade of nAChRs and support the hypothesis that it is one of the pharmacophores of this group of toxins. PMID:22024965

  19. α-Conotoxins Identify the α3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells.

    PubMed

    Hone, Arik J; McIntosh, J Michael; Azam, Layla; Lindstrom, Jon; Lucero, Linda; Whiteaker, Paul; Passas, Juan; Blázquez, Jesús; Albillos, Almudena

    2015-11-01

    Ligands that selectively inhibit human α3β2 and α6β2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3β4 and α6β4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3β2 than α3β4 and 165-fold more potent on human α6/α3β2β3 than α6/α3β4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with β2 ligand-binding sites. In contrast, the β4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained β4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3β4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, β2, and β4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits. PMID:26330550

  20. Identification of domains influencing assembly and ion channel properties in α7 nicotinic receptor and 5-HT3 receptor subunit chimaeras

    PubMed Central

    Gee, V J; Kracun, S; Cooper, S T; Gibb, A J; Millar, N S

    2007-01-01

    Background and purpose: Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR α7 subunit and the 5-HT3 receptor 3A subunit. Experimental approach: A series of subunit chimaeras containing α7 and 5-HT3A subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology. Key results: Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on α7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some α7/5-HT3A chimaeras. Conclusions and implications: Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties. PMID:17721553

  1. Recent Advances in Nicotinic Receptor Signaling in Alcohol Abuse and Alcoholism.

    PubMed

    Rahman, Shafiqur; Engleman, Eric A; Bell, Richard L

    2016-01-01

    Alcohol is the most commonly abused legal substance and alcoholism is a serious public health problem. It is a leading cause of preventable death in the world. The cellular and molecular mechanisms of alcohol reward and addiction are still not well understood. Emerging evidence indicates that unlike other drugs of abuse, such as nicotine, cocaine, or opioids, alcohol targets numerous channel proteins, receptor molecules, and signaling pathways in the brain. Previously, research has identified brain nicotinic acetylcholine receptors (nAChRs), a heterogeneous family of pentameric ligand-gated cation channels expressed in the mammalian brain, as critical molecular targets for alcohol abuse and dependence. Genetic variations encoding nAChR subunits have been shown to increase the vulnerability to develop alcohol dependence. Here, we review recent insights into the rewarding effects of alcohol, as they pertain to different nAChR subtypes, associated signaling molecules, and pathways that contribute to the molecular mechanisms of alcoholism and/or comorbid brain disorders. Understanding these cellular changes and molecular underpinnings may be useful for the advancement of brain nicotinic-cholinergic mechanisms, and will lead to a better translational and therapeutic outcome for alcoholism and/or comorbid conditions. PMID:26810002

  2. Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

    PubMed

    Zhang, Yixi; Wang, Xin; Yang, Baojun; Hu, Yuanyuan; Huang, Lixin; Bass, Chris; Liu, Zewen

    2015-11-01

    Target-site resistance is commonly caused by qualitative changes in insecticide target-receptors and few studies have implicated quantitative changes in insecticide targets in resistance. Here we show that resistance to imidacloprid in a selected strain of Nilaparvata lugens is associated with a reduction in expression levels of the nicotinic acetylcholine receptor (nAChR) subunit Nlα8. Synergism bioassays of the selected strain suggested resistance was conferred, in part, by a target-site mechanism. Sequencing of N. lugens nAChR subunit genes identified no mutations associated with resistance, however, a decrease in mRNA and protein levels of Nlα8 was observed during selection. RNA interference knockdown of Nlα8 decreased the sensitivity of N. lugens to imidacloprid, demonstrating that a decrease in Nlα8 expression is sufficient to confer resistance in vivo. Radioligand binding assays revealed that the affinity of the high-affinity imidacloprid-binding site of native nAChRs was reduced by selection, and reducing the amount of Nlα8 cRNA injected into Xenopus oocytes significantly decreased imidacloprid potency on recombinant receptors. Taken together, these results provide strong evidence that a decrease in Nlα8 levels confers resistance to imidacloprid in N. lugens, and thus provides a rare example of target-site resistance associated with a quantitative rather than qualitative change. In insects, target-site mutations often cause high resistance to insecticides, such as neonicotinoids acting on nicotinic acetylcholine receptors (nAChRs). Here we found that a quantitative change in target-protein level, decrease in mRNA and protein levels of Nlα8, contributed importantly to imidacloprid resistance in Nilaparvata lugens. This finding provides a new target-site mechanism of insecticide resistance. PMID:26259922

  3. The role of nicotinic acetylcholine receptors in autosomal dominant nocturnal frontal lobe epilepsy

    PubMed Central

    Becchetti, Andrea; Aracri, Patrizia; Meneghini, Simone; Brusco, Simone; Amadeo, Alida

    2015-01-01

    Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal epilepsy with attacks typically arising in the frontal lobe during non-rapid eye movement (NREM) sleep. It is characterized by clusters of complex and stereotyped hypermotor seizures, frequently accompanied by sudden arousals. Cognitive and psychiatric symptoms may be also observed. Approximately 12% of the ADNFLE families carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs). This is consistent with the widespread expression of these receptors, particularly the α4β2* subtype, in the neocortex and thalamus. However, understanding how mutant nAChRs lead to partial frontal epilepsy is far from being straightforward because of the complexity of the cholinergic regulation in both developing and mature brains. The relation with the sleep-waking cycle must be also explained. We discuss some possible pathogenetic mechanisms in the light of recent advances about the nAChR role in prefrontal regions as well as the studies carried out in murine models of ADNFLE. Functional evidence points to alterations in prefrontal GABA release, and the synaptic unbalance probably arises during the cortical circuit maturation. Although most of the available functional evidence concerns mutations on nAChR subunit genes, other genes have been recently implicated in the disease, such as KCNT1 (coding for a Na+-dependent K+ channel), DEPD5 (Disheveled, Egl-10 and Pleckstrin Domain-containing protein 5), and CRH (Corticotropin-Releasing Hormone). Overall, the uncertainties about both the etiology and the pathogenesis of ADNFLE point to the current gaps in our knowledge the regulation of neuronal networks in the cerebral cortex. PMID:25717303

  4. Nicotine improves the functional activity of late endothelial progenitor cells via nicotinic acetylcholine receptors.

    PubMed

    Yu, Min; Liu, Qian; Sun, Jing; Yi, Kaihong; Wu, Libiao; Tan, Xuerui

    2011-08-01

    The aim of this study is to investigate whether nicotinic acetylcholine receptors (nAChRs) are involved in the modulation of functional activity of late endothelial progenitor cells (EPCs) induced by nicotine. Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-acLDL) uptake and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1) binding. Expression of von Willbrand factor (vWF), kinase insert domain receptor (KDR), and α7 nAChR was detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 32 h with either vehicle or nicotine with or without pre-incubation of nAChR antagonism, mecamylamine, or α-bungarotoxin. The viability, migration, and in vitro vasculogenesis activity of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with 10 nmol/L nicotine enhanced viable, migratory, adhesive, and in vitro vasculogenesis capacity of late EPCs. The effect of nicotine on late EPCs can be attenuated by mecamylamine or α-bungarotoxin. In conclusion, nicotine improves the functional activity of late EPCs via nAChRs. PMID:21774635

  5. Nicotinic receptors in non-human primates: analysis of genetic and functional conservation with humans

    PubMed Central

    Shorey-Kendrick, Lyndsey E.; Ford, Matthew M.; Allen, Daicia C.; Kuryatov, Alexander; Lindstrom, Jon; Wilhelm, Larry; Grant, Kathleen A.; Spindel, Eliot R.

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) are highly conserved between humans and non-human primates. Conservation exists at the level of genomic structure, protein structure and epigenetics. Overall homology of nAChRs at the protein level is 98% in macaques versus 89% in mice, which is highly relevant for evaluating subtype-specific ligands that have different affinities in humans versus rodents. In addition to conservation at the protein level, there is high conservation of genomic structure in terms of intron and exon size and placement of CpG sites that play a key role in epigenetic regulation. Analysis of single nucleotide polymorphisms (SNPs) shows that while the majority of SNPs are not conserved between humans and macaques, some functional polymorphisms are. Most significantly, cynomolgus monkeys express a similar α5 nAChR Asp398Asn polymorphism to the human α5 Asp398Asn polymorphism that has been linked to greater nicotine addiction and smoking related disease. Monkeys can be trained to readily self-administer nicotine, and in an initial study we have demonstrated that cynomolgus monkeys bearing the α5 D398N polymorphism show a reduced behavioral sensitivity to oral nicotine and tend to consume it in a different pattern when compared to wild-type monkeys. Thus the combination of highly homologous nAChR, higher cortical functions and capacity for complex training makes non-human primates a unique model to study in vivo functions of nicotinic receptors. In particular, primate studies on nicotine addiction and evaluation of therapies to prevent or overcome nicotine addiction are likely to be highly predictive of treatment outcomes in humans. PMID:25661700

  6. Role of L- and N-type Ca2+ channels in muscarinic receptor-mediated facilitation of ACh and noradrenaline release in the rat urinary bladder.

