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Sample records for a7 nicotinic acetylcholine

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. 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…

  4. Alcohol's actions on neuronal nicotinic acetylcholine receptors.

    PubMed

    Davis, Tiffany J; de Fiebre, Christopher M

    2006-01-01

    Although it has been known for many years that alcoholism and tobacco addiction often co-occur, relatively little information is available on the biological factors that regulate the co-use and abuse of nicotine and alcohol. In the brain, nicotine acts at several different types of receptors collectively known as nicotinic acetylcholine receptors (nAChRs). Alcohol also acts on at least some of these receptors, enhancing the function of some nAChR subtypes and inhibiting the activity of others. Chronic alcohol and nicotine administration also lead to changes in the numbers of nAChRs. Natural variations (i.e., polymorphisms) in the genes encoding different nAChR subunits may be associated with individual differences in the sensitivity to some of alcohol's and nicotine's effects. Finally, at least one subtype of nAChR may help protect cells against alcohol-induced neurotoxicity.

  5. Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors.

    PubMed

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

    2010-09-06

    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. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  6. Nicotinic acetylcholine receptor from chick optic lobe.

    PubMed Central

    Norman, R I; Mehraban, F; Barnard, E A; Dolly, J O

    1982-01-01

    An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest of the chick brain which can also bind alpha-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the alpha-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors. Images PMID:6175967

  7. 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

  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.

  9. Embedded cholesterol in the nicotinic acetylcholine receptor.

    PubMed

    Brannigan, Grace; Hénin, Jérôme; Law, Richard; Eckenhoff, Roderic; Klein, Michael L

    2008-09-23

    The nicotinic acetylcholine receptor (nAChR) is a cation-selective channel central to both neuronal and muscular processes and is considered the prototype for ligand-gated ion channels, motivating a structural determination effort that spanned several decades [Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4 A resolution. J Mol Biol 346:967-989]. Purified nAChR must be reconstituted in a mixture containing cholesterol to function. Proposed modes of interaction between cholesterol and the protein range from specific binding to indirect membrane-mediated mechanisms. However, the underlying cause of nAChR sensitivity to cholesterol remains controversial, in part because the vast majority of functional studies were conducted before a medium resolution structure was reported. We show that the nAChR contains internal sites capable of containing cholesterol, whose occupation stabilizes the protein structure. We detect sites at the protein-lipid interface as conventionally predicted from functional data, as well as deeply buried sites that are not usually considered. Molecular dynamics simulations reveal that occupation of both superficial and deeply buried sites most effectively preserves the experimental structure; the structure collapses in the absence of bound cholesterol. In particular, we find that bound cholesterol directly supports contacts between the agonist-binding domain and the pore that are thought to be essential for activation of the receptor. These results likely apply to those other ion channels within the Cys-loop superfamily that depend on cholesterol, such as the GABA receptor.

  10. Endogenous acetylcholine modulates impulsive action via alpha4beta2 nicotinic acetylcholine receptors in rats.

    PubMed

    Tsutsui-Kimura, Iku; Ohmura, Yu; Izumi, Takeshi; Yamaguchi, Taku; Yoshida, Takayuki; Yoshioka, Mitsuhiro

    2010-09-01

    Nicotine has been well established as an impulsive action-inducing agent, but it remains unknown whether endogenous acetylcholine affects impulsive action via nicotinic acetylcholine receptors. In the present study, the 3-choice serial reaction time task (3-CSRTT), a simple and valid assessment of impulsive action, was employed. Male Wistar/ST rats were trained to detect and respond to 1-s flashes of light presented in one of three holes until stable performance was achieved. Following training on the 3-CSRTT, rats received intracerebroventricular injections of the preferential alpha4beta2 nicotinic acetylcholine receptor antagonist dihydro-beta-erythroidine (DHbetaE; 0, 3, 10, and 30 microg) or the selective alpha7 nicotinic acetylcholine receptor antagonist methyllycaconitine (MLA; 0, 3, 10, and 30 microg) 5 min before test sessions. Injection of 10 microg of DHbetaE significantly suppressed premature responses, an index of impulsive-like action, without changing other behavioral parameters. On the other hand, MLA infusions failed to affect impulsive-like action at any dose. These results suggest that the central alpha4beta2 nicotinic acetylcholine receptors that enable a provoking effect of endogenous acetylcholine play a critical role in impulsive action. Substances that modulate nicotinic acetylcholine receptors, especially the alpha4beta2 subtype, may be beneficial for the treatment of psychiatric disorders characterized by lack of inhibitory control. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  11. Modulation of nicotinic acetylcholine receptors by strychnine

    PubMed Central

    García-Colunga, Jesús; Miledi, Ricardo

    1999-01-01

    Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α1β1γδ, α1β1γ, and α1β1δ) and neuronal (α2β2 and α2β4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α1β1γδ > α2β4 > α2β2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex. PMID:10097172

  12. The wonderland of neuronal nicotinic acetylcholine receptors.

    PubMed

    Bertrand, Daniel; Terry, A V

    2018-05-01

    Nearly 30 years of experimental evidence supports the argument that ligands of nicotinic acetylcholine receptors (nAChRs) have potential as therapeutic agents. However, as in the famous Lewis Carroll novel "Alice in Wonderland", there have been many unexpected adventures along the pathway of development, and few nAChR ligands have been approved for any clinical condition to date with the exception of nicotine dependence. The recent failures of nAChR ligands in AD and schizophrenia clinical trials have reduced enthusiasm for this therapeutic strategy and many pharmaceutical companies have now abandoned this field of research. As with other clinical failures, multiple questions arise as to the basis for the failure. More generic questions focus on a potential translational gap between the animal models used and the human clinical condition they are meant to simulate, or the clinical trial mindset that large Ns have to be achieved for statistical power (often requiring multiple trial sites) as opposed to smaller patient cohorts at limited sites where conditions can be better controlled and replicated. More specific to the nAChR field are questions about subtype selectivity, dose selection, whether an agonist, antagonist, or allosteric modulator strategy is best, etc. The purpose of this review is to discuss each of these questions, but also to provide a brief overview of the remarkable progress that has been made over the last three decades in our understanding of this unique ligand-gated ion channel and how this new knowledge may help us improve drug development successes in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Topographical studies of the nicotinic acetylcholine receptor. [Torpedo californica

    SciTech Connect

    Middlemas, D.S.

    1987-01-01

    All four subunits of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with the photoactivated hydrophobic probe, (/sup 3/H)adamantanediazirine, which selectively labels regions of integral membrane proteins in contact with the hydrocarbon core of the lipid bilayer. All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with (/sup 3/H)cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. Since the photogenerated carbene is situated near the lipid-water interface, this probe has potential as a topographicmore » tool for mapping membrane protein structure. The labeling studies with both (/sup 3/H)adamantanediazirine and (/sup 3/H)cholesteryl diazoacetate support the concept that the acetylcholine receptor is a pseudosymmetric complex of homologous subunits, all of which interact with and span the membrane. The synthesis of the fluorine-containing agonists for the Torpedo californica nicotinic acetylcholine receptor, fluoroacetylcholine bromide and p-fluorophenyltrimethylammonium iodide, are described. It is demonstrated that both are agonists using a cation flux assay with acetylcholine receptor enriched membrane vesicles. The affinity cleavage reagent, p-thiocyanophenyltrimethylammonium iodide, specifically cleaves a peptide bond of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica. It is demonstrated that this reagent is an agonist using a cation flux assay. The cleavage is blocked by stoichiometric quantities of ..cap alpha..-bungarotoxin.« less

  14. Modal gating of muscle nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Vij, Ridhima

    Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (alpha188-alpha199, at the agonist binding site) generate heterogeneity in PO ('modes'). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except alphaY190 and alphaY198) and with different side chain substitutions at alphaP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the alphadelta agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (alphaY93, alphaW149, alphaY190, alphaY198 and deltaW57). Phenylalanine substitutions at all aromatic residues except alphaY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at deltaW57 on the complementary side but not at other aromatics. Also, by substituting four gamma subunit residues into the delta subunit on the complementary beta sheet, I found that

  15. 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

  16. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. They consist of five subunits arranged around a central ion channeL Since the subunit composition determines the functional and pharmacological properties of the receptor the presence of nAChR families comprising several subunit-encodinggenes provides a molecular basis for broad functional diversity. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their nematode andvertebrate counterparts. Thus, the fruit fly (Drosophila melanogaster), malaria mosquito (Anopheles gambiae), honey bee (Apis mellifera), silk worm (Bombyx mon) and the red flour beetle (Tribolium castaneum) possess 10-12 nAChR genes while human and the nematode Caenorhabditis elegans have 16 and 29 respectively. Although insect nAChRgene families are amongst the smallest known, receptor diversity can be considerably increased by the posttranscriptional processes alternative splicing and mRNA A-to-I editingwhich can potentially generate protein products which far outnumber the nAChR genes. These two processes can also generate species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit which may perform species-specific functions. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that target specific pest insects while sparing beneficial species.

  17. Brain nicotinic acetylcholine receptors are involved in stress-induced potentiation of nicotine reward in rats.

    PubMed

    Javadi, Parastoo; Rezayof, Ameneh; Sardari, Maryam; Ghasemzadeh, Zahra

    2017-07-01

    The aim of the present study was to examine the possible role of nicotinic acetylcholine receptors of the dorsal hippocampus (CA1 regions), the medial prefrontal cortex or the basolateral amygdala in the effect of acute or sub-chronic stress on nicotine-induced conditioned place preference. Our results indicated that subcutaneous administration of nicotine (0.2 mg/kg) induced significant conditioned place preference. Exposure to acute or sub-chronic elevated platform stress potentiated the response of an ineffective dose of nicotine. Pre-conditioning intra-CA1 (0.5-4 µg/rat) or intra-medial prefrontal cortex (0.2-0.3 µg/rat) microinjection of mecamylamine (a non-selective nicotinic acetylcholine receptor antagonist) reversed acute stress-induced potentiation of nicotine reward as measured in the conditioned place preference paradigm. By contrast, pre-conditioning intra-basolateral amygdala microinjection of mecamylamine (4 µg/rat) potentiated the effects of acute stress on nicotine reward. Our findings also showed that intra-CA1 or intra-medial prefrontal cortex, but not intra-basolateral amygdala, microinjection of mecamylamine (4 µg/rat) prevented the effect of sub-chronic stress on nicotine reward. These findings suggest that exposure to elevated platform stress potentiates the rewarding effect of nicotine which may be associated with the involvement of nicotinic acetylcholine receptors. It seems that there is a different contribution of the basolateral amygdala, the medial prefrontal cortex or the CA1 nicotinic acetylcholine receptors in stress-induced potentiation of nicotine-induced conditioned place preference.

  18. Potentiation of the actions of acetylcholine, epibatidine, and nicotine by methyllycaconitine at fetal muscle-type nicotinic acetylcholine receptors.

    PubMed

    Green, Benedict T; Welch, Kevin D; Cook, Daniel; Gardner, Dale R

    2011-07-15

    Methyllycaconitine (MLA) is a norditerpenoid alkaloid found in high abundance in toxic Delphinium (larkspur) species. It is a potent and selective antagonist of α(7)-nicotinic acetylcholine receptors, but has not been well investigated for activity aside from receptor antagonism. The aim of this study was to investigate the effects of MLA alone and in combination with acetylcholine, epibatidine, nicotine, and neostigmine for actions other than receptor antagonism in TE-671 cells expressing (α(1))(2)β(1)γδ nicotinic acetylcholine receptors. Ligand activity was assessed through measurements of membrane potential changes in TE-671 cells using a fluorescent membrane potential-sensitive dye and normalized to the maximum response to epibatidine (10μM). MLA was ineffective in changing cell membrane potential in the absence of other receptor agonists. However at nanomolar concentrations, it acted as a co-agonist to potentiate TE-671 cell responses to acetylcholine, epibatidine, nicotine, and neostigmine. These results suggest that the poisoning of cattle by norditerpenoid alkaloids found in larkspur may be more complex than previously determined. Published by Elsevier B.V.

  19. Acetylcholine excites neocortical pyramidal neurons via nicotinic receptors

    PubMed Central

    Hedrick, Tristan

    2015-01-01

    The neuromodulator acetylcholine (ACh) shapes neocortical function during sensory perception, motor control, arousal, attention, learning, and memory. Here we investigate the mechanisms by which ACh affects neocortical pyramidal neurons in adult mice. Stimulation of cholinergic axons activated muscarinic and nicotinic ACh receptors on pyramidal neurons in all cortical layers and in multiple cortical areas. Nicotinic receptor activation evoked short-latency, depolarizing postsynaptic potentials (PSPs) in many pyramidal neurons. Nicotinic receptor-mediated PSPs promoted spiking of pyramidal neurons. The duration of the increase in spiking was membrane potential dependent, with nicotinic receptor activation triggering persistent spiking lasting many seconds in neurons close to threshold. Persistent spiking was blocked by intracellular BAPTA, indicating that nicotinic ACh receptor activation evoked persistent spiking via a long-lasting calcium-activated depolarizing current. We compared nicotinic PSPs in primary motor cortex (M1), prefrontal cortex (PFC), and visual cortex. The laminar pattern of nicotinic excitation was not uniform but was broadly similar across areas, with stronger modulation in deep than superficial layers. Superimposed on this broad pattern were local differences, with nicotinic PSPs being particularly large and common in layer 5 of M1 but not layer 5 of PFC or primary visual cortex (V1). Hence, in addition to modulating the excitability of pyramidal neurons in all layers via muscarinic receptors, synaptically released ACh preferentially increases the activity of deep-layer neocortical pyramidal neurons via nicotinic receptors, thereby adding laminar selectivity to the widespread enhancement of excitability mediated by muscarinic ACh receptors. PMID:25589590

  20. The nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster: Dual role in nicotine addiction and lung cancer

    PubMed Central

    Improgo, Ma. Reina D.; Scofield, Michael D.; Tapper, Andrew R.; Gardner, Paul D.

    2010-01-01

    More than 1 billion people around the world smoke, with 10 million cigarettes sold every minute. Cigarettes contain thousands of harmful chemicals including the psychoactive compound, nicotine. Nicotine addiction is initiated by the binding of nicotine to nicotinic acetylcholine receptors, ligand-gated cation channels activated by the endogenous neurotransmitter, acetylcholine. These receptors serve as prototypes for all ligand-gated ion channels and have been extensively studied in an attempt to elucidate their role in nicotine addiction. Many of these studies have focused on heteromeric nicotinic acetylcholine receptors containing α4 and β2 subunits and homomeric nicotinic acetylcholine receptors containing the α7 subunit, two of the most abundant subtypes expressed in the brain. Recently however, a series of linkage analyses, candidate-gene analyses and genome-wide association studies have brought attention to three other members of the nicotinic acetylcholine receptor family: the α5, α3 and β4 subunits. The genes encoding these subunits lie in a genomic cluster that contains variants associated with increased risk for several diseases including nicotine dependence and lung cancer. The underlying mechanisms for these associations have not yet been elucidated but decades of research on the nicotinic receptor gene family as well as emerging data provide insight on how these receptors may function in pathological states. Here, we review this body of work, focusing on the clustered nicotinic acetylcholine receptor genes and evaluating their role in nicotine addiction and lung cancer. PMID:20685379

  1. Molecular recognition of the neurotransmitter acetylcholine by an acetylcholine binding protein reveals determinants of binding to nicotinic acetylcholine receptors.

    PubMed

    Olsen, Jeppe A; Balle, Thomas; Gajhede, Michael; Ahring, Philip K; Kastrup, Jette S

    2014-01-01

    Despite extensive studies on nicotinic acetylcholine receptors (nAChRs) and homologues, details of acetylcholine binding are not completely resolved. Here, we report the crystal structure of acetylcholine bound to the receptor homologue acetylcholine binding protein from Lymnaea stagnalis. This is the first structure of acetylcholine in a binding pocket containing all five aromatic residues conserved in all mammalian nAChRs. The ligand-protein interactions are characterized by contacts to the aromatic box formed primarily by residues on the principal side of the intersubunit binding interface (residues Tyr89, Trp143 and Tyr185). Besides these interactions on the principal side, we observe a cation-π interaction between acetylcholine and Trp53 on the complementary side and a water-mediated hydrogen bond from acetylcholine to backbone atoms of Leu102 and Met114, both of importance for anchoring acetylcholine to the complementary side. To further study the role of Trp53, we mutated the corresponding tryptophan in the two different acetylcholine-binding interfaces of the widespread α4β2 nAChR, i.e. the interfaces α4(+)β2(-) and α4(+)α4(-). Mutation to alanine (W82A on the β2 subunit or W88A on the α4 subunit) significantly altered the response to acetylcholine measured by oocyte voltage-clamp electrophysiology in both interfaces. This shows that the conserved tryptophan residue is important for the effects of ACh at α4β2 nAChRs, as also indicated by the crystal structure. The results add important details to the understanding of how this neurotransmitter exerts its action and improves the foundation for rational drug design targeting these receptors.