    PubMed Central

    Somogyi, G T; Zernova, G V; Tanowitz, M; de Groat, W C

    1997-01-01

    1. 3H-Noradrenaline (NA) and 14C-acetylcholine (ACh) released by electrical field stimulation were measured simultaneously in strips from the body of rat urinary bladder. 2. omega-Conotoxin GVIA (omega-CgTX; 20-100 nM) suppressed the non-facilitated transmitter release evoked by intermittent stimulation (IS), whereas nifedipine (1 microM) did not affect release. 3. Continuous electrical stimulation (CS) facilitated NA and ACh release via an atropine-sensitive mechanism. omega-CgTX reduced the facilitated release of NA (44% depression) but did not affect ACh release. Nifedipine depressed ACh release (43%) but not NA release. Combined administration of nifedipine and omega-CgTX (20 nM) produced a greater suppression of NA and ACh release (86 and 91%, respectively). 4. Maximal muscarinic facilitation of NA (5-fold) and ACh (17-fold) release occurred following administration of eserine, an anticholinesterase agent. Release of both NA and ACh was depressed by nifedipine (70 and 83%, respectively) but not by omega-CgTX. Combined application of omega-CgTX and nifedipine elicited a further depression of NA (95%) but not ACh release. 5. When NA and ACh release was facilitated with phorbol dibutyrate (0.5 microM), nifedipine inhibited ACh (67%) but not NA release, whereas omega-CgTX inhibited NA (73%) but not ACh release. Combined administration of both Ca2+ channel blockers did not elicit greater inhibition. 6. Bay K 8644, the L-type Ca2+ channel activator, increased ACh release in a dose-dependent manner (up to 5-fold) but did not significantly change NA release. 7. Both omega-CgTX (20-100 nM) and nifedipine (100 nM-1 microM) significantly decreased (50-80%) the neurally evoked contractions of the bladder strips. 8. It is concluded that L-type Ca2+ channels play a major role in muscarinic facilitation of NA and ACh release in the urinary bladder but are not essential for non-facilitated release. Other types of Ca2+ channels, including N-type, are involved to varying

  7. A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression.

    PubMed

    Watson, Gerald B; Chouinard, Scott W; Cook, Kevin R; Geng, Chaoxian; Gifford, Jim M; Gustafson, Gary D; Hasler, James M; Larrinua, Ignacio M; Letherer, Ted J; Mitchell, Jon C; Pak, William L; Salgado, Vincent L; Sparks, Thomas C; Stilwell, Geoff E

    2010-05-01

    Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dalpha6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dalpha6, providing convincing evidence that Dalpha6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dalpha6 alone, co-expression of Dalpha6 with an additional nAChR subunit, Dalpha5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR. PMID:19944756

  8. Effects of short- and long-term rat hind limb immobilization on spinal cord insulin-like growth factor-I and its receptor.

    PubMed

    Suliman, I A; Lindgren, J U; Elhassan, A M; Diab, K M; Adem, A

    2001-08-31

    In this study we investigated changes in the spinal cord insulin-like growth factor-I peptide (IGF-I) and its receptors (IGF-IR) after hind limb immobilization for 5 days, 2, 4, and 8 weeks. Moreover, effects on IGF-I and nicotinic cholinergic receptors (nAChRs) in two types of skeletal muscle were also investigated. IGF-I levels were measured by radioimmunoassay (RIA) whereas IGF-IR and nAChRs were measured by quantitative receptor autoradiography. Spinal cord IGF-I levels decreased significantly after 5 days, 2 and 4 weeks of immobilization, whereas IGF-IR increased significantly after 4 and 8 weeks compared to controls. In skeletal muscles, nAChRs increased significantly after 5 days and 2 weeks in the soleus (SOL) and tibialis anterior (TIB) muscles, respectively, and continued up to 8 weeks in both muscles. IGF-I concentration decrease significantly after 4 and 8 weeks in the SOL and TIB muscles, respectively. Despite the normal levels of IGF-I in both muscles at the early time points (5 days and 2 weeks), low levels of IGF-I were observed concurrently in the spinal cord ipsilateral to the immobilized limb. Our findings suggest that the early decrease in the IGF-I level and the late upregulation in the IGF-IR in the spinal cord might represent a nervous system response to disuse. PMID:11520489

  9. The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors.

    PubMed

    Wieskopf, Jeffrey S; Mathur, Jayanti; Limapichat, Walrati; Post, Michael R; Al-Qazzaz, Mona; Sorge, Robert E; Martin, Loren J; Zaykin, Dmitri V; Smith, Shad B; Freitas, Kelen; Austin, Jean-Sebastien; Dai, Feng; Zhang, Jie; Marcovitz, Jaclyn; Tuttle, Alexander H; Slepian, Peter M; Clarke, Sarah; Drenan, Ryan M; Janes, Jeff; Al Sharari, Shakir; Segall, Samantha K; Aasvang, Eske K; Lai, Weike; Bittner, Reinhard; Richards, Christopher I; Slade, Gary D; Kehlet, Henrik; Walker, John; Maskos, Uwe; Changeux, Jean-Pierre; Devor, Marshall; Maixner, William; Diatchenko, Luda; Belfer, Inna; Dougherty, Dennis A; Su, Andrew I; Lummis, Sarah C R; Imad Damaj, M; Lester, Henry A; Patapoutian, Ardem; Mogil, Jeffrey S

    2015-05-13

    Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain. PMID:25972004

  10. The Nicotinic α6 Subunit Gene Determines Variability in Chronic Pain Sensitivity via Cross-inhibition of P2X2/3 Receptors

    PubMed Central

    Wieskopf, Jeffrey S.; Mathur, Jayanti; Limapichat, Walrati; Post, Michael R.; Al-Qazzaz, Mona; Sorge, Robert E.; Martin, Loren J.; Zaykin, Dmitri V.; Smith, Shad B.; Freitas, Kelen; Austin, Jean-Sebastien; Dai, Feng; Zhang, Jie; Marcovitz, Jaclyn; Tuttle, Alexander H.; Slepian, Peter M.; Clarke, Sarah; Drenan, Ryan M.; Janes, Jeff; Sharari, Shakir Al; Segall, Samantha K.; Aasvang, Eske K.; Lai, Weike; Bittner, Reinhard; Richards, Christopher I.; Slade, Gary D.; Kehlet, Henrik; Walker, John; Maskos, Uwe; Changeux, Jean-Pierre; Devor, Marshall; Maixner, William; Diatchenko, Luda; Belfer, Inna; Dougherty, Dennis A.; Su, Andrew I.; Lummis, Sarah C.R.; Damaj, M. Imad; Lester, Henry A.; Patapoutian, Ardem; Mogil, Jeffrey S.

    2016-01-01

    Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (i.e., α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6’s role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical pain and temporomandibular pain. PMID:25972004

  11. Distinctive effects of nicotinic receptor intracellular-loop mutations associated with nocturnal frontal lobe epilepsy.

    PubMed

    Weltzin, Maegan M; Lindstrom, Jon M; Lukas, Ronald J; Whiteaker, Paul

    2016-03-01

    Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in α2, α4 and β2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for β2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, α4(R336H) and β2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on α4β2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations' effects at alternate isoforms: high-sensitivity [HS; (α4)2(β2)3] or low-sensitivity [LS; (α4)3(β2)2] via 1:10 or 30:1 [α4:β2] cRNA injection ratios, respectively. An unbiased (1:1 [α4:β2] cRNA) injection ratio was also used to study potential shifts in isoform expression. α4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). β2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. α4β2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using β2-subunit-specific [(125)I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the β2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between

  12. Insights into docking and scoring neuronal alpha4beta2 nicotinic receptor agonists using molecular dynamics simulations and QM/MM calculations.

    PubMed

    Sgrignani, Jacopo; Bonaccini, Claudia; Grazioso, Giovanni; Chioccioli, Matteo; Cavalli, Andrea; Gratteri, Paola

    2009-11-30

    A combined quantum mechanical (QM)-polarized docking and molecular dynamics approach to study the binding mode and to predict the binding affinity of ligands acting at the alpha4beta2-nAChR is presented. The results obtained in this study indicate that the quantum mechanical/molecular mechanics docking protocol well describes the charge-driven interactions occurring in the binding of nicotinic agonists, and it is able to represent the polarization effects on the ligand exerted by the surrounding atoms of the receptor at the binding site. This makes it possible to properly score agonists of alpha4beta2-nAChR and to reproduce the experimental binding affinity data with good accuracy, within a mean error of 2.2 kcal/mol. Moreover, applying the QM-polarized docking to an ensemble of nAChR conformations obtained from MD simulations enabled us to accurately capture nAChR-ligand induced-fit effects. PMID:19360794

  13. Role of α4- and α6-containing nicotinic receptors in the acquisition and maintenance of nicotine self-administration.

    PubMed

    Madsen, Heather B; Koghar, Harcharan S; Pooters, Tine; Massalas, Jim S; Drago, John; Lawrence, Andrew J

    2015-05-01

    Tobacco smoking is a major cause of death and disease and as such there is a critical need for the development of new therapeutic approaches to treat nicotine addiction. Here, we utilize genetic and pharmacological tools to further investigate the nicotinic acetylcholine receptor (nAChR) subtypes that support intravenous self-administration of nicotine. α4-S248F mice contain a point mutation within the α4 nAChR subunit which confers increased sensitivity to nicotine and resistance to mecamylamine. Here, we show that acute administration of mecamylamine (2 mg/kg, i.p.) reduces established nicotine self-administration (0.05 mg/kg/infusion) in wild-type (WT), but not in α4-S248F heterozygous mice, demonstrating a role for α4* nAChRs in the modulation of ongoing nicotine self-administration. Administration of N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), a selective α6β2* nAChR antagonist, dose dependently (5 and 10 mg/kg, i.p.) impairs the acquisition of nicotine self-administration and reduces established nicotine self-administration in WT mice when administered acutely (10 mg/kg, i.p.). This was not due to a general reduction in locomotor activity and the same dose of bPiDI did not affect operant responding for sucrose. bPiDI treatment (10 mg/kg, i.p.) also impaired both the acquisition and maintenance of nicotine self-administration in α4-S248F heterozygous mice. This provides further evidence for the involvement of α6β2* nAChRs in the reinforcing effects of nicotine that underlies its ability to support ongoing self-administration. Taken together, selective targeting of α6β2* or α4α6β2* nAChRs may prove to be an effective strategy for the development of smoking cessation therapies. PMID:24750355

  14. Single-channel properties of α3β4, α3β4α5 and α3β4β2 nicotinic acetylcholine receptors in mice lacking specific nicotinic acetylcholine receptor subunits

    PubMed Central

    Ciuraszkiewicz, Anna; Schreibmayer, Wolfgang; Platzer, Dieter; Orr-Urtreger, Avi; Scholze, Petra; Huck, Sigismund

    2013-01-01

    Previous attempts to measure the functional properties of recombinant nicotinic acetylcholine receptors (nAChRs) composed of known receptor subunits have yielded conflicting results. The use of knockout mice that lack α5, β2, α5β2 or α5β2α7 nAChR subunits enabled us to measure the single-channel properties of distinct α3β4, α3β4α5 and α3β4β2 receptors in superior cervical ganglion (SCG) neurons. Using this approach, we found that α3β4 receptors had a principal conductance level of 32.6 ± 0.8 pS (mean ± SEM) and both higher and lower secondary conductance levels. α3β4α5 receptors had the same conductance as α3β4 receptors, but differed from α3β4 receptors by having an increased channel open time and increased burst duration. By contrast, α3β4β2 receptors differed from α3β4 and α3β4α5 receptors by having a significantly smaller conductance level (13.6 ± 0.5 pS). After dissecting the single-channel properties of these receptors using our knockout models, we then identified these properties – and hence the receptors themselves – in wild-type SCG neurons. This study is the first to identify the single-channel properties of distinct neuronal nicotinic receptors in their native environment. PMID:23613527

  15. Mood and anxiety regulation by nicotinic acetylcholine receptors: A potential pathway to modulate aggression and related behavioral states.

    PubMed

    Picciotto, Marina R; Lewis, Alan S; van Schalkwyk, Gerrit I; Mineur, Yann S

    2015-09-01

    The co-morbidity between smoking and mood disorders is striking. Preclinical and clinical studies of nicotinic effects on mood, anxiety, aggression, and related behaviors, such as irritability and agitation, suggest that smokers may use the nicotine in tobacco products as an attempt to self-medicate symptoms of affective disorders. The role of nicotinic acetylcholine receptors (nAChRs) in circuits regulating mood and anxiety is beginning to be elucidated in animal models, but the mechanisms underlying the effects of nicotine on aggression-related behavioral states (ARBS) are still not understood. Clinical trials of nicotine or nicotinic medications for neurological and psychiatric disorders have often found effects of nicotinic medications on ARBS, but few trials have studied these outcomes systematically. Similarly, the increase in ARBS resulting from smoking cessation can be resolved by nicotinic agents, but the effects of nicotinic medications on these types of mental states and behaviors in non-smokers are less well understood. Here we review the literature on the role of nAChRs in regulating mood and anxiety, and subsequently on the closely related construct of ARBS. We suggest avenues for future study to identify how nAChRs and nicotinic agents may play a role in these clinically important areas. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25582289

  16. α6β2* and α4β2* Nicotinic Acetylcholine Receptors As Drug Targets for Parkinson's Disease

    PubMed Central

    Wonnacott, Susan

    2011-01-01

    Parkinson's disease is a debilitating movement disorder characterized by a generalized dysfunction of the nervous system, with a particularly prominent decline in the nigrostriatal dopaminergic pathway. Although there is currently no cure, drugs targeting the dopaminergic system provide major symptomatic relief. As well, agents directed to other neurotransmitter systems are of therapeutic benefit. Such drugs may act by directly improving functional deficits in these other systems, or they may restore aberrant motor activity that arises as a result of a dopaminergic imbalance. Recent research attention has focused on a role for drugs targeting the nicotinic cholinergic systems. The rationale for such work stems from basic research findings that there is an extensive overlap in the organization and function of the nicotinic cholinergic and dopaminergic systems in the basal ganglia. In addition, nicotinic acetylcholine receptor (nAChR) drugs could have clinical potential for Parkinson's disease. Evidence for this proposition stems from studies with experimental animal models showing that nicotine protects against neurotoxin-induced nigrostriatal damage and improves motor complications associated with l-DOPA, the “gold standard” for Parkinson's disease treatment. Nicotine interacts with multiple central nervous system receptors to generate therapeutic responses but also produces side effects. It is important therefore to identify the nAChR subtypes most beneficial for treating Parkinson's disease. Here we review nAChRs with particular emphasis on the subtypes that contribute to basal ganglia function. Accumulating evidence suggests that drugs targeting α6β2* and α4β2* nAChR may prove useful in the management of Parkinson's disease. PMID:21969327

  17. Conservation of neural nicotinic acetylcholine receptors from Drosophila to vertebrate central nervous systems.

    PubMed Central

    Bossy, B; Ballivet, M; Spierer, P

    1988-01-01

    Nicotinic acetylcholine receptors (nAChR) are found both in vertebrate and insect central nervous systems. We have isolated a Drosophila gene by crosshybridization with a vertebrate probe. Structural conservation of domains of the deduced protein and of intron/exon boundaries indicate that the Drosophila gene encodes an nAChR alpha-like subunit (ALS). That the Drosophila gene product most resembles the neuronal set of vertebrate nAChRs alpha-subunits is also indicated by the failure of an ALS-beta-galactosidase fusion protein to bind alpha-bungarotoxin on blots in contrast to vertebrate endplate alpha-subunit constructions. The ALS encoding gene exceeds 54 kb in length and the transcript has a very long and unusual 5' leader. As we found previously for a gene whose product is also involved in cholinergic synapses, acetylcholinesterase, the leader encodes short open reading frames, which might be involved in translation control. We also note the presence of opa repeats in the gene, as has been found for various Drosophila genes expressed in the nervous system. Images PMID:2840281

  18. Alterations of cortical pyramidal neurons in mice lacking high-affinity nicotinic receptors

    PubMed Central

    Ballesteros-Yáñez, Inmaculada; Benavides-Piccione, Ruth; Bourgeois, Jean-Pierre; Changeux, Jean-Pierre; DeFelipe, Javier

    2010-01-01

    The neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric membrane proteins involved in multiple cognitive processes, including attention, learning, and memory. The most abundant form of heterooligomeric nAChRs in the brain contains the β2- and α4- subunits and binds nicotinic agonists with high affinity. In the present study, we investigated in the mouse the consequences of the deletion of one of the nAChR components: the β2-subunit (β2−/−) on the microanatomy of cortical pyramidal cells. Using an intracellular injection method, complete basal dendritic arbors of 650 layer III pyramidal neurons were sampled from seven cortical fields, including primary sensory, motor, and associational areas, in both β2−/− and WT animals. We observed that the pyramidal cell phenotype shows significant quantitative differences among different cortical areas in mutant and WT mice. In WT mice, the density of dendritic spines was rather similar in all cortical fields, except in the prelimbic/infralimbic cortex, where it was significantly higher. In the absence of the β2-subunit, the most significant reduction in the density of spines took place in this high-order associational field. Our data suggest that the β2-subunit is involved in the dendritic morphogenesis of pyramidal neurons and, in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex. PMID:20534523

  19. Insight into the Binding Mode of Agonists of the Nicotinic Acetylcholine Receptor from Calculated Electron Densities

    PubMed Central

    Beck, Michael E; Gutbrod, Oliver; Matthiesen, Svend

    2015-01-01

    Insect nicotinic acetylcholine receptors (nAChRs) are among the most prominent and most economically important insecticide targets. Thus, an understanding of the modes of binding of respective agonists is important for the design of specific compounds with favorable vertebrate profiles. In the case of nAChRs, the lack of available high-resolution X-ray structures leaves theoretical considerations as the only viable option. Starting from classical homology and docking approaches, binding mode hypotheses are created for five agonists of the nAChR, covering insecticides in the main group 4 of the Insecticide Resistance Action Committee (IRAC) mode of action (MoA) classification, namely, neonicotinoids, nicotine, sulfoxaflor, and butenolides. To better understand these binding modes, the topologies of calculated electron densities of small-model systems are analyzed in the framework of the quantum theory of atoms in molecules. The theoretically obtained modes of binding are very much in line with the biology-driven IRAC MoA classification of the investigated ligands. PMID:26175091

  20. Alpha-7 and alpha-4 nicotinic receptor subunit immunoreactivity in genioglossus muscle motoneurons.

    PubMed

    Dehkordi, Ozra; Millis, Richard M; Dennis, Gary C; Coleman, Bernell R; Johnson, Sheree M; Changizi, Loubat; Ovid Trouth, C

    2005-02-15

    In the present study, immunohistochemistry combined with retrograde labeling techniques were used to determine if hypoglossal motoneurons (HMNs), retrogradely labeled after cholera toxin B subunit (CTB) injection to the genioglossus muscle in rats, show immunoreactivity for alpha-7 and alpha-4 subunits of nicotinic acetylcholine receptors (nAChRs). CTB-positive HMNs projecting to the genioglossus muscle were consistently labeled throughout the rostrocaudal extent of the hypoglossal nuclei with the greatest labeling at and caudal to area postrema. Alpha-7 subunit immunoreactivity was found in 39.44+/-5.10% of 870 CTB-labeled motoneurons and the alpha-4 subunit in 51.01+/-3.71% of 983 CTB-positive neurons. Rostrally, the number of genioglossal motoneurons demonstrating immunoreactivity for the alpha-7 subunit was 45.85+/-10.04% compared to 34.96+/-5.11% at and caudal to area postrema (P>0.1). The number of genioglossal motoneurons that showed immunoreactivity for the alpha-4 subunit was 55.03+/-4.83% at and caudal to area postrema compared to 42.98+/-3.90% in rostral areas (P=0.074). These results demonstrate that nAChR immunoreactivity is present in genioglossal motoneurons and suggest a role for alpha-7 and alpha-4 subunits containing nAChRs in the regulation of upper airway patency. PMID:15705531

  1. Molecular cloning and characterization of two nicotinic acetylcholine receptor β subunit genes from Apis cerana cerana.

    PubMed

    Yu, Xiaoli; Wang, Mian; Kang, Mingjiang; Liu, Li; Guo, Xingqi; Xu, Baohua

    2011-08-01

    Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect nervous system and are important targets for insecticides. In this study, we identified and characterized two novel β subunit genes (Accβ1 and Accβ2) from Apis cerana cerana. Homology analysis indicated that Accβ1 and Accβ2 possess characteristics that are typical of nAChR subunits although Accβ2 was distinct from Accβ1 and the other nAChR subunits, due to its unusual transmembrane structure and uncommon exon-intron boundary within the genomic region encoding the TM1 transmembrane domain. Analysis of the 5' flanking regions indicated that Accβ1 and Accβ2 possess different regulatory elements, suggesting that the genes might exhibit various expression and regulatory patterns. RT-PCR analysis demonstrated that Accβ2 was expressed at a much higher level than Accβ1 in the tissues of adult bees. During development, Accβ1 was highly expressed at the pupal stages, whereas Accβ2 was abundantly expressed at the larval stages. Furthermore, Accβ1 and Accβ2 were both induced by exposure to various insecticides and environmental stresses although Accβ2 was more responsive than Accβ1. These results indicate that Accβ1 and Accβ2 may have distinct roles in insect growth and development and that they may belong to separate regulatory pathways involved in the response to insecticides and environmental stresses. This report is the first description of the differences between the nAChR β subunit genes in the Chinese honey bee and establishes an initial foundation for further study. PMID:21618599

  2. Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival

    PubMed Central

    St-Pierre, Stéphanie; Jiang, Wei; Roy, Patrick; Champigny, Camille; LeBlanc, Éric; Morley, Barbara J.; Hao, Junwei; Simard, Alain R.