  2. Anionic Lipids Allosterically Modulate Multiple Nicotinic Acetylcholine Receptor Conformational Equilibria*

    PubMed Central

    daCosta, Corrie J. B.; Medaglia, Sarah A.; Lavigne, Nadine; Wang, Shuzhi; Carswell, Casey L.; Baenziger, John E.

    2009-01-01

    Anionic lipids influence the ability of the nicotinic acetylcholine receptor to gate open in response to neurotransmitter binding, but the underlying mechanisms are poorly understood. We show here that anionic lipids with relatively small headgroups, and thus the greatest ability to influence lipid packing/bilayer physical properties, are the most effective at stabilizing an agonist-activatable receptor. The differing abilities of anionic lipids to stabilize an activatable receptor stem from differing abilities to preferentially favor resting over both uncoupled and desensitized conformations. Anionic lipids thus modulate multiple acetylcholine receptor conformational equilibria. Our data suggest that both lipids and membrane physical properties act as classic allosteric modulators influencing function by interacting with and thus preferentially stabilizing different native acetylcholine receptor conformational states. PMID:19815550

  3. A computational model of the nicotinic acetylcholine binding site

    NASA Astrophysics Data System (ADS)

    Gálvez-ruano, Enrique; Iriepa-Canalda, Isabel; Morreale, Antonio; Lipkowitz, Kenny B.

    1999-01-01

    We have derived a model of the nicotinic acetylcholine binding site. This was accomplished by using three known agonists (acetylcholine, nicotine and epibatidine) as templates around which polypeptide side chains, found to be part of the receptor cavity from published molecular biology studies, are allowed to flow freely in molecular dynamics simulations and mold themselves around these templates. The resulting supramolecular complex should thus be a complement, both in terms of steric effects as well as electronic effects, to the agonists and it should be a good estimation of the true receptor cavity structure. The shapes of those minireceptor cavities equilibrated rapidly on the simulation time scale and their structural congruence is very high, implying that a satisfactory model of the nicotinic acetylcholine binding site has been achieved. The computational methodology was internally tested against two rigid and specific antagonists (dihydro-β-erytroidine and erysoidine), that are expected to give rise to a somewhat differently shaped binding site compared to that derived from the agonists. Using these antagonists as templates there were structural reorganizations of the initial receptor cavities leading to distinctly different cavities compared to agonists. This indicates that adequate times and temperatures were used in our computational protocols to achieve equilibrium structures for the agonists. Overall, both minireceptor geometries for agonists and antagonists are similar with the exception of one amino acid (ARG209).

  4. Acetylcholine excites neocortical pyramidal neurons via nicotinic receptors.

    PubMed

    Hedrick, Tristan; Waters, Jack

    2015-04-01

    The neuromodulator acetylcholine (ACh) shapes neocortical function during sensory perception, motor control, arousal, attention, learning, and memory. Here we investigate the mechanisms by which ACh affects neocortical pyramidal neurons in adult mice. Stimulation of cholinergic axons activated muscarinic and nicotinic ACh receptors on pyramidal neurons in all cortical layers and in multiple cortical areas. Nicotinic receptor activation evoked short-latency, depolarizing postsynaptic potentials (PSPs) in many pyramidal neurons. Nicotinic receptor-mediated PSPs promoted spiking of pyramidal neurons. The duration of the increase in spiking was membrane potential dependent, with nicotinic receptor activation triggering persistent spiking lasting many seconds in neurons close to threshold. Persistent spiking was blocked by intracellular BAPTA, indicating that nicotinic ACh receptor activation evoked persistent spiking via a long-lasting calcium-activated depolarizing current. We compared nicotinic PSPs in primary motor cortex (M1), prefrontal cortex (PFC), and visual cortex. The laminar pattern of nicotinic excitation was not uniform but was broadly similar across areas, with stronger modulation in deep than superficial layers. Superimposed on this broad pattern were local differences, with nicotinic PSPs being particularly large and common in layer 5 of M1 but not layer 5 of PFC or primary visual cortex (V1). Hence, in addition to modulating the excitability of pyramidal neurons in all layers via muscarinic receptors, synaptically released ACh preferentially increases the activity of deep-layer neocortical pyramidal neurons via nicotinic receptors, thereby adding laminar selectivity to the widespread enhancement of excitability mediated by muscarinic ACh receptors. Copyright © 2015 the American Physiological Society.

  5. Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced Actin Cytoskeletal Remodeling and Extracellular Matrix Degradation by Vascular Smooth Muscle Cells

    PubMed Central

    Gu, Zhizhan; Fonseca, Vera; Hai, Chi-Ming

    2012-01-01

    Cigarette smoking is a significant risk factor for atherosclerosis, which involves the invasion of vascular smooth muscle cells (VSMCs) from the media to intima. A hallmark of many invasive cells is actin cytoskeletal remodeling in the form of podosomes, accompanied by extracellular matrix (ECM) degradation. A7r5 VSMCs form podosomes in response to PKC activation. In this study, we found that cigarette smoke extract, nicotine, and the cholinergic agonist, carbachol, were similarly effective in inducing the formation of podosome rosettes in A7r5 VSMCs. α-Bungarotoxin and atropine experiments confirmed the involvement of nicotinic acetylcholine receptors (nAChRs). Western blotting and immunofluorescence experiments revealed the aggregation of nAChRs at podosome rosettes. Cycloheximide experiments and media exchange experiments suggested that autocrine factor(s) and intracellular phenotypic modulation are putative mechanisms. In situ zymography experiments indicated that, in response to PKC activation, nicotine-treated cells degraded ECM near podosome rosettes, and possibly endocytose ECM fragments to intracellular compartments. Invasion assay of human aortic smooth muscle cells indicated that nicotine and PKC activation individually and synergistically enhanced cell invasion through ECM. Results from this study suggest that nicotine enhances the ability of VSMCs to degrade and invade ECM. nAChR activation, actin cytoskeletal remodeling and phenotypic modulation are possible mechanisms. PMID:22940282

  6. Ethanol-nicotine interactions at alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors in rat cortical neurons.

    PubMed

    Marszalec, W; Aistrup, G L; Narahashi, T

    1999-03-01

    Numerous studies have indicated a correlation between ethanol intake and cigarette smoking in heavy drinkers. We have studied the underlying pharmacological basis of this relationship using cultured rat cortical neurons. These neurons express nicotinic receptors having characteristics similar to those described for the alpha4beta2 subunit combination. In the presence of alpha-bungarotoxin both acetylcholine (ACh) and nicotine evoked currents with respective EC50 values of 4.3 and 3.4 microM. The maximal nicotine-activated response, however, was only 56% that of the maximal ACh current. It was previously shown that 10 to 100 mM of ethanol potentiated ACh-mediated currents in these neurons. We demonstrate that 100 mM ethanol similarly potentiates currents evoked by 300 nM (40%) and 1 microM nicotine 61%). This suggests that an ethanol-induced potentiation of nicotinic currents may enhance the acute positive reinforcement associated with nicotine and could increase tobacco use during heavy ethanol intake. However, further experimentation indicated that the continuous perfusion of 30, 100, or 300 nM nicotine desensitizes ACh-evoked currents by 38, 54, and 62%, respectively, with little direct receptor-channel activation. The residual ACh currents of nicotine-desensitized receptor channels were potentiated by 100 mM ethanol to nearly the extent as were the undesensitized control responses. We propose that the opposing effect of ethanol on nicotine-induced desensitization could also explain the increased tobacco use observed with excessive drinking. Thus, ethanol has a dual effect regarding nicotine. It enhances acute nicotine-mediated receptor activation, although opposing the net effect of nicotine-induced receptor channel desensitization.

  7. 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.

  8. Nicotine promotes cell migration through alpha7 nicotinic acetylcholine receptor in gastric cancer cells.

    PubMed

    Lien, Yung-Chang; Wang, Weu; Kuo, Li-Jen; Liu, Jun-Jen; Wei, Po-Li; Ho, Yuan-Soon; Ting, Wen-Chien; Wu, Chih-Hsiung; Chang, Yu-Jia

    2011-09-01

    The objective was to study the mechanism of nicotine-enhanced migration of gastric cancer cells. Long-term cigarette smoking increases the risk of gastric cancer mortality. Tobacco-specific mitogen, nicotine, was reported to correlate with cancer progression on gastric cancer. Since metastasis is the major cause of cancer death, the influence of nicotine on the migration of gastric cancer cells remains to be determined. The influence of nicotine on migration of gastric cancer cells was evaluated by transwell assay and wound-healing migration assay. Receptor-mediated migration was studied by both inhibitor and small interfering RNA. Alpha7 nicotinic acetylcholine receptor, alpha7-nAChR, was identified in gastric cancer cell lines, AGS cells. Nicotine enhanced AGS cell migration in transwell assay and wound-healing migration assay in a dose-dependent manner. We used inhibitor and siRNA to demonstrate that alpha7-nAChR mediated nicotine-enhanced gastric cancer cell migration through downregulation E-cadherin and upregulation ZEB-1 and snail. Tobacco-specific mitogen, nicotine, enhanced gastric cancer metastasis through alpha7-nAChR and suppression of E-cadherin level-one of the hallmarks of epithelial to mesenchymal transition. Therefore, patients with gastric cancer should avoid smoking.

  9. Effects of antihistamines on the function of human α7-nicotinic acetylcholine receptors.

    PubMed

    Sadek, Bassem; Khanian, Seyedeh Soha; Ashoor, Abrar; Prytkova, Tatiana; Ghattas, Mohammad A; Atatreh, Noor; Nurulain, Syed M; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Oz, Murat

    2015-01-05

    Effects of the histamine H₁ receptor (H1R) antagonists (antihistamines), promethazine (PMZ), orphenadrine (ORP), chlorpheniramine (CLP), pyrilamine (PYR), diphenhydramine (DPH), citerizine (CTZ), and triprolidine (TRP) on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes were investigated. Antihistamines inhibited the α7-nicotinic acetylcholine receptor in the order PYR>CLP>TRP>PMZ>ORP≥DPH≥CTZ. Among the antihistamines, PYR showed the highest reversible inhibition of acetylcholine (100 µM)-induced responses with IC₅₀ of 6.2 µM. PYR-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. Specific binding of [¹²⁵I] α-bungarotoxin, a selective antagonist for α7-nicotinic acetylcholine receptor, was not changed in the presence of PYR suggesting a non-competitive inhibition of nicotinic receptors. In line with functional experiments, docking studies indicated that PYR can potentially bind allosterically with the α7 transmembrane domain. Our results indicate that the H₂-H₄ receptor antagonists tested in this study (10 µM) showed negligible inhibition of α7-nicotinic acetylcholine receptors. On the other hand, H₁ receptor antagonists inhibited the function of human α7-nicotinic acetylcholine receptor, with varying potencies. These results emphasize the importance of α7-nicotinic acetylcholine receptor for future pharmacological/toxicological profiling. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex

    PubMed Central

    Beiranvand, F; Zlabinger, C; Orr-Urtreger, A; Ristl, R; Huck, S; Scholze, P

    2014-01-01

    Background and purpose Nicotinic acetylcholine receptors (nACh receptors) play a central role in the habenulo-interpeduncular system. We studied nicotine-induced release of NA and ACh in the habenula and interpeduncular nucleus (IPN). Experimental approach The habenula and IPN were loaded with [3H]-choline or [3H]-NA and placed in superfusion chambers. [3H]-ACh release was also stimulated using nicotinic agonists, electrical pulses and elevated [KCl]o in hippocampal and cortical slices from rats, wild-type mice and mice lacking α5, α7, β2, or β4 nACh receptor subunits. Finally, we analysed nACh receptor subtypes in the IPN using immunoprecipitation. Key results Nicotine induced release of [3H]-ACh in the IPN of rats and mice. This release was calcium-dependent but not blocked by tetrodotoxin (TTX); moreover, [3H]-ACh release was abolished in β4-knockout mice but was unaffected in β2- and α5-knockout mice. In contrast, nicotine-induced release of [3H]-NA in the IPN and habenula was blocked by TTX and reduced in both β2-knockout and β4-knockout mice, and dose–response curves were right-shifted in α5-knockout mice. Although electrical stimuli triggered the release of both transmitters, [3H]-ACh release required more pulses delivered at a higher frequency. Conclusions and implications Our results confirm previous findings that β4-containing nACh receptors are critical for [3H]-ACh release in the mouse IPN. Experiments using α5-knockout mice also revealed that unlike in the hippocampus, nicotine-induced [3H]-NA release in the habenulo-interpeduncular system is altered in this knockout model. As α5-containing nACh receptors play a key role in nicotine intake, our results add NA to the list of transmitters involved in this mechanism. PMID:25041479

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

    SciTech Connect

    Soeda, Junpei; Morgan, Maelle; McKee, Chad

    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 inmore » 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

  13. Nicotinic acetylcholine receptor alpha7 subunit mediates migration of vascular smooth muscle cells toward nicotine.

    PubMed

    Li, Sheng; Zhao, Tiejun; Xin, Hong; Ye, Li-Hong; Zhang, Xiaodong; Tanaka, Hideyuki; Nakamura, Akio; Kohama, Kazuhiro

    2004-03-01

    GbaSM-4 cells, vascular smooth muscle cells (VSMCs) derived from brain basilar arteries, were shown to migrate toward d-nicotine by augmenting the actin cytoskeleton in their cell bodies and lamellipodia, and expression of nicotinic acetylcholine receptor (alpha7-nAChR) was detected in GbaSM-4 cells. Their chemotaxis was antagonized by an alpha7-nAChR antagonist of methyllycaconitine. It was also antagonized by inhibiting myosin light chain (MLC) kinase and by down-regulating MLC kinase. However, the changes in MLC phosphorylation were not associated with the nicotine treatment, suggesting the involvement of non-kinase activity of MLC kinase as reviewed by Gao et al. (IUBMB Life. 2001;51:337). This plot may work to induce arteriosclerosis during cigarette smoking.

  14. Phospholipase C activity affinity purifies with the Torpedo nicotinic acetylcholine receptor.

    PubMed

    Labriola, Jonathan M; daCosta, Corrie J B; Wang, Shuzhi; Figeys, Daniel; Smith, Jeffrey C; Sturgeon, R Michel; Baenziger, John E

    2010-04-02

    Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function.

  15. Developmental regulation of nicotinic acetylcholine receptors within midbrain dopamine neurons

    PubMed Central

    Azam, Layla; Chen, Yiling; Leslie, Frances M.