    2016-01-01

    It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers. PMID:26925951

  3. Prenatal restraint stress decreases the expression of alpha-7 nicotinic receptor in the brain of adult rat offspring.

    PubMed

    Baier, Carlos J; Pallarés, María E; Adrover, Ezequiela; Monteleone, Melisa C; Brocco, Marcela A; Barrantes, Francisco J; Antonelli, Marta C

    2015-01-01

    Prenatal stress (PS) strongly impacts fetal brain development and function in adulthood. In normal aging and Alzheimer's disease, there is hypothalamic-pituitary-adrenal axis dysfunction and loss of cholinergic neurons and neuronal nicotinic acetylcholine receptors (nAChRs). This study investigated whether prenatal restraint stress affects nAChR expression in the brain of adult offspring. For PS, pregnant dams were placed in a plastic restrainer for 45 min, three times daily during the last week of pregnancy; controls were undisturbed. Male offspring were analyzed at postnatal day (PND) 60 (n = 4 rats per group). Western blot (WB) and fluorescence microscopy showed that PS decreased α7-AChR subunit expression (∼50%) in the frontal cortex in the adult offspring. PS decreased significantly the number of α7-AChR-expressing cells in the medial prefrontal cortex (by ∼25%) and in the sensory-motor cortex (by ∼20%) without affecting the total cell number in those areas. No alterations were found in the hippocampus by quantitative polymerase chain reaction (qPCR), or WB analysis, but a detailed fluorescence microscopy analysis showed that PS affected α7-AChR mainly in the CA3 and dentate gyrus subfields: PS decreased α7-AChR subunit expression by ∼25 and ∼30%, respectively. Importantly, PS decreased the number of α7-AChR-expressing cells and the total cell number (by ∼15 and 20%, respectively) in the dentate gyrus. PS differently affected α4-AChR: PS impaired its mRNA expression in the frontal cortex (by ∼50%), without affecting protein levels. These results demonstrate that disturbances during gestation produce long-term alterations in the expression pattern of α7-AChR in rat brain. PMID:25798813

  4. Functional Contribution of α3L8′ to the Neuronal Nicotinic α3 Receptor

    PubMed Central

    Nieves-Cintrón, Madeline; Caballero-Rivera, Daniel; Silva, Walter I.; Navedo, Manuel F.; Lasalde-Dominicci, José A.

    2015-01-01

    The role of position L8′, located in transmembrane domain 1 of the neuronal nicotinic α3 subunit, was characterized by using two-electrode voltage clamp in Xenopus oocytes. Four amino acids (Ala, Ser, Phe, and Tyr) were inserted at this conserved position, and the mutant subunit was coexpressed with either wild-type β2 or β4 subunits. These substitutions led to significant alterations in the pharmacodynamic parameters of cholinergic agents, resulting in loss of function. Ala and Ser substitutions resulted in losses in agonist (ACh, nicotine, and DMPP) potency and intrinsic activity at both α3β2 and α3β4 receptors. Similarly, significant changes in antagonist potency were produced by the Ala and Ser substitutions. Phe and Tyr mutations did not alter the receptor's EC50 for ACh or nicotine but reduced the EC50 for DMPP at both receptors. The Phe mutation also reduced the intrinsic activity of all agonists tested at both receptors. The Tyr mutation, though, led to a decrease in intrinsic activity for all agonists at the α3β2 receptor, yet resulted in no changes for DMPP, a decrease for nicotine, and an increase for ACh at the α3β4 receptor. The most dramatic changes in the receptor's functional properties were produced by substitutions that introduced the largest changes in amino acid volume. Additional replacements (Gly, Thr, and Val) suggested an inverse correlation between amino acid volume at position α3L8′ and EC50 for α3β4 nAChRs; however, α3β2 nAChRs displayed a nonlinear correlation. These data demonstrate that structural alterations at position α3L8′ could propagate to the agonist-binding site. PMID:18615639

  5. Synthesis and Behavioral Studies of Chiral Cyclopropanes as Selective α4β2-Nicotinic Acetylcholine Receptor Partial Agonists Exhibiting an Antidepressant Profile. Part III.

    PubMed

    Onajole, Oluseye K; Vallerini, Gian Paolo; Eaton, J Brek; Lukas, Ronald J; Brunner, Dani; Caldarone, Barbara J; Kozikowski, Alan P

    2016-06-15

    We report the synthesis and biological characterization of novel derivatives of 3-[(1-methyl-2(S)-pyrrolidinyl)methoxy]-5-cyclopropylpyridine (4a-f and 5) as potent and highly selective α4β2-nicotinic acetylcholine receptor (nAChR) full or partial agonists. A systematic structure-activity study was carried out on the previously described compound 3b, particularly concerning its (2-methoxyethyl)cyclopropyl side-chain, in an effort to improve its metabolic stability while maintaining receptor selectivity. Compound 4d exhibited very similar subnanomolar binding affinity for α4β2- and α4β2*-nAChRs compared to 3b, and it showed excellent potency in activating high-sensitivity (HS) α4β2-nAChRs with an EC50 value of 8.2 nM. Testing of 4d in the SmartCube assay revealed that the compound has a combined antidepressant plus antipsychotic signature. In the forced swim test at a dose of 30 mg/kg given intraperitoneally, 4d was found to be as efficacious as sertraline, thus providing evidence of the potential use of the compound as an antidepressant. Additional promise for use of 4d in humans comes from pharmacokinetic studies in mice indicating brain penetration, and additional assays show compound stability in the presence of human microsomes and hepatocytes. Thus, 4d has a very favorable preclinical drug profile. PMID:27035276

  6. Subtype-selective nicotinic acetylcholine receptor agonists can improve cognitive flexibility in an attentional set shifting task.

    PubMed

    Wood, Christopher; Kohli, Shivali; Malcolm, Emma; Allison, Claire; Shoaib, Mohammed

    2016-06-01

    Nicotinic acetylcholine receptors (nAChRs) are considered to be viable targets to enhance cognition in patients diagnosed with schizophrenia. Activation of nAChRs with selective nicotinic receptor agonists may provide effective means to pharmacologically treat cognitive deficits observed in schizophrenia. Cognitive flexibility is one aspect of cognition, which can be assessed in a rodent model of the attentional set-shifting task (ASST). The aim of the present study was two-fold, firstly, to evaluate the efficacy of a series of subtype selective nAChR agonists, such as those that target α7 and α4β2 nAChR subtypes in non-compromised rodents. Secondly, nicotine as a prototypic agonist was evaluated for its effects to restore attentional deficits produced by sub-chronic ketamine exposure in the ASST. Male hooded Lister rats underwent habituation, consisting of a simple odour and medium discrimination with subsequent assessment 24 h later. In experimentally naïve rats, α7 subtype selective agonists, compound-A and SSR180711 along with PNU-120596, an α7 positive allosteric modulator (PAM), were compared against the β2* selective agonist, 5IA-85380. All compounds except for PNU-120596 were observed to significantly improve extra-dimensional (ED) shift performance, nicotine, 5IA-85380 and SSR180711 further enhanced the final reversal (REV3) stage of the task. In another experiment, sub-chronic ketamine treatment produced robust deficits during the ED and the REV3 stages of the discriminations; rodents required significantly more trials to reach criterion during these discriminations. These deficits were attenuated in rodents treated acutely with nicotine (0.1 mg/kg SC) 10 min prior to the ED shift. These results highlight the potential utility of targeting nAChRs to enhance cognitive flexibility, particularly the α7 and β2* receptor subtypes. The improvement with nicotine was much greater in rodents that were impaired following the sub-chronic ketamine

  7. Evidence for functional atypical nicotinic receptors that activate K+-dependent Cl- secretion in mouse tracheal epithelium.

    PubMed

    Hollenhorst, Monika I; Lips, Katrin S; Weitz, Ariane; Krasteva, Gabriela; Kummer, Wolfgang; Fronius, Martin

    2012-01-01

    The present study focused on the influence of nicotinic acetylcholine receptors (nAChR) on ion transport processes in mouse tracheal epithelium. RT-PCR experiments revealed expression of the α3, α4, α5, α7, α9, α10, β2, and β4 nAChR subunits in mouse tracheal epithelium. In Ussing chamber recordings of mouse tracheae, apically applied nicotine (100 μM) induced a dose-dependent increase of the transepithelial short-circuit current (EC(50): 14.6 μM). The nicotine-induced effect (I(NIC)) was attenuated by mecamylamine (25 μM, apical) and methyllycaconitine (1 μM, apical). The nAChR agonist 1.1-dimethyl-4-phenylpiperatinium iodide (DMPP) (100 μM) revealed apical and basolateral location of the receptors. I(NIC) was not affected by the sodium channel inhibitor amiloride (10 μM, apical) or the cystic fibrosis transmembrane conductance regulator inhibitor CFTR(inh)-172 (20 μM, apical) but was reduced by the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 μM, apical), the Na(+)/K(+)/2Cl(-) cotransporter inhibitor bumetanide (200 μM, basolateral), the potassium channel inhibitor Ba(2+) (5 mM, basolateral), and 4.4'-diisothiocyanatostilbene-2.2'-disulfonate (100 μM, apical), indicating a contribution of Ca(2+)-activated chloride channels and potassium channels. Removal of extracellular Na(+) (apical) or Ca(2+) (apical) did not influence I(NIC) but reduced the DMPP effect. Experiments with the Ca(2+)-ionophore A23187, a mix of 3-isobutyl-1-methylxanthine and forskolin, or the inositol-1,4,5-triphospate (IP(3)) receptor inhibitor 2-aminoethyl-diphenyl-borinate (75 μM, apical) decreased I(NIC), indicating a nicotine-mediated increase of intracellular Ca(2+) and cAMP levels involving the IP(3) signaling pathway. These findings indicate the activity of Ca(2+)-permeable nAChRs and alternative metabotropic pathways by nAChR activation that mediate Cl(-) and K(+) transport in tracheal epithelium. PMID:21852683

  8. Structure-function studies of the muscle nicotinic acetylcholine receptor by site-directed mutagenesis in the pore region

    NASA Astrophysics Data System (ADS)

    Zhang, Haiyun

    In nicotinic acetylcholine receptors (nAChRs), as in glycine, GABA A, serotonin 5-HT3, and GluCl glutamate receptors, a leucine residue at the approximate midpoint (the 9' position) of the M2 transmembrane domain is conserved across all known subunits. We expressed the embryonic mouse muscle nAChRs with varying numbers (m* s) of subunits (2 αs, 1 β, 1 γ, and 1 δ) mutated at this position in Xenopus oocytes and discovered that mutations to serine (Leu9'Ser) result in a tenfold higher receptor sensitivity to acetylcholine (ACh) for each subunit mutated. Moreover, increases of side-chain polarity increase the sensitivity to ACh when other natural and unnatural residues are incorporated into this position. The data also indicated an especially strong interaction between the γ and δ subunits in the pore region, suggesting a specific arrangement of subunits within the pentamer. Detailed single-channel kinetic studies reveal that Leu9'Ser AChRs have (1) longer voltage- relaxation time constants, (2) longer ACh-induced openings and bursts, and (3) more frequent spontaneous openings. These effects increase with m* s. Synthesized postsynaptic currents were produced with a piezoelectric micromanipulator that delivered brief ACh pulses to multi-channel patches. Their decay time constants were, as expected, similar to the channel burst duration. Thus, both longer and more frequent openings contribute to the >=104-fold increase in the receptor sensitivity to ACh from the wild-type receptor to the receptor with m*s=4; and the highly conserved 9' leucine is crucial for the brief synaptic events that are normally observed. We also explored the effects of ligand-binding domain mutations: γD174N and δD180N (aspartic acid (D) to asparagine (N)). Macroscopic dose-response relations revealed that these mutations decrease the receptor's sensitivity to ACh. The combined effect with Leu9'Ser, however, differs from that predicted from a linear or independent sum of effects from

  9. The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors

    PubMed Central

    Chatzidaki, Anna; D'Oyley, Jarryl M.; Gill-Thind, JasKiran K.; Sheppard, Tom D.; Millar, Neil S.

    2015-01-01

    Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9′ position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22′ position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties. PMID:25998276

  10. Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

    PubMed Central

    Spurny, Radovan; Debaveye, Sarah; Farinha, Ana; Veys, Ken; Vos, Ann M.; Gossas, Thomas; Atack, John; Bertrand, Sonia; Bertrand, Daniel; Danielson, U. Helena; Tresadern, Gary; Ulens, Chris

    2015-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential. PMID:25918415

  11. Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation.

    PubMed

    Imamura, Osamu; Arai, Masaaki; Dateki, Minori; Ogata, Toru; Uchida, Ryuji; Tomoda, Hiroshi; Takishima, Kunio

    2015-12-01

    Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer's disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil

  12. Intrinsically Low Open Probability of α7 Nicotinic Acetylcholine Receptors Can Be Overcome by Positive Allosteric Modulation and Serum Factors Leading to the Generation of Excitotoxic Currents at Physiological Temperatures

    PubMed Central

    Williams, Dustin K.; Peng, Can; Kimbrell, Matthew R.

    2012-01-01

    α7 nicotinic acetylcholine receptors (nAChRs) have been a puzzle since their discovery in brain and non-neuronal tissues. Maximal transient probability of an α7 nAChR being open with rapid agonist applications is only 0.002. The concentration dependence of α7 responses measured from transfected cells and Xenopus laevis oocytes shows the same disparity in potency estimations for peak currents and net charge, despite being studied at 1000-fold different time scales. In both cases the EC50 was approximately 10-fold lower for net charge than for peak currents. The equivalence of the data obtained at such disparate time scales indicates that desensitization of α7 is nearly instantaneous. At high levels of agonist occupancy, the receptor is preferentially converted to a ligand-bound nonconducting state, which can be destabilized by the positive allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596). Such currents can be sufficiently large to be cytotoxic to the α7-expressing cells. Both the potentiating effect of PNU-120596 and the associated cytotoxicity have a high temperature dependence that can be compensated for by serum factors. Therefore, despite reduced potentiation at body temperatures, use of type II positive allosteric modulators may put cells that naturally express high levels of α7 nAChRs, such as neurons in the hippocampus and hypothalamus, at risk. With a low intrinsic open probability and high propensity toward the induction of nonconducting ligand-bound states, it is likely that the well documented regulation of signal transduction pathways by α7 nAChRs in cells such as those that regulate inflammation may be independent of ion channel activation and associated with the nonconducting conformational states. PMID:22828799

  13. The prototoxin LYPD6B modulates heteromeric α3β4-containing nicotinic acetylcholine receptors, but not α7 homomers.

    PubMed

    Ochoa, Vanessa; George, Andrew A; Nishi, Rae; Whiteaker, Paul

    2016-03-01

    Prototoxins are a diverse family of membrane-tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain-containing (LYPD)-6B on α3β4-, α3α5β4-, and α7-containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human α3, β4, and α5 (D and N variants) subunits were expressed in Xenopus laevis oocytes and tested with or without LYPD6B. We used the 2-electrode voltage-clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist-induced current (Imax), and time constant (τ) of desensitization. For β4-α3-α3-β4-α3 and β4-α3-β4-α3-α3, LYPD6B decreased EC50 from 631 to 79 μM, reduced Imax by at least 59%, and decreased τ. For β4-α3-α5D-β4-α3 and β4-α3-β4-α-α5D, LYPD6B decreased Imax by 63 and 32%, respectively. Thus, LYPD6B acted only on (α3)3(β4)2 and (α3)2(α5D)(β4)2 and did not affect the properties of (α3)2(β4)3, α7, or (α3)2(α5N)(β4)2 nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (α3)3(β4)2 nAChRs to ACh while reducing ACh-induced whole-cell currents. LYPD6B also negatively modulates α3β4 nAChRs that include the α5D common human variant, but not the N variant associated with nicotine dependence. PMID:26586467

  14. Exposure to nicotine increases nicotinic acetylcholine receptor density in the reward pathway and binge ethanol consumption in C57BL/6J adolescent female mice.

    PubMed

    Locker, Alicia R; Marks, Michael J; Kamens, Helen M; Klein, Laura Cousino

    2016-05-01

    Nearly 80% of adult smokers begin smoking during adolescence. Binge alcohol consumption is also common during adolescence. Past studies report that nicotine and ethanol activate dopamine neurons in the reward pathway and may increase synaptic levels of dopamine in the nucleus accumbens through nicotinic acetylcholine receptor (nAChR) stimulation. Activation of the reward pathway during adolescence through drug use may produce neural alterations affecting subsequent drug consumption. Consequently, the effect of nicotine exposure on binge alcohol consumption was examined along with an assessment of the neurobiological underpinnings that drive adolescent use of these drugs. Adolescent C57BL/6J mice (postnatal days 35-44) were exposed to either water or nicotine (200μg/ml) for ten days. On the final four days, ethanol intake was examined using the drinking-in-the-dark paradigm. Nicotine-exposed mice consumed significantly more ethanol and displayed higher blood ethanol concentrations than did control mice. Autoradiographic analysis of nAChR density revealed higher epibatidine binding in frontal cortical regions in mice exposed to nicotine and ethanol compared to mice exposed to ethanol only. These data show that nicotine exposure during adolescence increases subsequent binge ethanol consumption, and may affect the number of nAChRs in regions of the brain reward pathway, specifically the frontal cortex. PMID:26428091

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

    PubMed

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

    2014-03-25

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

  16. Acetylcholinesterase (AChE) inhibition aggravates fasting-induced triglyceride accumulation in the mouse liver.

    PubMed

    Yokota, Shin-Ichi; Nakamura, Kaai; Ando, Midori; Kamei, Hiroyasu; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Shibata, Shigenobu

    2014-01-01

    Although fasting induces hepatic triglyceride (TG) accumulation in both rodents and humans, little is known about the underlying mechanism. Because parasympathetic nervous system activity tends to attenuate the secretion of very-low-density-lipoprotein-triglyceride (VLDL-TG) and increase TG stores in the liver, and serum cholinesterase activity is elevated in fatty liver disease, the inhibition of the parasympathetic neurotransmitter acetylcholinesterase (AChE) may have some influence on hepatic lipid metabolism. To assess the influence of AChE inhibition on lipid metabolism, the effect of physostigmine, an AChE inhibitor, on fasting-induced increase in liver TG was investigated in mice. In comparison with ad libitum-fed mice, 30 h fasting increased liver TG accumulation accompanied by a downregulation of sterol regulatory element-binding protein 1 (SREBP-1) and liver-fatty acid binding-protein (L-FABP). Physostigmine promoted the 30 h fasting-induced increase in liver TG levels in a dose-dependent manner, accompanied by a significant fall in plasma insulin levels, without a fall in plasma TG. Furthermore, physostigmine significantly attenuated the fasting-induced decrease of both mRNA and protein levels of SREBP-1 and L-FABP, and increased IRS-2 protein levels in the liver. The muscarinic receptor antagonist atropine blocked these effects of physostigmine on liver TG, serum insulin, and hepatic protein levels of SREBP-1 and L-FABP. These results demonstrate that AChE inhibition facilitated fasting-induced TG accumulation with up regulation of the hepatic L-FABP and SREBP-1 in mice, at least in part via the activation of muscarinic acetylcholine receptors. Our studies highlight the crucial role of parasympathetic regulation in fasting-induced TG accumulation, and may be an important source of information on the mechanism of hepatic disorders of lipid metabolism. PMID:25383314

  17. Key residues in the nicotinic acetylcholine receptor β2 subunit contribute to α-conotoxin LvIA binding.

    PubMed

    Zhangsun, Dongting; Zhu, Xiaopeng; Wu, Yong; Hu, Yuanyan; Kaas, Quentin; Craik, David J; McIntosh, J Michael; Luo, Sulan

    2015-04-10

    α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val(111) is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr(59) and Phe(119) of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity. PMID:25713061

  18. Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis.