    2007-01-01

    We have combined anatomical and functional methodologies to provide a comprehensive analysis of the properties of nicotinic acetylcholine receptors (nAChRs) on developing dopamine (DA) neurons. Double-labeling in situ hybridization was used to examine the expression of nAChR subunit mRNAs within developing midbrain DA neurons. As brain maturation progressed there was a change in the pattern of subunit mRNA expression within DA neurons, such that α3 and α4 subunits declined and α6 mRNA increased. Although there were strong similarities in subunit mRNA expression in substantia nigra (SNc) and ventral tegmental area (VTA), there was higher expression of α4 mRNA in SNc than VTA at gestational day (G)15, and of α5, α6 and β3 mRNAs during postnatal development. Using a superfusion neurotransmitter release paradigm to functionally characterize nicotine-stimulated release of [3H]DA from striatal slices, the properties of the nAChRs on DA terminals were also found to change with age. Functional nAChRs were detected on striatal terminals at G18. There was a decrease in maximal release in the first postnatal week, followed by an increase in nicotine efficacy and potency during the second and third postnatal weeks. In the transition from adolescence (postnatal days (P) 30 and 40) to adulthood, there was a complex pattern of functional maturation of nAChRs in ventral, but not dorsal, striatum. In males, but not females, there were significant changes in both nicotine potency and efficacy during this developmental period. These findings suggest that nAChRs may play critical functional roles throughout DA neuronal maturation. PMID:17197101

  16. Nicotinic acetylcholine receptor expression by directionally selective ganglion cells.

    PubMed

    Strang, Christianne E; Renna, Jordan M; Amthor, Franklin R; Keyser, Kent T

    2007-01-01

    Acetylcholine (ACh) enhances the preferred direction responses of directionally selective ganglion cells (DS GCs; Ariel & Daw, 1982; Ariel & Adolph, 1985) through the activation of nicotinic acetylcholine receptors (nAChRs; Ariel & Daw, 1982; Massey et al., 1997; Kittila & Massey, 1997). DS GCs appear to express at least two types of nAChRs, those that are sensitive to the partially subtype-specific antagonist methyllycaconitine (MLA), and those that are MLA-insensitive (Reed et al., 2002). Our purpose was to confirm the expression of alpha7 nAChRs by DS GCs and to assess the contributions of other nAChR subtypes to DS GC responses. Using choline as a nAChR partially subtype-specific agonist, we found that the majority of DS GCs demonstrated responses to choline while under synaptic blockade. The blockade or reduction of choline-induced responses by bath application of nanomolar (nM) concentrations of MLA provided direct evidence that the choline responses were mediated by alpha7 nAChRs. Because choline is a partial agonist for alpha3beta4 nAChRs (Alkondon et al., 1997), the residual choline responses are consistent with mediation by alpha3beta4 nAChRs. Additionally, a subset of DS GCs responded to nicotine but not to choline, indicating the expression of a third nAChR subtype. The pharmacological results were supported by single cell reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry experiments. The expression of alpha7 and specific non-alpha7 nAChR subtypes was correlated with the preferred direction. This indicates the possibility of differential responses to ACh depending on the direction of movement. This is the first description of differential expression of multiple nAChR subtypes by DS GCs.

  17. Fluorescent agonists for the Torpedo nicotinic acetylcholine receptor.

    PubMed

    Krieger, Florian; Mourot, Alexandre; Araoz, Romulo; Kotzyba-Hibert, Florence; Molgó, Jordi; Bamberg, Ernst; Goeldner, Maurice

    2008-05-05

    We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC-Gly-C6-choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl- and NBD-acylcholine analogues. Binding of DEAC-Gly-C6-choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the alphadelta-binding site and 15.0 nM for the alphagamma-binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug-screening studies.

  18. Caenorhabditis elegans nicotinic acetylcholine receptors are required for nociception.

    PubMed

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

    2014-03-01

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. 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

  20. Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex.

    PubMed

    Beiranvand, F; Zlabinger, C; Orr-Urtreger, A; Ristl, R; Huck, S; Scholze, P

    2014-12-01

    Nicotinic acetylcholine receptors (nACh receptors) play a central role in the habenulo-interpeduncular system. We studied nicotine-induced release of NA and ACh in the habenula and interpeduncular nucleus (IPN). The habenula and IPN were loaded with [(3) H]-choline or [(3) H]-NA and placed in superfusion chambers. [(3) H]-ACh release was also stimulated using nicotinic agonists, electrical pulses and elevated [KCl]o in hippocampal and cortical slices from rats, wild-type mice and mice lacking α5, α7, β2, or β4 nACh receptor subunits. Finally, we analysed nACh receptor subtypes in the IPN using immunoprecipitation. Nicotine induced release of [(3) H]-ACh in the IPN of rats and mice. This release was calcium-dependent but not blocked by tetrodotoxin (TTX); moreover, [(3) H]-ACh release was abolished in β4-knockout mice but was unaffected in β2- and α5-knockout mice. In contrast, nicotine-induced release of [(3) H]-NA in the IPN and habenula was blocked by TTX and reduced in both β2-knockout and β4-knockout mice, and dose-response curves were right-shifted in α5-knockout mice. Although electrical stimuli triggered the release of both transmitters, [(3) H]-ACh release required more pulses delivered at a higher frequency. Our results confirm previous findings that β4-containing nACh receptors are critical for [(3) H]-ACh release in the mouse IPN. Experiments using α5-knockout mice also revealed that unlike in the hippocampus, nicotine-induced [(3) H]-NA release in the habenulo-interpeduncular system is altered in this knockout model. As α5-containing nACh receptors play a key role in nicotine intake, our results add NA to the list of transmitters involved in this mechanism. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

  1. Galantamine, an Acetylcholinesterase Inhibitor and Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors, Attenuates Nicotine Taking and Seeking in Rats

    PubMed Central

    Hopkins, Thomas J; Rupprecht, Laura E; Hayes, Matthew R; Blendy, Julie A; Schmidt, Heath D

    2012-01-01

    Current smoking cessation pharmacotherapies have limited efficacy in preventing relapse and maintaining abstinence during withdrawal. Galantamine is an acetylcholinesterase inhibitor that also acts as a positive allosteric modulator of nicotinic acetylcholine receptors. Galantamine has recently been shown to reverse nicotine withdrawal-induced cognitive impairments in mice, which suggests that galantamine may function to prevent relapse in human smokers. However, there are no studies examining whether galantamine administration modulates nicotine self-administration and/or reinstatement of nicotine seeking in rodents. The present experiments were designed to determine the effects of galantamine administration on nicotine taking and reinstatement of nicotine-seeking behavior, an animal model of relapse. Moreover, the effects of galantamine on sucrose-maintained responding and sucrose seeking were also examined to determine whether galantamine's effects generalized to other reinforced behaviors. An inverted U-shaped dose-response curve was obtained when animals self-administered different unit doses of nicotine with the highest responding for 0.03 mg/kg per infusion of nicotine. Acute galantamine administration (5.0 mg/kg, i.p.) attenuated nicotine self-administration when animals were maintained on either a fixed-ratio 5 (FR5) or progressive ratio (PR) schedule of reinforcement. Galantamine administration also attenuated the reinstatement of nicotine-seeking behavior. No significant effects of galantamine on sucrose self-administration or sucrose reinstatement were noted. Acetylcholinesterase inhibitors have also been shown to produce nausea and vomiting in humans. However, at doses required to attenuate nicotine self-administration, no effects of galantamine on nausea/malaise as measured by pica were noted. These results indicate that increased extracellular acetylcholine levels and/or nicotinic acetylcholine receptor stimulation is sufficient to attenuate

  2. Subtypes of nicotinic acetylcholine receptors in nicotine reward, dependence, and withdrawal: Evidence from genetically modified mice

    PubMed Central

    Fowler, Christie D.; Arends, Michael A.; Kenny, Paul J.

    2009-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) can regulate the activity of many neurotransmitter pathways throughout the central nervous system and are considered important modulators of cognition and emotion. nAChRs also are the primary site of action in the brain for nicotine, the major addictive component of tobacco smoke. nAChRs consist of five membrane-spanning subunits (α and β isoforms) that can associate in various combinations to form functional nAChR ion channels. Because of a dearth of nAChR subtype-selective ligands, the precise subunit composition of the nAChRs that regulate the rewarding effects of nicotine and the development of nicotine dependence are unknown. However, the advent of mice with genetic nAChR subunit modifications has provided a useful experimental approach to assess the contribution of individual subunits in vivo. Here we review data generated from nAChR subunit knockout and genetically modified mice supporting a role for discrete nAChR subunits in nicotine reinforcement and dependence processes. Importantly, the rates of tobacco dependence are far higher in patients suffering from comorbid psychiatric illnesses compared with the general population, which may at least partly reflect disease-associated alterations in nAChR signaling. An understanding of the role of nAChRs in psychiatric disorders associated with high rates of tobacco addiction, therefore, may reveal novel insights into mechanisms of nicotine dependence. Thus, we also briefly review data generated from genetically modified mice to support a role for discrete nAChR subunits in anxiety disorders, depression, and schizophrenia. PMID:18690103

  3. Nicotine facilitates nicotinic acetylcholine receptor targeting to mitochondria but makes them less susceptible to selective ligands.

    PubMed

    Uspenska, Kateryna; Lykhmus, Olena; Gergalova, Galyna; Chernyshov, Volodymyr; Arias, Hugo R; Komisarenko, Sergiy; Skok, Maryna

    2017-08-24

    Several nicotinic acetylcholine receptor (nAChR) subtypes are expressed in mitochondria to regulate the internal pathway of apoptosis in ion channel-independent manner. However, the mechanisms of nAChR activation in mitochondria and targeting to mitochondria are still unknown. Nicotine has been shown to favor nAChR pentamer assembly, folding, and maturation on the way of biosynthesis. The idea of the present work was to determine whether nicotine affects the content, glycosylation, and function of mitochondrial nAChRs. Experiments were performed in isolated liver mitochondria from mice, that either consumed or not nicotine with the drinking water (200μL/L) for 7days. Mitochondria detergent lysates were studied by sandwich or lectin ELISA for the presence and carbohydrate composition of different nAChR subunits. Intact mitochondria were examined by flow cytometry for the binding of fluorescently labeled α-cobratoxin and were tested in functional assay of cytochrome c release under the effect of either Ca 2+ or wortmannin in the presence or absence of nAChR-selective ligands, including PNU-282987 (1nM), dihydro-β-erythroidine (DhβE, 1μM), PNU-120596 (0.3, 3, or 10μM) and desformylflustrabromine hydrochloride (dFBr, 0.001, 0.3, or 1μM). It was found that nicotine consumption increased the ratio of mitochondrial vs non-mitochondrial nAChRs in the liver, enhanced fucosylation of mitochondrial nAChRs, but prevented the binding of α-cobratoxin and the cytochrome c release-attenuating effects of nAChR-specific agonists, antagonists, or positive allosteric modulators. It is concluded that nicotine consumption in vivo favors nAChR glycosylation and trafficking to mitochondria but makes them less susceptible to the effects of specific ligands. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Acetylcholine promotes binding of α-conotoxin MII at α3 β2 nicotinic acetylcholine receptors.

    PubMed

    Sambasivarao, Somisetti V; Roberts, Jessica; Bharadwaj, Vivek S; Slingsby, Jason G; Rohleder, Conrad; Mallory, Chris; Groome, James R; McDougal, Owen M; Maupin, C Mark

    2014-02-10

    α-Conotoxin MII (α-CTxMII) is a 16-residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2-Cys8 and Cys3-Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand-binding affinity, homology models of the heteropentameric α3β2-nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3- and β2-subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3β2-nAChR for α-CTxMII with ACh bound to the receptor, and this was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. a2* Nicotinic Acetylcholine Receptors Influence Hippocampus-Dependent Learning and Memory in Adolescent Mice

    ERIC Educational Resources Information Center

    Lotfipour, Shahrdad; Mojica, Celina; Nakauchi, Sakura; Lipovsek, Marcela; Silverstein, Sarah; Cushman, Jesse; Tirtorahardjo, James; Poulos, Andrew; Elgoyhen, Ana Belén; Sumikawa, Katumi; Fanselow, Michael S.; Boulter, Jim

    2017-01-01

    The absence of a2* nicotinic acetylcholine receptors (nAChRs) in oriens lacunosum moleculare (OLM) GABAergic interneurons ablate the facilitation of nicotine-induced hippocampal CA1 long-term potentiation and impair memory. The current study delineated whether genetic mutations of a2* nAChRs ("Chrna2"[superscript L9'S/L9'S] and…

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

    PubMed

    Azam, Layla; McIntosh, J Michael

    2009-06-01

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

  7. Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

    PubMed

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

    2013-01-01

    Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca(2+)-dependent Cl(-) channels, since menthol inhibition remained unchanged by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing Ba(2+). Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [(125)I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca(2+) transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner.

  8. Menthol Binding and Inhibition of α7-Nicotinic Acetylcholine Receptors

    PubMed Central

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

    2013-01-01

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

  9. Plant toxins that affect nicotinic acetylcholine receptors: a review.

    PubMed

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

    2013-08-19

    Plants produce a wide variety of chemical compounds termed secondary metabolites that are not involved in basic metabolism, photosynthesis, or reproduction. These compounds are used as flavors, fragrances, insecticides, dyes, hallucinogens, nutritional supplements, poisons, and pharmaceutical agents. However, in some cases these secondary metabolites found in poisonous plants perturb biological systems. Ingestion of toxins from poisonous plants by grazing livestock often results in large economic losses to the livestock industry. The chemical structures of these compounds are diverse and range from simple, low molecular weight toxins such as oxalate in halogeton to the highly complex norditerpene alkaloids in larkspurs. While the negative effects of plant toxins on people and the impact of plant toxins on livestock producers have been widely publicized, the diversity of these toxins and their potential as new pharmaceutical agents for the treatment of diseases in people and animals has also received widespread interest. Scientists are actively screening plants from all regions of the world for bioactivity and potential pharmaceuticals for the treatment or prevention of many diseases. In this review, we focus the discussion to those plant toxins extensively studied at the USDA Poisonous Plant Research Laboratory that affect the nicotinic acetylcholine receptors including species of Delphinium (Larkspurs), Lupinus (Lupines), Conium (poison hemlock), and Nicotiana (tobaccos).

  10. Nicotine at clinically relevant concentrations affects atrial inward rectifier potassium current sensitive to acetylcholine.

    PubMed

    Bébarová, Markéta; Matejovič, Peter; Švecová, Olga; Kula, Roman; Šimurdová, Milena; Šimurda, Jiří

    2017-05-01

    Nicotine abuse is associated with variety of diseases including arrhythmias, most often atrial fibrillation (AF). Altered inward rectifier potassium currents including acetylcholine-sensitive current I K(Ach) are known to be related to AF pathogenesis. Since relevant data are missing, we aimed to investigate I K(Ach) changes at clinically relevant concentrations of nicotine. Experiments were performed by the whole cell patch clamp technique at 23 ± 1 °C on isolated rat atrial myocytes. Nicotine was applied at following concentrations: 4, 40 and 400 nM; ethanol at 20 mM (∼0.09%). Nicotine at 40 and 400 nM significantly activated constitutively active component of I K(Ach) with the maximum effect at 40 nM (an increase by ∼100%); similar effect was observed at -110 and -50 mV. Changes at 4 nM nicotine were negligible on average. Coapplication of 40 nM nicotine and 20 mM ethanol (which is also known to activate this current) did not show cumulative effect. In the case of acetylcholine-induced component of I K(Ach) , a dual effect of nicotine and its correlation with the current magnitude in control were apparent: the current was increased by nicotine in the cells showing small current in control and vice versa. The effect of 40 and 400 nM nicotine on acetylcholine-induced component of I K(Ach) was significantly different at -110 and -50 mV. We conclude that nicotine at clinically relevant concentrations significantly increased constitutively active component of I K(Ach) and showed a dual effect on its acetylcholine-induced component, similarly as ethanol. Synchronous application of nicotine and ethanol did not cause additive effect.

  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. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  12. Acetylcholine elongates neuronal growth cone filopodia via activation of nicotinic acetylcholine receptors.

    PubMed

    Zhong, Lei Ray; Estes, Stephen; Artinian, Liana; Rehder, Vincent

    2013-07-01

    In addition to acting as a classical neurotransmitter in synaptic transmission, acetylcholine (ACh) has been shown to play a role in axonal growth and growth cone guidance. What is not well understood is how ACh acts on growth cones to affect growth cone filopodia, structures known to be important for neuronal pathfinding. We addressed this question using an identified neuron (B5) from the buccal ganglion of the pond snail Helisoma trivolvis in cell culture. ACh treatment caused pronounced filopodial elongation within minutes, an effect that required calcium influx and resulted in the elevation of the intracellular calcium concentration ([Ca]i ). Whole-cell patch clamp recordings showed that ACh caused a reduction in input resistance, a depolarization of the membrane potential, and an increase in firing frequency in B5 neurons. These effects were mediated via the activation of nicotinic acetylcholine receptors (nAChRs), as the nAChR agonist dimethylphenylpiperazinium (DMPP) mimicked the effects of ACh on filopodial elongation, [Ca]i elevation, and changes in electrical activity. Moreover, the nAChR antagonist tubucurarine blocked all DMPP-induced effects. Lastly, ACh acted locally at the growth cone, because growth cones that were physically isolated from their parent neuron responded to ACh by filopodial elongation with a similar time course as growth cones that remained connected to their parent neuron. Our data revealed a critical role for ACh as a modulator of growth cone filopodial dynamics. ACh signaling was mediated via nAChRs and resulted in Ca influx, which, in turn, caused filopodial elongation. Copyright © 2013 Wiley Periodicals, Inc.

  13. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies.

    PubMed

    Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen

    2017-01-18

    Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    SciTech Connect

    Middleton, R.E.; Cohen, J.B.

    1991-07-16

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

  15. A Multi-Route Model of Nicotine-Cotinine Pharmacokinetics, Pharmacodynamics and Brain Nicotinic Acetylcholine Receptor Binding in Humans

    SciTech Connect

    Teeguarden, Justin G.; Housand, Conrad; Smith, Jordan N.

    2013-02-01

    The pharmacokinetics of nicotine, the pharmacologically active alkaloid in tobacco responsible for addiction, are well characterized in humans. We developed a physiologically based pharmacokinetic/pharmacodynamic model of nicotine pharmacokinetics, brain dosimetry and brain nicotinic acetylcholine receptor (nAChRs) occupancy. A Bayesian framework was applied to optimize model parameters against multiple human data sets. The resulting model was consistent with both calibration and test data sets, but in general underestimated variability. A pharmacodynamic model relating nicotine levels to increases in heart rate as a proxy for the pharmacological effects of nicotine accurately described the nicotine related changes in heart rate and the developmentmore » and decay of tolerance to nicotine. The PBPK model was utilized to quantitatively capture the combined impact of variation in physiological and metabolic parameters, nicotine availability and smoking compensation on the change in number of cigarettes smoked and toxicant exposure in a population of 10,000 people presented with a reduced toxicant (50%), reduced nicotine (50%) cigarette Across the population, toxicant exposure is reduced in some but not all smokers. Reductions are not in proportion to reductions in toxicant yields, largely due to partial compensation in response to reduced nicotine yields. This framework can be used as a key element of a dosimetry-driven risk assessment strategy for cigarette smoke constituents.« less

  16. Nicotinic acetylcholine receptor inhibitors derived from snake and snail venoms.

    PubMed

    Dutertre, Sébastien; Nicke, Annette; Tsetlin, Victor I

    2017-12-01

    The nicotinic acetylcholine receptor (nAChR) represents the prototype of ligand-gated ion channels. It is vital for neuromuscular transmission and an important regulator of neurotransmission. A variety of toxic compounds derived from diverse species target this receptor and have been of elemental importance in basic and applied research. They enabled milestone discoveries in pharmacology and biochemistry ranging from the original formulation of the receptor concept, the first isolation and structural analysis of a receptor protein (the nAChR) to the identification, localization, and differentiation of its diverse subtypes and their validation as a target for therapeutic intervention. Among the venom-derived compounds, α-neurotoxins and α-conotoxins provide the largest families and still represent indispensable pharmacological tools. Application of modified α-neurotoxins provided substantial structural and functional details of the nAChR long before high resolution structures were available. α-bungarotoxin represents not only a standard pharmacological tool and label in nAChR research but also for unrelated proteins tagged with a minimal α-bungarotoxin binding motif. A major advantage of α-conotoxins is their smaller size, as well as superior selectivity for diverse nAChR subtypes that allows their development into ligands with optimized pharmacological and chemical properties and potentially novel drugs. In the following, these two groups of nAChR antagonists will be described focusing on their respective roles in the structural and functional characterization of nAChRs and their development into research tools. In addition, we provide a comparative overview of the diverse α-conotoxin selectivities that can serve as a practical guide for both structure activity studies and subtype classification. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.' Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Circulating antibodies against nicotinic acetylcholine receptors in chagasic patients

    PubMed Central

    GOIN, J C; VENERA, G; BONINO, M BISCOGLIO DE JIMÉNEZ; STERIN-BORDA, L

    1997-01-01

    Human and experimental Chagas' disease causes peripheral nervous system damage involving neuromuscular transmission alterations at the neuromuscular junction. Additionally, autoantibodies directed to peripheral nerves and sarcolemmal proteins of skeletal muscle have been described. In this work, we analyse the ability of serum immunoglobulin factors associated with human chagasic infection to bind the affinity-purified nicotinic acetylcholine receptor (nAChR) from electric organs of Discopyge tschudii and to identify the receptor subunits involved in the interaction. The frequency of serum anti-nAChR reactivity assayed by dot-blot was higher in seropositive chagasic patients than in uninfected subjects. Purified IgG obtained from chagasic patients immunoprecipitated a significantly higher fraction of the solubilized nAChR than normal IgG. Furthermore, immunoblotting assays indicated that α and β are the main subunits involved in the interaction. Chagasic IgG was able to inhibit the binding of α-bungarotoxin to the receptor in a concentration-dependent manner, confirming the contribution of the α-subunit in the autoantibody-receptor interaction. The presence of anti-nAChR antibodies was detected in 73% of chagasic patients with impairment of neuromuscular transmission in conventional electromyographical studies, indicating a strong association between seropositive reactivity against nAChR and electromyographical abnormalities in chagasic patients. The chronic binding of these autoantibodies to the nAChR could induce a decrease in the population of functional nAChRs at the neuromuscular junction and consequently contribute to the electrophysiological neuromuscular alterations described in the course of chronic Chagas' disease. PMID:9367405

  18. Nicotinic acetylcholine receptors are sensors for ethanol in lung fibroblasts.

    PubMed

    Ritzenthaler, Jeffrey D; Roser-Page, Susanne; Guidot, David M; Roman, Jesse

    2013-06-01

    Chronic ethanol (EtOH) abuse in humans is known to independently increase the incidence of and mortality due to acute lung injury in at-risk individuals. However, the mechanisms by which EtOH affects lung cells remain incompletely elucidated. In earlier work, we reported that EtOH increased the expression in lung fibroblasts of fibronectin, a matrix glycoprotein implicated in lung injury and repair. This effect was blocked by α-bungarotoxin, a neurotoxin that binds certain nicotinic acetylcholine receptors (nAChRs) thereby implicating nAChRs in this process. Here, we examine the identity of these receptors. Mouse lung fibroblasts were stimulated with EtOH (60 mM) or acetylcholine (100 to 500 μM) and evaluated for the expression of fibronectin and nAChRs. Inhibitors to nAChRs or the antioxidant N-acetyl cysteine (NAC) were used to assess changes in fibronectin expression. Animals exposed to EtOH for up to 6 weeks were used to evaluate the expression of nAChRs in vivo. First, in EtOH-treated fibroblasts, we observed increased expression of α4 and α9 nAChR subunits. Second, we found that acetylcholine, a natural ligand for nAChRs, mimicked the effects of EtOH. Dihydro-β-erythroidin hydrobromide, a competitive inhibitor of α4 nAChR, blocked the increase in fibronectin expression and cell proliferation. Furthermore, EtOH-induced fibronectin expression was inhibited in cells silenced for α4 nAChR. However, EtOH-treated cells showed increased α-bungarotoxin binding suggesting that α4 nAChR mediates the effects of EtOH via a ligand-independent pathway. Knowing there are several important cysteine residues near the ligand-binding site of α4 nAChRs, we tested the antioxidant NAC and found that it too blocked the induction of fibronectin expression by EtOH. Also, fibroblasts exposed to oxidant stress showed increased fibronectin expression that was blocked with α-bungarotoxin. Finally, we showed increased expression of α4 nAChRs in the lung tissue of mice and

  19. Nicotinic acetylcholine receptor (nAChR) mediated dopamine release in larval Drosophila melanogaster.

    PubMed

    Pyakurel, Poojan; Shin, Mimi; Venton, B Jill

    2018-03-01

    Acetylcholine is an excitatory neurotransmitter in the central nervous system of insects and the nicotinic acetylcholine receptor (nAChR) is a target for neonicotinoid insecticides. Functional insect nAChRs are difficult to express in host cells, and hence difficult to study. In mammals, acetylcholine and nicotine evoke dopamine release, but the extent to which this mechanism is conserved in insects is unknown. In intact larval ventral nerve cords (VNCs), we studied dopamine evoked by acetylcholine, nicotine, or neonicotinoids. Using fast-scan cyclic voltammetry, we confirmed dopamine was measured by its cyclic voltammogram and also by feeding Drosophila the synthesis inhibitor, 3-iodotyrosine, which lowered the evoked dopamine response. Acetylcholine (1.8 pmol) evoked on average 0.43 ± 0.04 μM dopamine. Dopamine release significantly decreased after incubation with α-bungarotoxin, demonstrating the release is mediated by nAChR, but atropine, a muscarinic AChR antagonist, had no effect. Nicotine (t 1/2  = 71 s) and the neonicotinoids nitenpyram and imidacloprid (t 1/2  = 86 s, 121 s respectively) also evoked dopamine release, which lasted longer than acetylcholine-stimulated release (t 1/2  = 19 s). Nicotine-stimulated dopamine was significantly lower in the presence of sodium channel blocker, tetrodotoxin, showing that the release is exocytotic. Drosophila that have mutations in the nAChR subunit α1 or β2 have significantly lower neonicotinoid-stimulated release but no changes in nicotine-stimulated release. This work demonstrates that nAChR agonists mediate dopamine release in Drosophila larval VNC and that mutations in nAChR subunits affect how insecticides stimulate dopamine release. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES.

    PubMed

    Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K

    2014-06-05

    A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5-10mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2- interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES✩

    PubMed Central

    Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K

    2013-01-01

    A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5–10 mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2− interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors. PMID:22732654

  2. L-theanine inhibits nicotine-induced dependence via regulation of the nicotine acetylcholine receptor-dopamine reward pathway.

    PubMed

    Di, Xiaojing; Yan, Jingqi; Zhao, Yan; Chang, Yanzhong; Zhao, Baolu

    2012-12-01

    In this study, the inhibitory effect of L-theanine, an amino acid derivative of tea, on the rewarding effects of nicotine and its underlying mechanisms of action were studied. We found that L-theanine inhibited the rewarding effects of nicotine in a conditioned place preference (CPP) model of the mouse and reduced the excitatory status induced by nicotine in SH-SY5Y cells to the same extent as the nicotine receptor inhibitor dihydro-beta-erythroidine (DHβE). Further studies using high performance liquid chromatography, western blotting and immunofluorescence staining analyses showed that L-theanine significantly inhibited nicotine-induced tyrosine hydroxylase (TH) expression and dopamine production in the midbrain of mice. L-theanine treatment also reduced the upregulation of the α(4), β(2) and α(7) nicotine acetylcholine receptor (nAChR) subunits induced by nicotine in mouse brain regions that related to the dopamine reward pathway, thus decreasing the number of cells that could react to nicotine. In addition, L-theanine treatment inhibited nicotine-induced c-Fos expression in the reward circuit related areas of the mouse brain. Knockdown of c-Fos by siRNA inhibited the excitatory status of cells but not the upregulation of TH induced by nicotine in SH-SY5Y cells. Overall, the present study showed that L-theanine reduced the nicotine-induced reward effects via inhibition of the nAChR-dopamine reward pathway. These results may offer new therapeutic strategies for treatment of tobacco addiction.

  3. INHIBITORY EFFECTS OF VOLATILE ORGANIC COMPOUNDS ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS.

    EPA Science Inventory

    INHIBITORY EFFECTS OF VOLATILE ORGANIC COMPOUNDS ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS.
    A.S. Bale*; P.J. Bushnell; C.A. Meacham; T.J. Shafer
    Neurotoxicology Division, NHEERL, ORD, US Environmental Protection Agency, Research Triangle Park, NC, USA
    Toluene (TOL...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. Multiple inhibitory actions of lidocaine on Torpedo nicotinic acetylcholine receptors transplanted to Xenopus oocytes.

    PubMed

    Alberola-Die, Armando; Martinez-Pinna, Juan; González-Ros, José Manuel; Ivorra, Isabel; Morales, Andrés

    2011-06-01

    Lidocaine is a local anaesthetic that blocks sodium channels, but also inhibits several ligand-gated ion-channels. The aim of this work was to unravel the mechanisms by which lidocaine blocks Torpedo nicotinic receptors transplanted to Xenopus oocytes. Acetylcholine-elicited currents were reversibly blocked by lidocaine, in a concentration dependent manner. At doses lower than the IC(50) , lidocaine blocked nicotinic receptors only at negative potentials, indicating an open-channel blockade; the binding site within the channel was at about 30% of the way through the electrical field across the membrane. In the presence of higher lidocaine doses, nicotinic receptors were blocked both at positive and negative potentials, acetylcholine dose-response curve shifted to the right and lidocaine pre-application, before its co-application with acetylcholine, enhanced the current inhibition, indicating all together that lidocaine also blocked resting receptors; besides, it increased the current decay rate. When lidocaine, at low doses, was co-applied with 2-(triethylammonio)-N-(2,6-dimethylphenyl) acetamide bromide, edrophonium or 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, which are quaternary-ammonium molecules that also blocked nicotinic receptors, there was an additive inhibitory effect, indicating that these molecules bound to different sites within the channel pore. These results prove that lidocaine blocks nicotinic receptors by several independent mechanisms and evidence the diverse and complex modulation of this receptor by structurally related molecules. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  6. Probing for and Quantifying Agonist Hydrogen Bonds in α6β2 Nicotinic Acetylcholine Receptors.

    PubMed

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

    2017-04-04

    Designing subtype-selective agonists for neuronal nicotinic acetylcholine receptors is a challenging and significant goal aided by intricate knowledge of each subtype's binding patterns. We previously reported that in α6β2 receptors, acetylcholine makes a functional cation-π interaction with Trp149, but nicotine and TC299423 do not, suggesting a distinctive binding site. This work explores hydrogen binding at the backbone carbonyl associated with α6β2 Trp149. Substituting residue i + 1, Thr150, with its α-hydroxy analogue (Tah) attenuates the carbonyl's hydrogen bond accepting ability. At α6(T150Tah)β2, nicotine shows a 24-fold loss of function, TC299423 shows a modest loss, and acetylcholine shows no effect. Nicotine was further analyzed via a double-mutant cycle analysis utilizing N'-methylnicotinium, which indicated a hydrogen bond in α6β2 with a ΔΔG of 2.6 kcal/mol. Thus, even though nicotine does not make the conserved cation-π interaction with Trp149, it still makes a functional hydrogen bond to its associated backbone carbonyl.