    PubMed

    Chernyavsky, Alex; Chen, Yumay; Wang, Ping H; Grando, Sergei A

    2015-11-01

    The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, β2 and β4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity. PMID:25998908

  19. Organization of the mouse 5-HT3 receptor gene and functional expression of two splice variants.

    PubMed

    Werner, P; Kawashima, E; Reid, J; Hussy, N; Lundström, K; Buell, G; Humbert, Y; Jones, K A

    1994-10-01

    The structure of the mouse 5-HT3 receptor gene, 5-HT3R-A, is most similar to nicotinic acetylcholine receptor (nAChR) genes, in particular to the gene encoding the neuronal nAChR subunit alpha 7. These genes share among other things the location of three adjacent introns, suggesting that 5-HT3R-A and nAChR genes arose from a common precursor gene. The alternative use of two adjacent splice acceptor sites in intron 8 creates, in addition to the original 5-HT3R-A cDNA (5-HT3R-AL), a shorter isoform (5-HT3R-AS) which lacks six codons in the segment that translates into the major intracellular domain. This splice consensus sequence is not found in human genomic DNA. In mouse, we demonstrate by RNAse protection assay that 5-HT3R-AS mRNA is approximately 5 times more abundant than 5-HT3R-AL mRNA in both neuroblastoma cell lines and neuronal tissues. We used the Semliki Forest virus expression system for electrophysiological characterization of 5-HT3R-AS and 5-HT3R-AL in mammalian cells. No differences in electrophysiological characteristics, such as voltage dependence, desensitization kinetics, or unitary conductance were found between homomeric 5-HT3R-AS and 5-HT3R-AL receptors. Their properties are very similar to those of 5-HT3 receptors in mouse neuroblastoma cell lines. PMID:7854052

  20. Understanding the Bases of Function and Modulation of α7 Nicotinic Receptors: Implications for Drug Discovery.

    PubMed

    Corradi, Jeremías; Bouzat, Cecilia

    2016-09-01

    The nicotinic acetylcholine receptor (nAChR) belongs to a superfamily of pentameric ligand-gated ion channels involved in many physiologic and pathologic processes. Among nAChRs, receptors comprising the α7 subunit are unique because of their high Ca(2+) permeability and fast desensitization. nAChR agonists elicit a transient ion flux response that is further sustained by the release of calcium from intracellular sources. Owing to the dual ionotropic/metabotropic nature of α7 receptors, signaling pathways are activated. The α7 subunit is highly expressed in the nervous system, mostly in regions implicated in cognition and memory and has therefore attracted attention as a novel drug target. Additionally, its dysfunction is associated with several neuropsychiatric and neurologic disorders, such as schizophrenia and Alzheimer's disease. α7 is also expressed in non-neuronal cells, particularly immune cells, where it plays a role in immunity, inflammation, and neuroprotection. Thus, α7 potentiation has emerged as a therapeutic strategy for several neurologic and inflammatory disorders. With unique activation properties, the receptor is a sensitive drug target carrying different potential binding sites for chemical modulators, particularly agonists and positive allosteric modulators. Although macroscopic and single-channel recordings have provided significant information about the underlying molecular mechanisms and binding sites of modulatory compounds, we know just the tip of the iceberg. Further concerted efforts are necessary to effectively exploit α7 as a drug target for each pathologic situation. In this article, we focus mainly on the molecular basis of activation and drug modulation of α7, key pillars for rational drug design. PMID:27190210

  1. Pharmacological characterization of [(125)I]CHIBA-1006 binding, a new radioligand for α7 nicotinic acetylcholine receptors, to rat brain membranes.

    PubMed

    Wu, Jin; Toyohara, Jun; Tanibuchi, Yuko; Fujita, Yuko; Zhang, Jichun; Chen, Hongxian; Matsuo, Masaaki; Wang, Rong Fu; Hashimoto, Kenji

    2010-11-11

    The α7 nicotinic acetylcholine receptors (nAChRs) play an important role in the pathophysiology of neuropsychiatric diseases such as schizophrenia and Alzheimer's disease. However, there are currently no suitable small molecule radioligands for imaging α7 nAChRs in the brain. In this study, we synthesized the novel radioligand [(125)I]4-iodophenyl 1,4-diazaicyclo[3.2.2]nonane-4-carboxylate ([(125)I]CHIBA-1006), a iodine-derivative of the selective α7 nAChR agonist SSR180711, and studied the characterization of [(125)I]CHIBA-1006 binding to rat brain membranes. The assays of [(125)I]CHIBA-1006 binding to rat brain membranes were performed at 4°C. The presence of a single saturable high-affinity binding component for [(125)I]CHIBA-1006 in the rat brain was shown. Scatchard analysis revealed an apparent equilibrium dissociation constant (K(d)) of 88.2±21.4nM and a maximal number of binding sites (B(max)) of 65.4±6.8fmol/mg protein (mean±SEM, n=4). The specific binding of [(125)I]CHIBA-1006 was inhibited by a number of α7 nAChR-selective ligands (e.g., unlabeled CHIBA-1006, SSR180711, CHIBA-1001, MG624 and A844606), suggesting a similarity among α7 nAChR pharmacological profiles. In contrast, α-bungarotoxin, MLA, and nicotine showed very weak affinity for [(125)I]CHIBA-1006 binding. The regional distribution of [(125)I]CHIBA-1006 binding to crude membranes from dissected regions of the rat brain was different from that of [(125)I]α-bungarotoxin binding, suggesting that [(125)I]CHIBA-1006 binding sites may not be identical to [(125)I]α-bungarotoxin binding sites in the rat brain. The present findings suggest that [(125)I]CHIBA-1006 would be a useful new small molecule radioligand for α7 nAChRs in the brain. PMID:20816767

  2. Α4β2 and α7 nicotinic acetylcholine receptor binding predicts choice preference in two cost benefit decision-making tasks.

    PubMed

    Mendez, I A; Damborsky, J C; Winzer-Serhan, U H; Bizon, J L; Setlow, B

    2013-01-29

    Nicotinic receptors have been linked to a wide range of cognitive and behavioral functions, but surprisingly little is known about their involvement in cost benefit decision making. The goal of these experiments was to determine how nicotinic acetylcholine receptor (nAChR) expression is related to two forms of cost benefit decision making. Male Long Evans rats were tested in probability- and delay-discounting tasks, which required discrete trial choices between a small reward and a large reward associated with varying probabilities of omission and varying delays to reward delivery, respectively. Following testing, radioligand binding to α4β2 and α7 nAChR subtypes in brain regions implicated in cost benefit decision making was examined. Significant linear relationships were observed between choice of the large delayed reward in the delay discounting task and α4β2 receptor binding in both the dorsal and ventral hippocampus. Additionally, trends were found suggesting that choice of the large costly reward in both discounting tasks was inversely related to α4β2 receptor binding in the medial prefrontal cortex and nucleus accumbens shell. Similar trends suggested that choice of the large delayed reward in the delay discounting task was inversely related to α4β2 receptor binding in the orbitofrontal cortex, nucleus accumbens core, and basolateral amygdala, as well as to α7 receptor binding in the basolateral amygdala. These data suggest that nAChRs (particularly α4β2) play both unique and common roles in decisions that require consideration of different types of reward costs. PMID:23159316

  3. α4β2* and α7 Nicotinic Acetylcholine Receptor Binding Predicts Choice Preference in Two Cost Benefit Decision Making Tasks

    PubMed Central

    Mendez, Ian A.; Damborsky, Joanne C.; Winzer-Serhan, Ursula H.; Bizon, Jennifer L.; Setlow, Barry

    2012-01-01

    Nicotinic receptors have been linked to a wide range of cognitive and behavioral functions, but surprisingly little is known about their involvement in cost benefit decision making. The goal of these experiments was to determine how nicotinic acetylcholine receptor (nAChR) expression is related to two forms of cost benefit decision making. Male Long Evans rats were tested in probability and delay discounting tasks, which required discrete trial choices between a small reward and a large reward associated with varying probabilities of omission and varying delays to reward delivery, respectively. Following testing, radioligand binding to α4β2* and α7 nAChR subtypes in brain regions implicated in cost benefit decision making was examined. Significant linear relationships were observed between choice of the large delayed reward in the delay discounting task and α4β2* receptor binding in both dorsal and ventral hippocampus. Additionally, trends were found suggesting that choice of the large costly reward in both discounting tasks was inversely related to α4β2* receptor binding in the medial prefrontal cortex and nucleus accumbens shell. Similar, trends suggested that choice of the large delayed reward in the delay discounting task was inversely related to α4β2* receptor binding in the orbitofrontal cortex, nucleus accumbens core, and basolateral amygdala, as well as to α7 receptor binding in basolateral amygdala. These data suggest that nAChRs (particularly α4β2*) play both unique and common roles in decisions that require consideration of different types of reward costs. PMID:23159316

  4. Mechanisms of inhibition and potentiation of α4β2 nicotinic acetylcholine receptors by members of the Ly6 protein family.

    PubMed

    Wu, Meilin; Puddifoot, Clare A; Taylor, Palmer; Joiner, William J

    2015-10-01

    α4β2 nicotinic acetylcholine receptors (nAChRs) are abundantly expressed throughout the central nervous system and are thought to be the primary target of nicotine, the main addictive substance in cigarette smoking. Understanding the mechanisms by which these receptors are regulated may assist in developing compounds to selectively interfere with nicotine addiction. Here we report previously unrecognized modulatory properties of members of the Ly6 protein family on α4β2 nAChRs. Using a FRET-based Ca(2+) flux assay, we found that the maximum response of α4β2 receptors to agonist was strongly inhibited by Ly6h and Lynx2 but potentiated by Ly6g6e. The mechanisms underlying these opposing effects appear to be fundamentally distinct. Receptor inhibition by Lynx2 was accompanied by suppression of α4β2 expression at the cell surface, even when assays were preceded by chronic exposure of cells to an established chaperone, nicotine. Receptor inhibition by Lynx2 also was resistant to pretreatment with extracellular phospholipase C, which cleaves lipid moieties like those that attach Ly6 proteins to the plasma membrane. In contrast, potentiation of α4β2 activity by Ly6g6e was readily reversible by pretreatment with phospholipase C. Potentiation was also accompanied by slowing of receptor desensitization and an increase in peak currents. Collectively our data support roles for Lynx2 and Ly6g6e in intracellular trafficking and allosteric potentiation of α4β2 nAChRs, respectively. PMID:26276394

  5. Differential modulation of brain nicotinic acetylcholine receptor function by cytisine, varenicline, and two novel bispidine compounds: emergent properties of a hybrid molecule.