  7. Effect of galantamine on the human α7 neuronal nicotinic acetylcholine receptor, the Torpedo nicotinic acetylcholine receptor and spontaneous cholinergic synaptic activity

    PubMed Central

    Texidó, Laura; Ros, Esteve; Martín-Satué, Mireia; López, Susana; Aleu, Jordi; Marsal, Jordi; Solsona, Carles

    2005-01-01

    Various types of anticholinesterasic agents have been used to improve the daily activities of Alzheimer's disease patients. It was recently demonstrated that Galantamine, described as a molecule with anticholinesterasic properties, is also an allosteric enhancer of human α4β2 neuronal nicotinic receptor activity. We explored its effect on the human α7 neuronal nicotinic acetylcholine receptor (nAChR) expressed in Xenopus oocytes. Galantamine, at a concentration of 0.1 μM, increased the amplitude of acetylcholine (ACh)-induced ion currents in the human α7 nAChR expressed in Xenopus oocytes, but caused inhibition at higher concentrations. The maximum effect of galantamine, an increase of 22% in the amplitude of ACh-induced currents, was observed at a concentration of 250 μM Ach. The same enhancing effect was obtained in oocytes transplanted with Torpedo nicotinic acetylcholine receptor (AChR) isolated from the electric organ, but in this case the optimal concentration of galantamine was 1 μM. In this case, the maximum effect of galantamine, an increase of 35% in the amplitude of ACh-induced currents, occurred at a concentration of 50 μM ACh. Galantamine affects not only the activity of post-synaptic receptors but also the activity of nerve terminals. At a concentration of 1 μM, quantal spontaneous events, recorded in a cholinergic synapse, increased their amplitude, an effect which was independent of the anticholinesterasic activity associated with this compound. The anticholinesterasic effect was recorded in preparations treated with a galantamine concentration of 10 μM. In conclusion, our results show that galantamine enhances human α7 neuronal nicotinic ACh receptor activity. It also enhances muscular AChRs and the size of spontaneous cholinergic synaptic events. However, only a very narrow range of galantamine concentrations can be used for enhancing effects. PMID:15834443

  8. Molecular Determinants of α3β4 Nicotinic Acetylcholine Receptors Inhibition by Triterpenoids.

    PubMed

    Eom, Sanung; Kim, Yoon Suh; Lee, Sung Bae; Noh, Shinhwa; Yeom, Hye Duck; Bae, Hyunsu; Lee, Jun-Ho

    2018-01-01

    In a previous work, we reported the regulatory role of the triterpenoids on 5-hydroxytryptamine (5-HT) 3 A receptors activity in Xenopus laevis oocytes (Eur. J. Pharmacol., 615, 2009, Lee et al.). In the present report, we studied the modulation of triterpenoids on the activity of the human nicotinic acetylcholine receptor type α3β4. Two-electrode voltage clamp experiments were used to test acetylcholine mediated inward current (I ACh ). Treatment with triterpenoids (dehydroeburicoic acid, 6α-hydroxypolyporenic acid C and pachymic acid) inhibited I ACh in a concentration dependent and reversible manner. The IC 50 values for pachymic acid, dehydroeburicoic acid, and 6α-hydroxypolyporenic acid C were 14.9, 37.7, and 20.9 µM, respectively. The inhibitory regulation of I ACh by each triterpenoid showed in a non-competitive manner on the activity of α3β4 nicotinic acetylcholine receptors. These results show that triterpenoids (pachymic acid, dehydroeburicoic acid, 6α-hydroxypolyporenic acid C) can be used as agents to modulate the activity of nicotinic acetylcholine receptor type α3β4. Furthermore, molecular docking studies of 6α-hydroxypolyporenic acid C on α3β4 nicotinic acetylcholine receptors in silico showed that this molecule interacted predominantly with residues at cavities in the α3 subunit and β4 subunit. This docking assays indicated four potential binding sites for this ligand in the extracellular region at sensor domain of α3β4 nicotinic acetylcholine receptors. In point mutagenesis of those whose alanine substitution, 6α-hydroxypolyporenic acid C potency decreased on W25A of α3 subunit or N109A of β4 subunit in both mutants. The double mutation of W25A of α3 subunit and N109A of β4 subunit was significantly attenuated inhibitory effects by 6α-hydroxypolyporenic acid C. All taken together, this study revealed that molecular basis of α3β4 nicotinic acetylcholine receptors by triterpenoids and provides a novel potent interaction

  9. Ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppressing nicotinic acetylcholine receptor-ion channels in cultured bovine adrenal medullary cells.

    PubMed

    Li, Xiaojia; Toyohira, Yumiko; Horisita, Takafumi; Satoh, Noriaki; Takahashi, Keita; Zhang, Han; Iinuma, Munekazu; Yoshinaga, Yukari; Ueno, Susumu; Tsutsui, Masato; Sata, Takeyoshi; Yanagihara, Nobuyuki

    2015-12-01

    Ikarisoside A is a natural flavonol glycoside derived from plants of the genus Epimedium, which have been used in Traditional Chinese Medicine as tonics, antirheumatics, and aphrodisiacs. Here, we report the effects of ikarisoside A and three other flavonol glycosides on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that ikarisoside A (1-100 μM), but not icariin, epimedin C, or epimedoside A, concentration-dependently inhibited the secretion of catecholamines induced by acetylcholine, a physiological secretagogue and agonist of nicotinic acetylcholine receptors. Ikarisoside A had little effect on catecholamine secretion induced by veratridine and 56 mM K(+). Ikarisoside A (1-100 μM) also inhibited (22)Na(+) influx and (45)Ca(2+) influx induced by acetylcholine in a concentration-dependent manner similar to that of catecholamine secretion. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, ikarisoside A (0.1-100 μM) directly inhibited the current evoked by acetylcholine. It also suppressed (14)C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine at 1-100 μM and 10-100 μM, respectively. The present findings suggest that ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

  10. Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

    PubMed

    Vulfius, Catherine A; Kasheverov, Igor E; Starkov, Vladislav G; Osipov, Alexey V; Andreeva, Tatyana V; Filkin, Sergey Yu; Gorbacheva, Elena V; Astashev, Maxim E; Tsetlin, Victor I; Utkin, Yuri N

    2014-01-01

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

  11. Nicotinic and muscarinic acetylcholine receptors shape ganglion cell response properties

    PubMed Central

    Long, Ye; Gavrikov, Konstantin E.; Amthor, Franklin R.; Keyser, Kent T.

    2014-01-01

    The purpose of this study was to evaluate the expression patterns of nicotinic and muscarinic ACh receptors (nAChRs and mAChRs, respectively) in relation to one another and to understand their effects on rabbit retinal ganglion cell response properties. Double-label immunohistochemistry revealed labeled inner-retinal cell bodies and complex patterns of nAChR and mAChR expression in the inner plexiform layer. Specifically, the expression patterns of m1, m4, and m5 muscarinic receptors overlapped with those of non-α7 and α7 nicotinic receptors in presumptive amacrine and ganglion cells. There was no apparent overlap in the expression patterns of m2 muscarinic receptors with α7 nicotinic receptors or of m3 with non-α7 nicotinic receptors. Patch-clamp recordings demonstrated cell type-specific effects of nicotinic and muscarinic receptor blockade. Muscarinic receptor blockade enhanced the center responses of brisk-sustained/G4 On and G4 Off ganglion cells, whereas nicotinic receptor blockade suppressed the center responses of G4 On-cells near the visual streak but enhanced the center responses of nonstreak G4 On-cells. Blockade of muscarinic or nicotinic receptors suppressed the center responses of brisk-sustained Off-cells and the center light responses of subsets of brisk-transient/G11 On- and Off-cells. Only nicotinic blockade affected the center responses of G10 On-cells and G5 Off-cells. These data indicate that physiologically and morphologically identified ganglion cell types have specific patterns of AChR expression. The cholinergic receptor signatures of these cells may have implications for understanding visual defects in disease states that result from decreased ACh availability. PMID:25298382

  12. Preclinical evidence that activation of mesolimbic alpha 6 subunit containing nicotinic acetylcholine receptors supports nicotine addiction phenotype.

    PubMed

    Brunzell, Darlene H

    2012-11-01

    Nicotine is a major psychoactive ingredient in tobacco yet very few individuals quit smoking with the aid of nicotine replacement therapy. Targeted therapies with more selective action at nicotinic acetylcholine receptors (nAChRs) that contain a β2 subunit (β2*nAChRs; *denotes assembly with other subunits) have enjoyed significantly greater success, but exhibit potential for unwanted cardiac, gastrointestinal, and emotive side effects. This literature review focuses on the preclinical evidence that suggests that subclasses of β2*nAChRs that assemble with the α6 subunit may provide an effective target for tobacco cessation. α6β2*nAChRs have a highly selective pattern of neuroanatomical expression in catecholaminergic nuclei including the ventral tegmental area and its projection regions. α6β2*nAChRs promote dopamine (DA) neuron activity and DA release in the mesolimbic dopamine system, a brain circuitry that is well-studied for its contributions to addiction behavior. A combination of genetic and pharmacological studies indicates that activation of α6β2*nAChRs is necessary and sufficient for nicotine psychostimulant effects and nicotine self-administration. α6β2*nAChRs support maintenance of nicotine use, support the conditioned reinforcing effects of drug-associated cues, and regulate nicotine withdrawal. These data suggest that α6β2*nAChRs represent a critical pool of high affinity β2*nAChRs that regulates nicotine dependence phenotype and suggest that inhibition of these receptors may provide an effective strategy for tobacco cessation therapy.

  13. Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

    PubMed

    Raymond Delpech, Valérie; Ihara, Makoto; Coddou, Claudio; Matsuda, Kazuhiko; Sattelle, David B

    2003-11-01

    Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken alpha7, chicken alpha4beta2 and the Drosophila melanogaster/chicken hybrid receptors SAD/beta2 and ALS/beta2. No agonist action of NTX (0.1-100 microM) was observed on chicken alpha7, chicken alpha4beta2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (alpha7) or heteromeric (alpha4beta2) nicotinic AChRs. Block by NTX of the chicken alpha7, chicken alpha4beta2 and the SAD/beta2 and ALS/beta2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.

  14. 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.

  15. Agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action

    SciTech Connect

    Forman, S.A.; Firestone, L.L.; Miller, K.W.

    1987-05-19

    Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring /sup 86/Rb/sup +/ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: (1) integrated net ion efflux (in 10 s) from untreated vesicles, (2) integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with ..cap alpha..-bungarotoxin, and (3) initial rates of efflux (5-100 ms) from vesicles that were partially blocked with ..cap alpha..-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10/sup 8/-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel openingmore » at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum /sup 86/Rb/sup +/ efflux by 50% (K/sub B/ values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, K/sub B/ values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times K/sub B/ did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. The authors conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.« less

  16. Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

    PubMed Central

    Gill, JasKiran K.; Savolainen, Mari; Young, Gareth T.; Zwart, Ruud; Sher, Emanuele; Millar, Neil S.

    2011-01-01

    Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular “orthosteric” binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site. PMID:21436053

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

    PubMed Central

    Tomizawa, Motohiro; Casida, John E

    1999-01-01

    The major nitroimine insecticide imidacloprid (IMI) and the nicotinic analgesics epibatidine and ABT-594 contain the 6-chloro-3-pyridinyl moiety important for high activity and/or selectivity. ABT-594 has considerable nicotinic acetylcholine receptor (AChR) subtype specificity which might carry over to the chloropyridinyl insecticides. This study considers nine IMI analogues for selectivity in binding to immuno-isolated α1, α3 and α7 containing nicotinic AChRs and to purported α4β2 nicotinic AChRs.α1- and α3-Containing nicotinic AChRs (both immuno-isolated by mAb 35, from Torpedo and human neuroblastoma SH-SY5Y cells, respectively) are between two and four times more sensitive to DN-IMI than to (−)-nicotine.With immuno-isolated α3 nicotinic AChRs, the tetrahydropyrimidine analogues of IMI with imine or nitromethylene substituents are 3–4 fold less active than (−)-nicotine. The structure-activity profile with α3 nicotinic AChRs from binding assays is faithfully reproduced in agonist potency as induction of 86rubidium ion efflux in intact cells.α7-Containing nicotinic AChRs of SH-SY5Y cells (immuno-isolated by mAb 306) and rat brain membranes show maximum sensitivity to the tetrahydropyrimidine analogue of IMI with the nitromethylene substituent.The purported α4β2 nicotinic AChRs [mouse (Chao & Casida, 1997) and rat brain] are similar in sensitivity to DN-IMI, the tetrahydropyrimidine nitromethylene and nicotine.The commercial insecticides (IMI, acetamiprid and nitenpyram) have low to moderate potency at the α3 and purported α4β2 nicotinic AChRs and are essentially inactive at α1 and α7 nicotinic AChRs.In conclusion, the toxicity of the analogues and metabolites of nicotinoid insecticides in mammals may involve action at multiple receptor subtypes with selectivity conferred by minor structural changes. PMID:10369463

  18. Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors

    PubMed Central

    Moaddel, Ruin; Abdrakhmanova, Galia; Kozak, Joanna; Jozwiak, Krzysztof; Toll, Lawrence; Jimenez, Lucita; Rosenberg, Avraham; Tran, Thao; Xiao, Yingxian; Zarate, Carlos A.; Wainer, Irving W.

    2012-01-01

    The effect of the (R,S)-ketamine metabolites (R,S)-norketamine, (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)- hydroxynorketamine on the activity of α7 and α3β4 neuronal nicotinic acetylcholine receptors was investigated using patch-clamp techniques. The data indicated that (R,S)-dehydronorketamine inhibited acetylcholine-evoked currents in α7-nicotinic acetylcholine receptor, IC50 = 55 ± 6 nM, and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-norketamine also inhibited α7-nicotinic acetylcholine receptor function at concentrations ≤1μM, while (R,S)-ketamine was inactive at these concentrations. The inhibitory effect of (R,S)-dehydronorketamine was voltage-independent and the compound did not competitively displace selective α7-nicotinic acetylcholine receptor ligands [125I]-α-bungarotoxin and [3H]-epibatidine indicating that (R,S)-dehydronorketamine is a negative allosteric modulator of the α7-nicotinic acetylcholine receptor. (R,S)-Ketamine and (R,S)-norketamine inhibited (S)-nicotine-induced whole-cell currents in cells expressing α3β4-nicotinic acetylcholine receptor, IC50 3.1 and 9.1μM, respectively, while (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were weak inhibitors, IC50 >100μM. The binding affinities of (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine at the NMDA receptor were also determined using rat brain membranes and the selective NMDA receptor antagonist [3H]-MK-801. The calculated Ki values were 38.95 μM for (S)-dehydronorketamine, 21.19 μM for (2S,6S)-hydroxynorketamine and > 100 μM for (2R,6R)-hydroxynorketamine. The results suggest that the inhibitory activity of ketamine metabolites at the α7-nicotinic acetylcholine receptor may contribute to the clinical effect of the drug. PMID:23183107

  19. Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors.

    PubMed

    Moaddel, Ruin; Abdrakhmanova, Galia; Kozak, Joanna; Jozwiak, Krzysztof; Toll, Lawrence; Jimenez, Lucita; Rosenberg, Avraham; Tran, Thao; Xiao, Yingxian; Zarate, Carlos A; Wainer, Irving W

    2013-01-05

    The effect of the (R,S)-ketamine metabolites (R,S)-norketamine, (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine on the activity of α7 and α3β4 neuronal nicotinic acetylcholine receptors was investigated using patch-clamp techniques. The data indicated that (R,S)-dehydronorketamine inhibited acetylcholine-evoked currents in α7-nicotinic acetylcholine receptor, IC(50) = 55 ± 6 nM, and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-norketamine also inhibited α7-nicotinic acetylcholine receptor function at concentrations ≤ 1 μM, while (R,S)-ketamine was inactive at these concentrations. The inhibitory effect of (R,S)-dehydronorketamine was voltage-independent and the compound did not competitively displace selective α7-nicotinic acetylcholine receptor ligands [(125)I]-α-bungarotoxin and [(3)H]-epibatidine indicating that (R,S)-dehydronorketamine is a negative allosteric modulator of the α7-nicotinic acetylcholine receptor. (R,S)-Ketamine and (R,S)-norketamine inhibited (S)-nicotine-induced whole-cell currents in cells expressing α3β4-nicotinic acetylcholine receptor, IC(50) 3.1 and 9.1 μM, respectively, while (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were weak inhibitors, IC(50) >100 μM. The binding affinities of (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine at the NMDA receptor were also determined using rat brain membranes and the selective NMDA receptor antagonist [(3)H]-MK-801. The calculated K(i) values were 38.95 μM for (S)-dehydronorketamine, 21.19 μM for (2S,6S)-hydroxynorketamine and>100 μM for (2R,6R)-hydroxynorketamine. The results suggest that the inhibitory activity of ketamine metabolites at the α7-nicotinic acetylcholine receptor may contribute to the clinical effect of the drug. Published by Elsevier B.V.

  20. Mammalian Nicotinic Acetylcholine Receptors: From Structure to Function

    PubMed Central

    Albuquerque, Edson X.; Pereira, Edna F. R.; Alkondon, Manickavasagom; Rogers, Scott W.

    2009-01-01

    The classical studies of nicotine by Langley at the turn of the 20th century introduced the concept of a “receptive substance,” from which the idea of a “receptor” came to light. Subsequent studies aided by the Torpedo electric organ, a rich source of muscle-type nicotinic receptors (nAChRs), and the discovery of α-bungarotoxin, a snake toxin that binds pseudo-irreversibly to the muscle nAChR, resulted in the muscle nAChR being the best characterized ligand-gated ion channel hitherto. With the advancement of functional and genetic studies in the late 1980s, the existence of nAChRs in the mammalian brain was confirmed and the realization that the numerous nAChR subtypes contribute to the psychoactive properties of nicotine and other drugs of abuse and to the neuropathology of various diseases, including Alzheimer’s, Parkinson’s, and schizophrenia, has since emerged. This review provides a comprehensive overview of these findings and the more recent revelations of the impact that the rich diversity in function and expression of this receptor family has on neuronal and nonneuronal cells throughout the body. Despite these numerous developments, our understanding of the contributions of specific neuronal nAChR subtypes to the many facets of physiology throughout the body remains in its infancy. PMID:19126755

  1. Alpha 7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to 5-fluorouracil.

    PubMed

    Chen, Wei-Yu; Huang, Chien-Yu; Cheng, Wan-Li; Hung, Chin-Sheng; Huang, Ming-Te; Tai, Cheng-Jeng; Liu, Yen-Nien; Chen, Chi-Long; Chang, Yu-Jia

    2015-12-01

    Gastric cancer is the second most common cause of cancer mortality worldwide. Most gastric cancer patients are asymptomatic until the advanced stages, for which current therapeutic treatments are suboptimal. 5-Fluorouracil (5-FU), an antimetabolite agent, is widely used in gastric cancer therapy. However, the presence of drug resistance in gastric cancer patients reduces the cytotoxic activity of 5-FU. In gastric cancer, no research has yet been conducted to analyze the effect of alpha 7-nicotinic acetylcholine receptor (A7-nAChR) on the therapeutic response to 5-FU. In this study, we generated A7-nAChR knockdown (A7-nAChR-KD) AGS cells by a small interfering RNA (siRNA) technique in gastric cancer cells. The anti-proliferative effects of 5-FU were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and cell cycle determination. We found that A7-nAChR-KD cells were more resistant to 5-FU treatment compared with the scrambled control cells according to the MTT assay. The apoptotic cell population was increased more in scrambled control cells treated with 5-FU than A7-nAChR-KD cells according to the cell cycle distribution and TUNEL assays. We analyzed expression levels of survival and apoptosis-associated proteins (pAkt, Akt, Mcl-1, Bcl-2, Bad, and Bax) altered by 5-FU treatment. Survival and antiapoptosis signaling (pAkt, Akt, Mcl-1 and Bcl-2) was downregulated, and the proapoptotic proteins (Bad and Bax) were upregulated in 5-FU-treated control cells but expression levels of Bcl-2, Bad, and Bad were not altered in 5-FU-treated A7-nAChR-KD cells. This is consistent with A7-nAChR-KD cells exhibiting more resistance to 5-FU treatment. In our study, we carried out an in vitro study on AGS gastric cancer cell line to elucidate the anticancer efficacy and molecular mechanisms of A7-nAChR silencing on 5-FU-induced cell death. The results clearly showed

  2. Nicotinic acetylcholine receptors in attention circuitry: the role of layer VI neurons of prefrontal cortex.

    PubMed

    Proulx, Eliane; Piva, Matthew; Tian, Michael K; Bailey, Craig D C; Lambe, Evelyn K

    2014-04-01

    Cholinergic modulation of prefrontal cortex is essential for attention. In essence, it focuses the mind on relevant, transient stimuli in support of goal-directed behavior. The excitation of prefrontal layer VI neurons through nicotinic acetylcholine receptors optimizes local and top-down control of attention. Layer VI of prefrontal cortex is the origin of a dense feedback projection to the thalamus and is one of only a handful of brain regions that express the α5 nicotinic receptor subunit, encoded by the gene chrna5. This accessory nicotinic receptor subunit alters the properties of high-affinity nicotinic receptors in layer VI pyramidal neurons in both development and adulthood. Studies investigating the consequences of genetic deletion of α5, as well as other disruptions to nicotinic receptors, find attention deficits together with altered cholinergic excitation of layer VI neurons and aberrant neuronal morphology. Nicotinic receptors in prefrontal layer VI neurons play an essential role in focusing attention under challenging circumstances. In this regard, they do not act in isolation, but rather in concert with cholinergic receptors in other parts of prefrontal circuitry. This review urges an intensification of focus on the cellular mechanisms and plasticity of prefrontal attention circuitry. Disruptions in attention are one of the greatest contributing factors to disease burden in psychiatric and neurological disorders, and enhancing attention may require different approaches in the normal and disordered prefrontal cortex.

  3. Activation of nicotinic receptors can contribute to endothelium-dependent relaxations to acetylcholine in the rat aorta.

    PubMed

    Zou, Qian; Leung, Susan W S; Vanhoutte, Paul M

    2012-06-01

    Acetylcholine causes endothelium-dependent relaxations in the rat aorta. Both muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are expressed in endothelial cells. It is generally accepted that mAChRs are responsible for the endothelium-dependent relaxations evoked by acetylcholine. The present study was designed to investigate whether nAChRs can also be involved in such responses evoked by the cholinergic transmitter. Rings with or without endothelium of aortae of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats were suspended in organ chambers for the measurement of isometric tension. In WKY aortae the muscarinic antagonist atropine abolished the relaxations to increasing concentrations of acetylcholine, confirming that mAChRs are responsible mainly for the response under control conditions. In SHR aortae, atropine caused only partial inhibition of the endothelium-dependent relaxations to acetylcholine; the remaining decreases in tension were inhibited by the nicotinic antagonist mecamylamine, which did not significantly affect the response in the absence of atropine in either SHR or WKY preparations. Thus, when mAChRs are inhibited, nAChRs mediate relaxation to the cholinergic transmitter in the SHR but not the WKY aorta. Nicotine, a direct agonist of the nicotinic receptor, induced endothelium-dependent relaxations in both SHR and WKY rats via the activation of α7-nAChRs, but not by mecamylamine-sensitive nicotinic receptors (α3 subtype). The acetylcholine-induced, atropine-insensitive relaxations and those to nicotine both involve the phosphatidylinositol 3-kinase/AKT pathway. The present study demonstrates that the activation of nAChRs can contribute to acetylcholine-induced, endothelium-dependent relaxations in the aortae of hypertensive animals and suggests that these receptors may contribute to the endothelium-dependent regulation of vascular tone.

  4. Chronic Nicotine Modifies Skeletal Muscle Na,K-ATPase Activity through Its Interaction with the Nicotinic Acetylcholine Receptor and Phospholemman

    PubMed Central

    Chibalin, Alexander V.; Heiny, Judith A.; Benziane, Boubacar; Prokofiev, Alexander V.; Vasiliev, Alexander V.; Kravtsova, Violetta V.; Krivoi, Igor I.

    2012-01-01

    Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21–31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV) while the activity of the α1 isoform decreased (−4.4 mV). Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM), measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser63 and Ser68. Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM. PMID:22442718

  5. Prior nicotine self-administration attenuates subsequent dopaminergic deficits of methamphetamine in rats: Role of nicotinic acetylcholine receptors

    PubMed Central

    Baladi, Michelle G; Nielsen, Shannon M; McIntosh, J. Michael; Hanson, Glen R; Fleckenstein, Annette E

    2015-01-01

    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 1): attenuates short-term dopaminergic damage induced by methamphetamine and 2) 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

  6. Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia and airway mucus formation in vitro and in vivo

    PubMed Central

    Gundavarapu, Sravanthi; Wilder, Julie A.; Mishra, Neerad C.; Rir-sima-ah, Jules; Langley, Raymond J.; Singh, Shashi P.; Saeed, Ali Imran; Jaramillo, Richard J.; Gott, Katherine M.; Peña-Philippides, Juan Carlos; Harrod, Kevin S.; McIntosh, J. Michael; Buch, Shilpa; Sopori, Mohan L.

    2012-01-01

    Background Airway mucus hypersecretion is a key pathophysiological feature in number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. Objectives Characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. Methods IL-13 and gamma-aminobutyric acid receptors (GABAARs) are implicated in airway mucus. We examined the role of IL-13 and GABAARs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells, and secondhand cigarette smoke and/or ovalbumin-induced mucus formation in vivo. Results Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABAAR antagonist picrotoxin (PIC). Airway epithelial cells express α7/α9/α10 nicotinic acetylcholine receptors (nAChRs) and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells and/or in mouse airways. Conclusions Nicotine-induced airway mucus formation is independent of IL-13 and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various pro-mucoid agents, including IL-13. In the absence of nicotine, acetylcholine may be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABAARα2 in the MUC5AC pathway. Acetylcholine and α-7-nAChRs may serve as therapeutic targets to control airway mucus. PMID:22578901

  7. Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors.

    PubMed

    Maus, A D; Pereira, E F; Karachunski, P I; Horton, R M; Navaneetham, D; Macklin, K; Cortes, W S; Albuquerque, E X; Conti-Fine, B M

    1998-11-01

    We demonstrated previously that human skin keratinocytes express acetylcholine receptors (AChRs) sensitive to acetylcholine and nicotine, which regulate cell adhesion and motility. We demonstrate here that human and rodent bronchial epithelial cells (BECs) express AChRs similar to those expressed by keratinocytes and by some neurons. Patch-clamp experiments demonstrated that the BEC AChRs are functional, and they are activated by acetylcholine and nicotine. They are blocked by kappa-bungarotoxin, a specific antagonist of the AChR isotypes expressed by neurons in ganglia. Their ion-gating properties are consistent with those of AChR isotypes expressed in ganglia, formed by alpha3, alpha5, and beta2 or beta4 subunits. Reverse transcription-polymerase chain reaction and in situ hybridization experiments demonstrated the presence in BECs of mRNA transcripts for all those AChR subunits, both in cell cultures and in tissue sections, whereas we could not detect transcripts for the alpha2, alpha4, alpha6, and beta3 AChR subunits. The expression of alpha3 and alpha5 proteins in BEC in vivo was verified by the binding of subunit-specific antibodies to sections of trachea. Mecamylamine and kappa-bungarotoxin, which are cholinergic antagonists able to block the ganglionic alpha3 AChRs, caused a reversible change of the cell shape of cultured, confluent human BECs. This resulted in a reduction of the area covered by the cell and in cell/cell detachment. The presence of AChRs sensitive to nicotine on the lining of the airways raises the possibility that the high concentrations of nicotine resulting from tobacco smoking will cause an abnormal activation, a desensitization, or both of the bronchial AChRs. This may mediate or facilitate some of the toxic effects of cigarette smoking in the respiratory system.

  8. Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.

    PubMed

    Nguyen, V T; Hall, L L; Gallacher, G; Ndoye, A; Jolkovsky, D L; Webber, R J; Buchli, R; Grando, S A

    2000-04-01

    A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

  9. Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival.

    PubMed

    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.

  10. Cyclic imine toxins from dinoflagellates: a growing family of potent antagonists of the nicotinic acetylcholine receptors.

    PubMed

    Molgó, Jordi; Marchot, Pascale; Aráoz, Rómulo; Benoit, Evelyne; Iorga, Bogdan I; Zakarian, Armen; Taylor, Palmer; Bourne, Yves; Servent, Denis

    2017-08-01

    We present an overview of the toxicological profile of the fast-acting, lipophilic macrocyclic imine toxins, an emerging family of organic compounds associated with algal blooms, shellfish contamination and neurotoxicity. Worldwide, shellfish contamination incidents are expanding; therefore, the significance of these toxins for the shellfish food industry deserves further study. Emphasis is directed to the dinoflagellate species involved in their production, their chemical structures, and their specific mode of interaction with their principal natural molecular targets, the nicotinic acetylcholine receptors, or with the soluble acetylcholine-binding protein, used as a surrogate receptor model. The dinoflagellates Karenia selliformis and Alexandrium ostenfeldii / A. peruvianum have been implicated in the biosynthesis of gymnodimines and spirolides, while Vulcanodinium rugosum is the producer of pinnatoxins and portimine. The cyclic imine toxins are characterized by a macrocyclic skeleton comprising 14-27 carbon atoms, flanked by two conserved moieties, the cyclic imine and the spiroketal ring system. These phycotoxins generally display high affinity and broad specificity for the muscle type and neuronal nicotinic acetylcholine receptors, a feature consistent with their binding site at the receptor subunit interfaces, composed of residues highly conserved among all nAChRs, and explaining the diverse toxicity among animal species. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

  11. The anthelmintic levamisole is an allosteric modulator of human neuronal nicotinic acetylcholine receptors.

    PubMed

    Levandoski, Mark M; Piket, Barbara; Chang, Jane

    2003-06-13

    L-[-]-2,3,5,6-Tetrahydro-6-phenylimidazo[2,1b]-thiazole hydrochloride (levamisole) is an anthelmintic that targets the nicotinic acetylcholine receptors of parasitic nematodes. We report here the effects of levamisole on human neuronal alpha 3 beta 2 and alpha 3 beta 4 nicotinic receptors, heterologously expressed in Xenopus oocytes and studied with the voltage clamp method. Applied alone, levamisole was a very weak partial agonist for the two subunit combinations. When co-applied with acetylcholine, micromolar concentrations of levamisole potentiated responses, while millimolar concentrations inhibited them; these effects were complex functions of both acetylcholine and levamisole concentrations. The differences in the levamisole effects on the two receptor combinations suggest that the effects are mediated by the beta subunit. Several combinations of agonist and anthelmintic gave the dual potentiation/inhibition behavior, suggesting that the modulatory effects are general. Levamisole inhibition showed macroscopic characteristics of open channel block. Several results led us to conclude that levamisole potentiation occurs through noncompetitive binding to the receptor. We propose pseudo-site binding for noncompetitive potentiation by levamisole.

  12. 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

  13. Hyoscine butylbromide potently blocks human nicotinic acetylcholine receptors in SH-SY5Y cells.

    PubMed

    Weiser, Thomas; Just, Stefan

    2009-02-06

    Hyoscine butylbromide (HBB; tradenames: Buscopan/Buscapina is an antispasmodic drug for the treatment of abdominal pain associated with gastrointestinal cramping. As a hyoscine derivative, this compound competitively inhibits muscarinic acetylcholine (ACh) receptors on smooth muscle cells in the gastrointestinal tract. Preliminary investigations suggested that it might also inhibit nicotinic ACh receptors. This study investigated the effect of HBB on nicotinic ACh receptor-mediated membrane currents in SH-SY5Y cells. ACh and nicotine application-induced comparable membrane currents with EC(50) values of 25.9+/-0.6 and 40.1+/-0.4microM, respectively. When coapplied with 100microM ACh, HBB concentration-dependently suppressed currents with an IC(50) value of 0.19+/-0.04microM, and was approximately seven-times more potent than the ganglionic blocker, hexamethonium (IC(50)=1.3+/-0.3microM). Increasing the agonist concentration to 5mM did not affect the amount of block by HBB, which suggests a non-competitive mode of action. These functional in vitro data demonstrate for the first time that HBB blocks neuronal nicotinic ACh receptors in the same concentration range as it inhibits muscarinic ACh receptors. If one hypothesizes that HBB might also affect nicotinic receptors in autonomic neurons in vivo (e. g. in the enteric nervous system), this effect could contribute to its spasmolytic activity.

  14. A behavioral economic analysis of the value-enhancing effects of nicotine and varenicline and the role of nicotinic acetylcholine receptors in male and female rats.

    PubMed

    Barrett, Scott T; Geary, Trevor N; Steiner, Amy N; Bevins, Rick A

    2018-04-09

    Reinforcement value enhancement by nicotine of non-nicotine rewards is believed to partially motivate smoking behavior. Recently, we showed that the value-enhancing effects of nicotine are well characterized by reinforcer demand models and that the value-enhancing effects of the smoking-cessation aid bupropion (Zyban) are distinct from those of nicotine and differ between the sexes. The present study evaluated potential sex differences in the enhancement effects of nicotine and varenicline (Chantix) using a reinforcer demand methodology. The role of α4β2* and α7 nicotinic acetylcholine receptors (nAChRs) in the enhancing effects of nicotine and varenicline is also evaluated. Male and female rats (n=12/sex) were trained to lever press maintained by sensory reinforcement by visual stimulus (VS) presentations. Changes in the VS value following nicotine and varenicline administration were assessed using an established reinforcer demand approach. Subsequently, the effects of antagonism of α4β2* and α7 nAChRs on varenicline and nicotine-induced enhancement active lever-pressing were assessed using a progressive ratio schedule. Nicotine and varenicline enhanced VS demand equivalently between the sexes as evaluated by reinforcer demand. However, α4β2* receptor antagonism attenuated value enhancement by nicotine and varenicline in females, but only of nicotine in males.

  15. Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors

    PubMed Central

    Kirsch, Glenn E.; Fedorov, Nikolai B.; Kuryshev, Yuri A.; Liu, Zhiqi; Orr, Michael S.