    PubMed

    Peng, Can; Stokes, Clare; Mineur, Yann S; Picciotto, Marina R; Tian, Chengju; Eibl, Christoph; Tomassoli, Isabelle; Guendisch, Daniela; Papke, Roger L

    2013-11-01

    Partial agonist therapies for the treatment of nicotine addiction and dependence depend on both agonistic and antagonistic effects of the ligands, and side effects associated with other nAChRs greatly limit the efficacy of nicotinic partial agonists. We evaluated the in vitro pharmacological properties of four partial agonists, two current smoking cessation drugs, varenicline and cytisine, and two novel bispidine compounds, BPC and BMSP, by using defined nAChR subtypes expressed in Xenopus laevis oocytes and human embryonic kidney 293 cells. Similar to varenicline and cytisine, BPC and BMSP are partial agonists of α4β2 nAChRs, although BMSP produced very little activation of these receptors. Unlike varenicline and cytisine, BPC and BMSP showed desired low activity. BPC produced mecamylamine-sensitive steady-state activation of α4* receptors that was not evident with BMSP. We evaluated the modulation of α4*- and α7-mediated responses in rat lateral geniculate nucleus (LGN) neurons and hippocampal stratum radiatum (SR) interneurons, respectively. The LGN neurons were sensitive to a very low concentration of varenicline, and the SR interneuron responses were also sensitive to varenicline at a submicromolar concentration. Although 300 nM BPC strongly inhibited the ACh-evoked responses of LGN neurons, it did not inhibit the α7 currents of SR interneurons. Similar results were observed with 300 nM BMSP. Additionally, the bispidine compounds were efficacious in the mouse tail suspension test, demonstrating that they affect receptors in the brain when delivered systemically. Our data indicate that BPC and BMSP are promising α4β2* partial agonists for pharmacotherapeutics. PMID:23959137

  6. Differential Modulation of Brain Nicotinic Acetylcholine Receptor Function by Cytisine, Varenicline, and Two Novel Bispidine Compounds: Emergent Properties of a Hybrid Molecule

    PubMed Central

    Peng, Can; Stokes, Clare; Mineur, Yann S.; Picciotto, Marina R.; Tian, Chengju; Eibl, Christoph; Tomassoli, Isabelle; Guendisch, Daniela

    2013-01-01

    Partial agonist therapies for the treatment of nicotine addiction and dependence depend on both agonistic and antagonistic effects of the ligands, and side effects associated with other nAChRs greatly limit the efficacy of nicotinic partial agonists. We evaluated the in vitro pharmacological properties of four partial agonists, two current smoking cessation drugs, varenicline and cytisine, and two novel bispidine compounds, BPC and BMSP, by using defined nAChR subtypes expressed in Xenopus laevis oocytes and human embryonic kidney 293 cells. Similar to varenicline and cytisine, BPC and BMSP are partial agonists of α4β2 nAChRs, although BMSP produced very little activation of these receptors. Unlike varenicline and cytisine, BPC and BMSP showed desired low activity. BPC produced mecamylamine-sensitive steady-state activation of α4* receptors that was not evident with BMSP. We evaluated the modulation of α4*- and α7-mediated responses in rat lateral geniculate nucleus (LGN) neurons and hippocampal stratum radiatum (SR) interneurons, respectively. The LGN neurons were sensitive to a very low concentration of varenicline, and the SR interneuron responses were also sensitive to varenicline at a submicromolar concentration. Although 300 nM BPC strongly inhibited the ACh-evoked responses of LGN neurons, it did not inhibit the α7 currents of SR interneurons. Similar results were observed with 300 nM BMSP. Additionally, the bispidine compounds were efficacious in the mouse tail suspension test, demonstrating that they affect receptors in the brain when delivered systemically. Our data indicate that BPC and BMSP are promising α4β2* partial agonists for pharmacotherapeutics. PMID:23959137

  7. Effects of varenicline on alpha4-containing nicotinic acetylcholine receptor expression and cognitive performance in mice.

    PubMed

    Lange-Asschenfeldt, Christian; Schäble, Sandra; Suvorava, Tatsiana; Fahimi, Ehsan Gholamreza; Bisha, Marion; Stermann, Torben; Henning, Uwe; Kojda, Georg

    2016-08-01

    Nicotinic acetylcholine receptor (nAChR) subtypes containing the α4 subunit, particularly α4β2 nAChRs, play an important role in cognitive functioning. The impact of the smoking cessation aid varenicline, a selective partial α4β2 nAChR agonist, on (1) changes of central protein and mRNA expression of this receptor and (2) on memory deficits in a mouse model of cognitive impairment was investigated. Protein and mRNA expression of both the α4 and β2 receptor subunits in mouse brain endothelial and hippocampal cells as well as hippocampus and neocortex tissues were determined by western blot and realtime PCR, respectively. The β2 antibody showed low specificity, though. Tissues were examined following a 2-week oral treatment with various doses of varenicline (0.01, 0.1, 1, 3 mg/kg/day) or vehicle. In addition, episodic memory of mice was assessed following this treatment with an object recognition task using (1) normal mice and (2) animals with anticholinergic-induced memory impairment (i.p. injection of 0.5 mg/kg scopolamine). Varenicline dose-dependently increased protein expression of both the α4 and β2 subunit in cell cultures and brain tissues, respectively, but had no effect on mRNA expression of both subunits. Scopolamine injection induced a significant reduction of object memory in vehicle-treated mice. By contrast, cognitive performance was not altered by scopolamine in varenicline-treated mice. In conclusion, a 2-week oral treatment with varenicline prevented memory impairment in the scopolamine mouse model. In parallel, protein, but not mRNA expression was upregulated, suggesting a posttranscriptional mechanism. Our findings suggest a beneficial effect of varenicline on cognitive dysfunction. PMID:27012889

  8. [Desensitization of the nicotinic acetylcholine receptor].

    PubMed

    Quiñonez, M; Rojas, L

    1994-01-01

    In biological membranes, ionic channels act speeding up ion movements. Each ionic channel is excited by a specific stimulus (i.e. electric, mechanical, chemical, etc.). Chemically activated ionic channels (CAIC), such as the nicotinic acetylcholine receptor (nAChR), suffer desensitization when the receptor site is still occupied by the agonist molecule. The desensitized CAIC is a non functional channel state regarded as a particular case of receptors rundown. CAIC desensitization only involve reduced activity and not their membrane elimination. Desensitization is important to control synaptic transmission and the development of the nervous system. In this review we discuss results related to its production, modulation and some aspects associated to models that consider it. Finally, an approach combining molecular biology and electrophysiology techniques to understand desensitization and its importance in biological systems is presented. PMID:8525756

  9. Structural and functional changes induced in the nicotinic acetylcholine receptor by membrane phospholipids.

    PubMed

    Fernández-Carvajal, Asia M; Encinar, José A; Poveda, José Antonio; de Juan, Entilio; Martínez-Pinna, Juan; Ivorra, Isabel; Ferragut, José Antonio; Morales, Andrés; González-Ros, José Manuel

    2006-01-01

    Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of

  10. Angiotensin AT1 and AT2 receptor antagonists modulate nicotine-evoked [³H]dopamine and [³H]norepinephrine release.

    PubMed

    Narayanaswami, Vidya; Somkuwar, Sucharita S; Horton, David B; Cassis, Lisa A; Dwoskin, Linda P

    2013-09-01

    Tobacco smoking is the leading preventable cause of death in the United States. A major negative health consequence of chronic smoking is hypertension. Untoward addictive and cardiovascular sequelae associated with chronic smoking are mediated by nicotine-induced activation of nicotinic receptors (nAChRs) within striatal dopaminergic and hypothalamic noradrenergic systems. Hypertension involves both brain and peripheral angiotensin systems. Activation of angiotensin type-1 receptors (AT1) release dopamine and norepinephrine. The current study determined the role of AT1 and angiotensin type-2 (AT2) receptors in mediating nicotine-evoked dopamine and norepinephrine release from striatal and hypothalamic slices, respectively. The potential involvement of nAChRs in mediating effects of AT1 antagonist losartan and AT2 antagonist, 1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid (PD123319) was evaluated by determining their affinities for α4β2* and α7* nAChRs using [³H]nicotine and [³H]methyllycaconitine binding assays, respectively. Results show that losartan concentration-dependently inhibited nicotine-evoked [³H]dopamine and [³H]norepinephrine release (IC₅₀: 3.9 ± 1.2 and 2.2 ± 0.7 μM; Imax: 82 ± 3 and 89 ± 6%, respectively). In contrast, PD123319 did not alter nicotine-evoked norepinephrine release, and potentiated nicotine-evoked dopamine release. These results indicate that AT1 receptors modulate nicotine-evoked striatal dopamine and hypothalamic norepinephrine release. Furthermore, AT1 receptor activation appears to be counteracted by AT2 receptor activation in striatum. Losartan and PD123319 did not inhibit [³H]nicotine or [³H]methyllycaconitine binding, indicating that these AT1 and AT2 antagonists do not interact with the agonist recognition sites on α4β2* and α7* nAChRs to mediate these effects of nicotine. Thus, angiotensin receptors contribute to the effects of

  11. Angiotensin AT1 and AT2 receptor antagonists modulate nicotine-evoked [3H]dopamine and [3H]norepinephrine release

    PubMed Central

    Narayanaswami, Vidya; Somkuwar, Sucharita S.; Horton, David B.; Cassis, Lisa A.; Dwoskin, Linda P.

    2014-01-01

    Tobacco smoking is the leading preventable cause of death in the United States. A major negative health consequence of chronic smoking is hypertension. Untoward addictive and cardiovascular sequelae associated with chronic smoking are mediated by nicotine-induced activation of nicotinic receptors (nAChRs) within striatal dopaminergic and hypothalamic noradrenergic systems. Hypertension involves both brain and peripheral angiotensin systems. Activation of angiotensin type-1 receptors (AT1) release dopamine and norepinephrine. The current study determined the role of AT1 and angiotensin type-2 (AT2) receptors in mediating nicotine-evoked dopamine and norepinephrine release from striatal and hypothalamic slices, respectively. The potential involvement of nAChRs in mediating effects of AT1 antagonist losartan and AT2 antagonist, 1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid (PD123319) was evaluated by determining their affinities for α4β2* and α7* nAChRs using [3H]nicotine and [3H]methyllycaconitine binding assays, respectively. Results show that losartan concentration-dependently inhibited nicotine-evoked [3H]dopamine and [3H]norepinephrine release (IC50: 3.9±1.2 and 2.2±0.7 μM; Imax: 82±3 and 89±6%, respectively). In contrast, PD123319 did not alter nicotine-evoked norepinephrine release, and potentiated nicotine-evoked dopamine release. These results indicate that AT1 receptors modulate nicotine-evoked striatal dopamine and hypothalamic norepinephrine release. Furthermore, AT1 receptor activation appears to be counteracted by AT2 receptor activation in striatum. Losartan and PD123319 did not inhibit [3H]nicotine or [3H]methyllycaconitine binding, indicating that these AT1 and AT2 antagonists do not interact with the agonist recognition sites on α4β2* and α7* nAChRs to mediate these effects of nicotine. Thus, angiotensin receptors contribute to the effects of nicotine on

  12. Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations.