    2016-01-01

    Abstract The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and β-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3β4, α3β4α5, α4β2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity. PMID:27505073

  16. Neuronal nicotinic acetylcholine receptors are important targets for alcohol reward and dependence.

    PubMed

    Wu, Jie; Gao, Ming; Taylor, Devin H

    2014-03-01

    Neuronal nicotinic acetylcholine receptors are important targets for alcohol reward and dependence. Alcoholism is a serious public health problem and has been identified as the third major cause of preventable mortality in the world. Worldwide, about 2 billion people consume alcohol, with 76.3 million having diagnosable alcohol use disorders. Alcohol is currently responsible for the death of 4% of adults worldwide (about 2.5 million deaths each year), and this number will be significantly increased by 2020 unless effective action is taken. Alcohol is the most commonly abused substance by humans. Ethanol (EtOH) is the intoxicating agent in alcoholic drinks that can lead to abuse and dependence. Although it has been extensively studied, the mechanisms of alcohol reward and dependence are still poorly understood. The major reason is that, unlike other addictive drugs (eg, morphine, cocaine or nicotine) that have specific molecular targets, EtOH affects much wider neuronal functions. These functions include phospholipid membranes, various ion channels and receptors, synaptic and network functions, and intracellular signaling molecules. The major targets in the brain that mediate EtOH's effects remain unclear. This knowledge gap results in a therapeutic barrier in the treatment of alcoholism. Interestingly, alcohol and nicotine are often co-abused, which suggests that neuronal nicotinic acetylcholine receptors (nAChRs), the molecular targets for nicotine, may also contribute to alcohol's abusive properties. Here, we briefly summarize recent lines of evidence showing how EtOH modulates nAChRs in the mesolimbic pathway, which provides a perspective that nAChRs are important targets mediating alcohol abuse.

  17. Nereistoxin: a naturally occurring toxin with redox effects on neuronal nicotinic acetylcholine receptors in chick retina.

    PubMed

    Xie, Y; Lane, W V; Loring, R H

    1993-02-01

    Nereistoxin (NTX; 4-N,N-dimethylamino-1,2-dithiolane) is previously reported to block both muscle and neuronal nicotinic acetylcholine receptors, and reversibly inhibit radioligand binding to Torpedo nicotinic receptors. Here, we studied redox effects of NTX on neuronal nicotinic receptors in chick retinas by electrophysiological recordings and by [125I]neuronal bungarotoxin binding. NTX blocked retinal responses to the nicotinic agonist dimethylphenylpiperazinium (300 microM, 2 sec) with an IC50 of 3.5 microM. NTX inhibition was selective for nicotinic receptors, long lasting and not reversible upon washing. The nonselective oxidizing compound dithiobis(nitrobenzoic acid) (1 mM) transiently and repetitively reversed NTX (100 microM) inhibition (85% recovery). After application of the alkylating agent bromoacetylcholine (2 or 100 microM, with 2 microM neostigmine), dithiobis(nitrobenzoic acid) could no longer restore nicotinic function. d-Tubocurarine (300 microM) equally protected against alkylation with bromoacetylcholine (2 microM) after dithiothreitol (2 mM) or NTX treatment. The action of NTX differs from that of dithiothreitol because the agonist dimethylphenylpiperazinium (30 microM) protects against inactivation by dithiothreitol, but not by NTX. NTX reversibly inhibited [125I]neuronal bungarotoxin binding to chick retinal homogenates (IC50 = 16 microM). The present study suggests that nereistoxin or a metabolite is a potent antagonist as well as a selective reducing agent for nicotinic receptors in chick retina. As a dithiolane, this latter action by nereistoxin remains to be explained.

  18. Molecular characterization of off-target activities of telithromycin: a potential role for nicotinic acetylcholine receptors.

    PubMed

    Bertrand, Daniel; Bertrand, Sonia; Neveu, Estelle; Fernandes, Prabhavathi

    2010-12-01

    Adverse effects have limited the clinical use of telithromycin. Preferential inhibition of the nicotinic acetylcholine receptors (nAChR) at the neuromuscular junction (α3β2 and NMJ), the ciliary ganglion of the eye (α3β4 and α7), and the vagus nerve innervating the liver (α7) could account for the exacerbation of myasthenia gravis, the visual disturbance, and the liver failure seen with telithromycin use. The studies presented here enable the prediction of expected side effects of macrolides in development, such as solithromycin (CEM-101).

  19. 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

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

    PubMed Central

    Ton, Hoai T.; Smart, Amanda E.; Aguilar, Brittany L.; Olson, Thao T.

    2015-01-01

    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 Ca2+ imaging, 86Rb+ 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 [3H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action. PMID:25964258

  1. Menthol Suppresses Nicotinic Acetylcholine Receptor Functioning in Sensory Neurons via Allosteric Modulation

    PubMed Central

    Wilhelm, M.; Swandulla, D.

    2012-01-01

    In this study, we have investigated how the function of native and recombinant nicotinic acetylcholine receptors (nAChRs) is modulated by the monoterpenoid alcohol from peppermint (−) menthol. In trigeminal neurons (TG), we found that nicotine (75 μM)-activated whole-cell currents through nAChRs were reversibly reduced by menthol in a concentration-dependent manner with an IC50 of 111 μM. To analyze the mechanism underlying menthol's action in more detail, we used single channel and whole-cell recordings from recombinant human α4β2 nAChR expressed in HEK tsA201 cells. Here, we found a shortening of channel open time and a prolongation of channel closed time, and an increase in single channel amplitude leading in summary to a reduction in single channel current. Furthermore, menthol did not affect nicotine's EC50 value for currents through recombinant human α4β2 nAChRs but caused a significant reduction in nicotine's efficacy. Taken together, these findings indicate that menthol is a negative allosteric modulator of nAChRs. PMID:22281529

  2. In vivo interactions between α7 nicotinic acetylcholine receptor and nuclear peroxisome proliferator-activated receptor-α: Implication for nicotine dependence.

    PubMed

    Jackson, Asti; Bagdas, Deniz; Muldoon, Pretal P; Lichtman, Aron H; Carroll, F Ivy; Greenwald, Mark; Miles, Michael F; Damaj, M Imad

    2017-05-15

    Chronic tobacco use dramatically increases health burdens and financial costs. Limitations of current smoking cessation therapies indicate the need for improved molecular targets. The main addictive component of tobacco, nicotine, exerts its dependency effects via nicotinic acetylcholine receptors (nAChRs). Activation of the homomeric α7 nAChR reduces nicotine's rewarding properties in conditioned place preference (CPP) test and i.v. self-administration models, but the mechanism underlying these effects is unknown. Recently, the nuclear receptor peroxisome proliferator-activated receptor type-α (PPARα) has been implicated as a downstream signaling target of the α7 nAChR in ventral tegmental area dopamine cells. The present study investigated PPARα as a possible mediator of the effect of α7 nAChR activation in nicotine dependence. Our results demonstrate the PPARα antagonist GW6471 blocks actions of the α7 nAChR agonist PNU282987 on nicotine reward in an unbiased CPP test in male ICR adult mice. These findings suggests that α7 nAChR activation attenuates nicotine CPP in a PPARα-dependent manner. To evaluate PPARα activation in nicotine dependence we used the selective and potent PPARα agonist, WY-14643 and the clinically used PPARα activator, fenofibrate, in nicotine CPP and we observed attenuation of nicotine preference, but fenofibrate was less potent. We also studied PPARα in nicotine dependence by evaluating its activation in nicotine withdrawal. WY-14643 reversed nicotine withdrawal signs whereas fenofibrate had modest efficacy. This suggests that PPARα plays a role in nicotine reward and withdrawal and that further studies are warranted to elucidate its function in mediating the effects of α7 nAChRs in nicotine dependence. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Effects of blockade of α4β2 and α7 nicotinic acetylcholine receptors on cue-induced reinstatement of nicotine-seeking behaviour in rats

    PubMed Central

    Liu, Xiu

    2013-01-01

    Exposure to environmental stimuli conditioned to nicotine consumption critically contributes to the high relapse rates of tobacco smoking. Our previous work demonstrated that non-selective blockade of nicotinic acetylcholine receptors (nAChRs) reversed the cue-induced reinstatement of nicotine seeking, indicating a role for cholinergic neurotransmission in the mediation of the conditioned incentive properties of nicotine cues. The present study further examined the relative roles of the two major nAChR subtypes, α4β2 and α7, in the cue-induced reinstatement of nicotine seeking. Male Sprague–Dawley rats were trained to intravenously self-administer nicotine (0.03 mg/kg/infusion, free base) on a fixed-ratio 5 schedule of reinforcement. A nicotine-conditioned cue was established by associating a sensory stimulus with each nicotine infusion. After nicotine-maintained responding was extinguished by withholding the nicotine infusion and its paired cue, reinstatement test sessions were conducted with re-presentation of the cue but without the availability of nicotine. Thirty minutes before the tests, the rats were administered the α4β2-selective antagonist dihydro-β-erythroidine (DHβE) and α7-selective antagonist methyllycaconitine (MLA). Pretreatment with MLA, but not DHβE, significantly reduced the magnitude of the cue-induced reinstatement of responses on the active, previously nicotine-reinforced lever. In different sets of rats, MLA altered neither nicotine self-administration nor cue-induced reinstatement of food seeking. These results demonstrate that activation of α7 nAChRs participates in the mediation of the conditioned incentive properties of nicotine cues and suggest that α7 nAChRs may be a promising target for the development of medications for the prevention of cue-induced smoking relapse. PMID:23953129

  4. The Duration of Nicotine Withdrawal-Associated Deficits in Contextual Fear Conditioning Parallels Changes in Hippocampal High Affinity Nicotinic Acetylcholine Receptor Upregulation

    PubMed Central

    Gould, Thomas J.; Portugal, George S.; André, Jessica M.; Tadman, Matthew P.; Marks, Michael J.; Kenney, Justin W.; Yildirim, Emre; Adoff, Michael

    2012-01-01

    A predominant symptom of nicotine withdrawal is cognitive deficits, yet understanding of the neural basis for these deficits is limited. Withdrawal from chronic nicotine disrupts contextual learning in mice and this deficit is mediated by direct effects of nicotine in the hippocampus. Chronic nicotine treatment upregulates nicotinic acetylcholine receptors (nAChR); however, it is unknown whether upregulation is related to the observed withdawal-induced cognitive deficits. If a relationship between altered learning and nAChR levels exists, changes in nAChR levels after cessation of nicotine treatment should match the duration of learning deficits. To test this hypothesis, mice were chronically administered 6.3 mg/kg/day (freebase) nicotine for 12 days and trained in contextual fear conditioning on day 11 or between 1 to 16 days after withdrawal of treatment. Changes in [125I]-epibatidine binding at cytisine-sensitive and cytisine-resistant nAChRs and chronic nicotine-related changes in α4, α7, and β2 nAChR subunit mRNA expression were assessed. Chronic nicotine had no behavioral effect but withdrawal produced deficits in contextual fear conditioning that lasted 4 days. Nicotine withdrawal did not disrupt cued fear conditioning. Chronic nicotine upregulated hippocampal cytisine-sensitive nAChR binding; upregulation continued after cessation of nicotine administration and the duration of upregulation during withdrawal paralleled the duration of behavioral changes. Changes in binding in cortex and cerebellum did not match behavioral changes. No changes in α4, α7, and β2 subunit mRNA expression were seen with chronic nicotine. Thus, nicotine withdrawal-related deficits in contextual learning are time-limited changes that are associated with temporal changes in upregulation of high-affinity nAChR binding. PMID:22285742

  5. Effects of paraformaldehyde fixation on nicotinic acetylcholine receptor binding in adult and developing rat brain sections.

    PubMed

    Huang, Luping Z; Winzer-Serhan, Ursula H

    2006-06-15

    In vitro receptor autoradiography requires unfixed tissue sections, but incubation and washing procedures often result in substantial tissue damage in sections from developing brain, hindering quantitative and qualitative analysis. Formaldehyde fixation greatly preserves morphology. However, fixation can interfere with pharmacological properties of receptors, increase in non-specific background labeling, or even destroy ligand binding sites. Two mild fixation protocols, 0.2% paraformaldehyde (pFA) and pFA vapor fixation, were compared for their ability to improve tissue morphology in postnatal day 7 (P7) brain slices and maintain binding of [125I]-epibatidine and [125I]-alpha-bungarotoxin to heteromeric and homomeric nicotinic acetylcholine receptors, respectively. Fixation greatly improved the ability of P7 brain slices to withstand incubation and washing procedures during binding, resulting in minimal or no loss of tissue after prior 0.2% pFA or vapor fixation, respectively. In adults, distribution pattern of [125I]-epibatidine was identical in fixed and unfixed slices, with no difference in total and non-specific labeling. Distribution of [125I]-alphaBTX labeling was similarly unaffected by 0.2% pFA fixation, but vapor fixation increased total and non-specific binding signal. Thus, mild fixations greatly improve tissue quality during receptor binding procedures and can preserve pharmacological properties of nicotinic acetylcholine receptors. However, different receptors or ligands might exhibit differential sensitivity to fixation protocols.

  6. Effects of neuronal nicotinic acetylcholine receptor allosteric modulators in animal behavior studies.

    PubMed

    Pandya, Anshul A; Yakel, Jerrel L

    2013-10-15

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation-conducting transmembrane channels from the cys-loop receptor superfamily. The neuronal subtypes of these receptors (e.g. the α7 and α4β2 subtypes) are involved in neurobehavioral processes such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and a number of cognitive functions like learning and memory. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders, and behavioral studies in animals are useful models to assess the effects of compounds that act on these receptors. Allosteric modulators are ligands that bind to the receptors at sites other than the orthosteric site where acetylcholine, the endogenous agonist for the nAChRs, binds. While conventional ligands for the neuronal nAChRs have been studied for their behavioral effects in animals, allosteric modulators for these receptors have only recently gained attention, and research on their behavioral effects is growing rapidly. Here we will discuss the behavioral effects of allosteric modulators of the neuronal nAChRs. Published by Elsevier Inc.

  7. Effects of neuronal nicotinic acetylcholine receptor allosteric modulators in animal behavior studies

    PubMed Central

    Pandya, Anshul. A.; Yakel, Jerrel L.

    2013-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation-conducting transmembrane channels from the cys-loop receptor superfamily. The neuronal subtypes of these receptors (e.g. the α7 and α4β2 subtypes) are involved in neurobehavioral processes such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and a number of cognitive functions like learning and memory. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders, and behavioral studies in animals are useful models to assess the effects of compounds that act on these receptors. Allosteric modulators are ligands that bind to the receptors at sites other than the orthosteric site where acetylcholine, the endogenous agonist for the nAChRs, binds. While conventional ligands for the neuronal nAChRs have been studied for their behavioral effects in animals, allosteric modulators for these receptors have only recently gained attention, and research on their behavioral effects is growing rapidly. Here we will discuss the behavioral effects of allosteric modulators of the neuronal nAChRs. PMID:23732296

  8. Monkey adrenal chromaffin cells express α6β4* nicotinic acetylcholine receptors.

    PubMed

    Hernández-Vivanco, Alicia; Hone, Arik J; Scadden, Mick L; Carmona-Hidalgo, Beatriz; McIntosh, J Michael; Albillos, Almudena

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) that contain α6 and β4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6β4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the α3β4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express α6β4* receptors. PCR was used to show the presence of transcripts for α6 and β4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6β4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and β2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6β4* nAChRs.

  9. Monkey Adrenal Chromaffin Cells Express α6β4* Nicotinic Acetylcholine Receptors

    PubMed Central

    Scadden, Mick´l; Carmona-Hidalgo, Beatriz; McIntosh, J. Michael; Albillos, Almudena

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) that contain α6 and β4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6β4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the α3β4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express α6β4* receptors. PCR was used to show the presence of transcripts for α6 and β4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6β4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and β2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6β4* nAChRs. PMID:24727685

  10. Brain α4β2 nicotinic acetylcholine receptors are involved in the secretion of noradrenaline and adrenaline from adrenal medulla in rats.