    PubMed

    Mathé-Allainmat, Monique; Swale, Daniel; Leray, Xavier; Benzidane, Yassine; Lebreton, Jacques; Bloomquist, Jeffrey R; Thany, Steeve H

    2013-12-01

    We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors. PMID:23884575

  13. Imaging of α7 nicotinic acetylcholine receptors in brain and cerebral vasculature of juvenile pigs with [18F]NS14490

    PubMed Central

    2014-01-01

    Background The α7 nicotinic acetylcholine receptor (nAChR) is an important molecular target in neuropsychiatry and oncology. Development of applicable highly specific radiotracers has been challenging due to comparably low protein expression. To identify novel ligands as candidates for positron emission tomography (PET), a library of diazabicyclononane compounds was screened regarding affinity and specificity towards α7 nAChRs. From these, [18F]NS14490 has been shown to yield reliable results in organ distribution studies; however, the radiosynthesis of [18F]NS14490 required optimization and automation to obtain the radiotracer in quantities allowing dynamic PET studies in piglets. Methods Automated radiosynthesis of [18F]NS14490 has been performed by [18F]fluorination with the tosylate precursor in the TRACERlab™ FX F-N synthesis module (Waukesha, WI, USA). After optimization, the radiochemical yield of [18F]NS14490 was consistently approximately 35%, and the total synthesis time was about 90 min. The radiotracer was prepared with >92% radiochemical purity, and the specific activity at the end of the synthesis was 226 ± 68 GBq μmol−1. PET measurements were performed in young pigs to investigate the metabolic stability and cerebral binding of [18F]NS14490 without and with administration of the α7 nAChR partial agonist NS6740 in baseline and blocking conditions. Results The total distribution volume relative to the metabolite-corrected arterial input was 3.5 to 4.0 mL g−1 throughout the telencephalon and was reduced to 2.6 mL g−1 in animals treated with NS6740. Assuming complete blockade, this displacement indicated a binding potential (BPND) of approximately 0.5 in the brain of living pigs. In addition, evidence for specific binding in major brain arteries has been obtained. Conclusion [18F]NS14490 is not only comparable to other preclinically investigated PET radiotracers for imaging of α7 nAChR in brain but also could be a potential PET

  14. SSR591813, a novel selective and partial alpha4beta2 nicotinic receptor agonist with potential as an aid to smoking cessation.

    PubMed

    Cohen, C; Bergis, O E; Galli, F; Lochead, A W; Jegham, S; Biton, B; Leonardon, J; Avenet, P; Sgard, F; Besnard, F; Graham, D; Coste, A; Oblin, A; Curet, O; Voltz, C; Gardes, A; Caille, D; Perrault, G; George, P; Soubrie, P; Scatton, B

    2003-07-01

    (5aS,8S,10aR)-5a,6,9,10-Tetrahydro,7H,11H-8,10a-methanopyrido[2',3':5,6]pyrano[2,3-d]azepine (SSR591813) is a novel compound that binds with high affinity to the rat and human alpha4beta2 nicotinic acetylcholine receptor (nAChR) subtypes (Ki = 107 and 36 nM, respectively) and displays selectivity for the alpha4beta2 nAChR (Ki, human alpha3beta4 > 1000, alpha3beta2 = 116; alpha1beta1deltagamma > 6000 nM and rat alpha7 > 6000 nM). Electrophysiological experiments indicate that SSR591813 is a partial agonist at the human alpha4beta2 nAChR subtype (EC50 = 1.3 micro M, IA =19% compared with the full agonist 1,1-dimethyl-4-phenyl-piperazinium). In vivo findings from microdialysis and drug discrimination studies confirm the partial intrinsic activity of SSR591813. The drug increases dopamine release in the nucleus accumbens shell (30 mg/kg i.p.) and generalizes to nicotine or amphetamine (10-20 mg/kg i.p.) in rats, with an efficacy approximately 2-fold lower than that of nicotine. Pretreatment with SSR591813 (10 mg/kg i.p.) reduces the dopamine-releasing and discriminative effects of nicotine. SSR591813 shows activity in animal models of nicotine dependence at doses devoid of unwanted side effects typically observed with nicotine (hypothermia and cardiovascular effects). The compound (10 mg/kg i.p.) also prevents withdrawal signs precipitated by mecamylamine in nicotine-dependent rats and partially blocks the discriminative cue of an acute precipitated withdrawal. SSR591813 (20 mg/kg i.p.) reduces i.v. nicotine self-administration and antagonizes nicotine-induced behavioral sensitization in rats. The present results confirm important role for alpha4beta2 nAChRs in mediating nicotine dependence and suggest that SSR591813, a partial agonist at this particular nAChR subtype, may have therapeutic potential in the clinical management of smoking cessation. PMID:12682217

  15. Enhanced Sensitivity of α3β4 Nicotinic Receptors in Enteric Neurons after Long-Term Morphine: Implication for Opioid-Induced Constipation.

    PubMed

    Gade, Aravind R; Kang, Minho; Khan, Fayez; Grider, John R; Damaj, M Imad; Dewey, William L; Akbarali, Hamid I

    2016-06-01

    Opioid-induced constipation is a major side effect that persists with long-term opioid use. Previous studies demonstrated that nicotine-induced contractions are enhanced after long-term morphine exposure in guinea pig ileum. In the present study, we examined whether the increased sensitivity to nicotine could be observed in single enteric neurons after long-term morphine exposure, determined the subunits in mouse enteric neurons, and examined the effect of nicotine in reversing opioid-induced constipation. Nicotine (0.03-1 mM) dose-dependently induced inward currents from a holding potential of -60 mV in isolated single enteric neurons from the mouse ileum. The amplitude of the currents, but not the potency to nicotine, was significantly increased in neurons receiving long-term (16-24 h) but not short-term (10 min) exposure to morphine. Quantitative mRNA analysis showed that nicotinic acetylcholine receptor (nAChR) subunit expression in the mouse ileum was α3 ≥ β2 > β4 > α5 > α4 > β3 > α6. Nicotine-induced currents were obtained in neurons from α7, β2, α5, and α6 knockout mice. The currents were, however, inhibited by mecamylamine (10 μM) and the α3β4 blocker α-conotoxin AuIB (3 μM), suggesting that nicotine-induced currents were mediated by the α3β4 subtype of nAChRs on enteric neurons. Conversely, NS3861, a partial agonist at α3β4 nAChR, enhanced fecal pellet expulsion in a dose-dependent manner in mice that received long-term, but not short-term, morphine treatment. Overall, our findings suggest that the efficacy of nAChR agonists on enteric neurons is enhanced after long-term morphine exposure, and activation of the α3β4 subtype of nAChR reverses chronic, but not acute, morphine-induced constipation. PMID:27068812

  16. Similarities between the Binding Sites of SB-206553 at Serotonin Type 2 and Alpha7 Acetylcholine Nicotinic Receptors: Rationale for Its Polypharmacological Profile

    PubMed Central

    Möller-Acuña, Patricia; Contreras-Riquelme, J. Sebastián; Rojas-Fuentes, Cecilia; Nuñez-Vivanco, Gabriel; Alzate-Morales, Jans; Iturriaga-Vásquez, Patricio; Arias, Hugo R.; Reyes-Parada, Miguel

    2015-01-01

    Evidence from systems biology indicates that promiscuous drugs, i.e. those that act simultaneously at various protein targets, are clinically better in terms of efficacy, than those that act in a more selective fashion. This has generated a new trend in drug development called polypharmacology. However, the rational design of promiscuous compounds is a difficult task, particularly when the drugs are aimed to act at receptors with diverse structure, function and endogenous ligand. In the present work, using docking and molecular dynamics methodologies, we established the most probable binding sites of SB-206553, a drug originally described as a competitive antagonist of serotonin type 2B/2C metabotropic receptors (5-HT2B/2CRs) and more recently as a positive allosteric modulator of the ionotropic α7 nicotinic acetylcholine receptor (nAChR). To this end, we employed the crystal structures of the 5-HT2BR and acetylcholine binding protein as templates to build homology models of the 5-HT2CR and α7 nAChR, respectively. Then, using a statistical algorithm, the similarity between these binding sites was determined. Our analysis showed that the most plausible binding sites for SB-206553 at 5-HT2Rs and α7 nAChR are remarkably similar, both in size and chemical nature of the amino acid residues lining these pockets, thus providing a rationale to explain its affinity towards both receptor types. Finally, using a computational tool for multiple binding site alignment, we determined a consensus binding site, which should be useful for the rational design of novel compounds acting simultaneously at these two types of highly different protein targets. PMID:26244344

  17. Similarities between the Binding Sites of SB-206553 at Serotonin Type 2 and Alpha7 Acetylcholine Nicotinic Receptors: Rationale for Its Polypharmacological Profile.

    PubMed

    Möller-Acuña, Patricia; Contreras-Riquelme, J Sebastián; Rojas-Fuentes, Cecilia; Nuñez-Vivanco, Gabriel; Alzate-Morales, Jans; Iturriaga-Vásquez, Patricio; Arias, Hugo R; Reyes-Parada, Miguel

    2015-01-01

    Evidence from systems biology indicates that promiscuous drugs, i.e. those that act simultaneously at various protein targets, are clinically better in terms of efficacy, than those that act in a more selective fashion. This has generated a new trend in drug development called polypharmacology. However, the rational design of promiscuous compounds is a difficult task, particularly when the drugs are aimed to act at receptors with diverse structure, function and endogenous ligand. In the present work, using docking and molecular dynamics methodologies, we established the most probable binding sites of SB-206553, a drug originally described as a competitive antagonist of serotonin type 2B/2C metabotropic receptors (5-HT2B/2CRs) and more recently as a positive allosteric modulator of the ionotropic α7 nicotinic acetylcholine receptor (nAChR). To this end, we employed the crystal structures of the 5-HT2BR and acetylcholine binding protein as templates to build homology models of the 5-HT2CR and α7 nAChR, respectively. Then, using a statistical algorithm, the similarity between these binding sites was determined. Our analysis showed that the most plausible binding sites for SB-206553 at 5-HT2Rs and α7 nAChR are remarkably similar, both in size and chemical nature of the amino acid residues lining these pockets, thus providing a rationale to explain its affinity towards both receptor types. Finally, using a computational tool for multiple binding site alignment, we determined a consensus binding site, which should be useful for the rational design of novel compounds acting simultaneously at these two types of highly different protein targets. PMID:26244344