    PubMed

    Shimizu, Takahiro; Tanaka, Kenjiro; Hasegawa, Takashi; Yokotani, Kunihiko

    2011-03-11

    Recently, we reported that intracerebroventricularly (i.c.v.) administered (±)-epibatidine (a non-selective agonist of nicotinic acetylcholine receptors) elevates plasma noradrenaline and adrenaline through brain nicotinic acetylcholine receptor-mediated mechanisms in rats. In the present study, we characterized the receptors involved in these responses using selective agonists and antagonists of nicotinic acetylcholine receptor subtypes in anesthetized rats. (±)-Epibatidine (5 and 10nmol/animal, i.c.v.) and (-)-nicotine (250 and 500nmol/animal, i.c.v.) both elevated plasma noradrenaline and adrenaline (adrenaline>noradrenaline) but the former was more efficient than the latter. The (±)-epibatidine (5nmol/animal, i.c.v.)-induced elevation of plasma catecholamines was reduced by dihydro-β-erythroidine (a selective antagonist of α4β2 nicotinic acetylcholine receptors) (100 and 300nmol/animal, i.c.v.), while methyllycaconitine (a selective antagonist of α7 nicotinic acetylcholine receptors) (100 and 300nmol/animal, i.c.v.) had no effect on the (±)-epibatidine-induced responses. RJR-2403 (a selective agonist of α4β2 nicotinic acetylcholine receptors) (2.5 and 5μmol/animal, i.c.v.) elevated plasma noradrenaline and adrenaline (adrenaline>noradrenaline), while PNU-282987 (a selective agonist of α7 nicotinic acetylcholine receptors) (2.5 and 5μmol/animal, i.c.v.) had no effect. Furthermore, the RJR-2403 (5μmol/animal, i.c.v.)-induced responses were abolished by acute bilateral adrenalectomy. Immunohistochemical procedures demonstrated the expression of α4 and β2 nicotinic acetylcholine receptor subunits on the spinally projecting hypothalamic paraventricular neurons. Taken together, brain α4β2 nicotinic acetylcholine receptors seem to be involved in the secretion of noradrenaline and adrenaline from adrenal medulla in rats. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells.

    PubMed

    Rasmussen, Andreas H; Strøbæk, Dorte; Dyhring, Tino; Jensen, Marianne L; Peters, Dan; Grunnet, Morten; Timmermann, Daniel B; Ahring, Philip K

    2014-01-13

    Nicotinic acetylcholine receptors (nAChR's) containing the α6 subunit (α6) are putative drug targets of relevance to Parkinson's disease and nicotine addiction. However, heterologous expression of α6 receptors has proven challenging which has stifled drug discovery efforts. Here, we investigate potential new avenues for achieving functional α6 receptor expression. Combinations of chimeric and mutated α6, β2 and β3 subunits were co-expressed in the human HEK293 cell line and receptor expression was assessed using Ca(2+)-imaging (FLIPR™) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric α6/α3 subunit construct in combination with β2 and β3(V9'S) gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the β3(V9'S):β2:α6/α3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wild-type β3, significant functional expression of α6/α3β2β3 was also demonstrated. Comparing the acetylcholine concentration-response relationship of α6/α3β2β3(V9'S) to that of α6/α3β2β3 revealed the β3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca(2+)-imaging experiments showed preservation of basic α6 receptor pharmacology. Our results establish that α6/α3β2β3(V9'S) replicate several basic features of native α6 receptors but also highlight several caveats associated with using this construct and may therefore provide guidance for future drug hunting efforts. © 2013 Published by Elsevier B.V.

  12. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function

    PubMed Central

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K.; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V.; Craig, Patricio O.; Millar, Neil S.; Bouzat, Cecilia

    2017-01-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits. PMID:28069778

  13. Positive allosteric modulation of α4β2 nicotinic acetylcholine receptors as a new approach to smoking reduction: evidence from a rat model of nicotine self-administration.

    PubMed

    Liu, Xiu

    2013-11-01

    The α4β2 subtype of nicotinic acetylcholine receptors (nAChRs) plays a central role in the mediation of nicotine reinforcement. Positive allosteric modulators (PAMs) at α4β2 nAChRs facilitate the intrinsic efficiency of these receptors, although they do not directly activate the receptors. α4β2 PAMs are hypothesized to reduce nicotine self-administration in subjects engaged in routine nicotine consumption. The present study tested this hypothesis using a rat model of nicotine self-administration. Male Sprague-Dawley rats were trained in daily 1-h sessions to intravenously self-administer nicotine (0.03 mg/kg per infusion, free base) on a fixed-ratio 5 schedule. The effects of the α4β2 PAM desformylflustrabromine (dFBr), α4β2 agonist 5-iodo-A-85380, and acetylcholinesterase inhibitor galantamine on nicotine intake were examined. The ability of dFBr and 5-iodo-A-85380 to substitute for nicotine was also assessed. dFBr and 5-iodo-A-85380 dose-dependently reduced nicotine self-administration without changing lever responses for food. Galantamine decreased the self-administration of nicotine and food at high doses. Unlike 5-iodo-A-85380, dFBr failed to substitute for nicotine in supporting self-administration behavior. These results demonstrated the effectiveness of dFBr in reducing nicotine intake and the inability of dFBr to support self-administration behavior. These findings suggest that positive allosteric modulation of α4β2 nAChRs may be a promising target for the treatment of nicotine addiction. Moreover, α4β2 PAMs, in contrast to agonist medications, may have clinical advantages because they may have little liability for abuse because of their lack of reinforcing actions on their own.

  14. Nicotine promotes cell proliferation and induces resistance to cisplatin by α7 nicotinic acetylcholine receptor‑mediated activation in Raw264.7 and El4 cells.

    PubMed

    Wang, Yan Yan; Liu, Yao; Ni, Xiao Yan; Bai, Zhen Huan; Chen, Qiong Yun; Zhang, Ye; Gao, Feng Guang

    2014-03-01

    Although nicotine is a risk factor for carcinogenesis and atherosclerosis, epidemiological data indicate that nicotine has therapeutic benefits in treating Alzheimer's disease. Our previous studies also showed that nicotine-treated dendritic cells have potential antitumor effects. Hence, the precise effects of nicotine on the biological characterizations of cells are controversial. The aim of the present study was to assess the roles of α7 nicotinic acetylcholine receptors (nAChRs), Erk1/2-p38-JNK and PI3K-Akt pathway in nicotine-mediated proliferation and anti-apoptosis effects. The results firstly showed that nicotine treatment clearly augmented cell viability and upregulated PCNA expression in both Raw264.7 and El4 cells. Meanwhile, nicotine afforded protection against cisplatin-induced toxicity through inhibiting caspase-3 activation and upregulating anti-apoptotic protein expression. Further exploration demonstrated that nicotine efficiently abolished cisplatin-promoted mitochondria translocation of Bax and the release of cytochrome c. The pretreatment of α-bungarotoxin and tubocurarine chloride significantly attenuated nicotine-augmented cell viability, abolished caspase-3 activation and α7 nAChR upregulation. Both Erk-JNK-p38 and PI3K-Akt signaling pathways could be activated by nicotine treatment in Raw264.7 and El4 cells. Notably, when Erk-JNK and PI3K-Akt activities were inhibited, nicotine-augmented cell proliferation and anti-apoptotic effects were abolished accordingly. The results presented here indicate that nicotine could achieve α7 nAChR-mediated proliferation and anti-apoptotic effects by activating Erk-JNK and PI3K-Akt pathways respectively, providing potential therapeutic molecules to deal with smoking-associated human diseases.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. The Actions of Piperidine Alkaloids at Fetal Muscle-Type and Autonomic-Type Nicotinic Acetylcholine Receptors

    USDA-ARS?s Scientific Manuscript database

    Piperidine alkaloids are found in many species of plants including Conium maculatum, Nicotiana spp., and Lupinus spp. A pharmacodynamic comparison was made of the alkaloids ammodendrine, anabasine, anabaseine, and coniine in; SH-SY5Y cells which express autonomic-type nicotinic acetylcholine recept...

  17. Chain length dependence of the interactions of bisquaternary ligands with the Torpedo nicotinic acetylcholine receptor.

    PubMed

    Carter, Chris R J; Cao, Liren; Kawai, Hideki; Smith, Peter A; Dryden, William F; Raftery, Michael A; Dunn, Susan M J

    2007-02-01

    The interactions of a series of bisholine esters [(CH3)3N+CH(2)CH2OCO-(CH2)n-COOCH2CH2N+(CH3)3] with the Torpedo nicotinic acetylcholine receptor have been investigated. In equilibrium binding studies, [3H]-suberyldicholine (n=6) binds to an equivalent number of sites as [3H]-acetylcholine and with similar affinity (KD approximately 15 nM). In competition studies, all bischoline esters examined displaced both radioligands in an apparently simple competitive manner. Estimated dissociation constants (KI) showed clear chain length dependence. Short chain molecules (n6) had high affinity similar to suberyldicholine. Functional responses were measured by either rapid flux techniques using Torpedo membrane vesicles or voltage-clamp analyses of recombinant receptors expressed in Xenopus oocytes. Both approaches revealed that suberyldicholine (EC50 approximately 3.4 microM) is 14-25-fold more potent than acetylcholine. However, suberyldicholine elicited only about 45% of the maximum response of the natural ligand, i.e., it is a partial agonist. The potency of this bischoline series increased with chain length. Whereas the shorter ligands (nacetylcholine, longer ligands (n>or=4) had similar (or higher) potency to suberyldicholine. Ligand efficacy had an approximately bell-shaped dependence on chain length and compounds where nor=8 were very poor partial agonists. Based on estimates of interonium distances, we suggest that bisquaternary ligands can interact with multiple binding sites on the nAChR and, depending on the conformational state of the receptor, these sites are 15-20A apart.

  18. 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

  19. Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors in the nucleus accumbens shell regulate progressive ratio responding maintained by nicotine.

    PubMed

    Brunzell, Darlene H; Boschen, Karen E; Hendrick, Elizabeth S; Beardsley, Patrick M; McIntosh, J Michael

    2010-02-01

    Beta2 subunit containing nicotinic acetylcholine receptors (beta2(*)nAChRs; asterisk ((*)) denotes assembly with other subunits) are critical for nicotine self-administration and nicotine-associated dopamine (DA) release that supports nicotine reinforcement. The alpha6 subunit assembles with beta2 on DA neurons where alpha6beta2(*)nAChRs regulate nicotine-stimulated DA release at neuron terminals. Using local infusion of alpha-conotoxin MII (alpha-CTX MII), an antagonist with selectivity for alpha6beta2(*)nAChRs, the purpose of these experiments was to determine if alpha6beta2(*)nAChRs in the nucleus accumbens (NAc) shell are required for motivation to self-administer nicotine. Long-Evans rats lever-pressed for 0.03 mg/kg, i.v., nicotine accompanied by light+tone cues (NIC) or for light+tone cues unaccompanied by nicotine (CUEonly). Following extensive training, animals were tested under a progressive ratio (PR) schedule that required an increasing number of lever presses for each nicotine infusion and/or cue delivery. Immediately before each PR session, rats received microinfusions of alpha-CTX MII (0, 1, 5, or 10 pmol per side) into the NAc shell or the overlying anterior cingulate cortex. alpha-CTX MII dose dependently decreased break points and number of infusions earned by NIC rats following infusion into the NAc shell but not the anterior cingulate cortex. Concentrations of alpha-CTX MII that were capable of attenuating nicotine self-administration did not disrupt locomotor activity. There was no effect of infusion on lever pressing in CUEonly animals and NAc infusion alpha-CTX MII did not affect locomotor activity in an open field. These data suggest that alpha6beta2(*)nAChRs in the NAc shell regulate motivational aspects of nicotine reinforcement but not nicotine-associated locomotor activation.

  20. Super agonist actions of clothianidin and related compounds on the SAD beta 2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes.

    PubMed

    Ihara, Makoto; Matsuda, Kazuhiko; Shimomura, Masaru; Sattelle, David B; Komai, Koichiro

    2004-03-01

    To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SAD beta 2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist action.

  1. Recruitment of α7 nicotinic acetylcholine receptor to caveolin-1-enriched lipid rafts is required for nicotine-enhanced Escherichia coli K1 entry into brain endothelial cells.

    PubMed

    Chi, Feng; Wang, Lin; Zheng, Xueye; Jong, Ambrose; Huang, Sheng-He

    2011-08-01

    We investigate how the α7 nicotinic acetylcholine receptor (α7 nAChR), an essential regulator of inflammation, contributes to the α7 agonist nicotine-enhanced Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMECs) through lipid rafts/caveolae-mediated signaling. α7 nAChR-mediated signaling and bacterial invasion were defined by lipid raft fractionation, immunofluorescence microscopy and siRNA knockdown. Nicotine-enhanced bacterial invasion was dose-dependently inhibited by two raft-disrupting agents, nystatin and filipin. Significant accumulation of the lipid raft marker GM3 was observed in HBMEC induced by E. coli K1 and nicotine. The recruitment of α7 nAChR and related signaling molecules, including vimentin, and Erk1/2, to caveolin-1 enriched lipid rafts was increased upon treatment with E44 or E44 plus nicotine. Erk1/2 activation (phosphorylation), which is required for α7 nAChR-mediated signaling and E44 invasion, was associated with lipid rafts and nicotine-enhanced bacterial infection. Furthermore, E44 invasion, E44/nicotine-induced activation of Erk1/2 and clustering of α7 nAChR and caveolin-1 was specifically blocked by both siRNAs. α7 nAChR-mediated signaling through lipid rafts/caveolae is required for nicotine-enhanced E. coli K1 invasion of HBMEC.

  2. 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

    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 proliferationmore » 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.« less

  3. Nicotine promotes cell proliferation via alpha7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells.

    PubMed

    Wong, Helen Pui Shan; Yu, Le; Lam, Emily Kai Yee; Tai, Emily Kin Ki; Wu, William Ka Kei; Cho, Chi Hin

    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(1)- and beta(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 (DbetaH) 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 alpha7-nicotinic acetylcholine receptor (alpha7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an alpha7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and DbetaH expression as well as adrenaline production. Taken together, through the action on alpha7-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 alpha7-nAChR and beta-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer.

  4. Structural characterization of binding mode of smoking cessation drugs to nicotinic acetylcholine receptors through study of ligand complexes with acetylcholine-binding protein.

    PubMed

    Rucktooa, Prakash; Haseler, Claire A; van Elk, René; Smit, August B; Gallagher, Timothy; Sixma, Titia K

    2012-07-06

    Smoking cessation is an important aim in public health worldwide as tobacco smoking causes many preventable deaths. Addiction to tobacco smoking results from the binding of nicotine to nicotinic acetylcholine receptors (nAChRs) in the brain, in particular the α4β2 receptor. One way to aid smoking cessation is by the use of nicotine replacement therapies or partial nAChR agonists like cytisine or varenicline. Here we present the co-crystal structures of cytisine and varenicline in complex with Aplysia californica acetylcholine-binding protein and use these as models to investigate binding of these ligands binding to nAChRs. This analysis of the binding properties of these two partial agonists provides insight into differences with nicotine binding to nAChRs. A mutational analysis reveals that the residues conveying subtype selectivity in nAChRs reside on the binding site complementary face and include features extending beyond the first shell of contacting residues.

  5. An allosteric binding site of the α7 nicotinic acetylcholine receptor revealed in a humanized acetylcholine-binding protein

    PubMed Central

    Delbart, Florian; Brams, Marijke; Gruss, Fabian; Noppen, Sam; Peigneur, Steve; Boland, Sandro; Chaltin, Patrick; Brandao-Neto, Jose; von Delft, Frank; Touw, Wouter G.; Joosten, Robbie P.; Liekens, Sandra; Tytgat, Jan; Ulens, Chris

    2018-01-01

    Nicotinic acetylcholine receptors (nAChRs) belong to the family of pentameric ligand-gated ion channels and mediate fast excitatory transmission in the central and peripheral nervous systems. Among the different existing receptor subtypes, the homomeric α7 nAChR has attracted considerable attention because of its possible implication in several neurological and psychiatric disorders, including cognitive decline associated with Alzheimer's disease or schizophrenia. Allosteric modulators of ligand-gated ion channels are of particular interest as therapeutic agents, as they modulate receptor activity without affecting normal fluctuations of synaptic neurotransmitter release. Here, we used X-ray crystallography and surface plasmon resonance spectroscopy of α7-acetylcholine–binding protein (AChBP), a humanized chimera of a snail AChBP, which has 71% sequence similarity with the extracellular ligand-binding domain of the human α7 nAChR, to investigate the structural determinants of allosteric modulation. We extended previous observations that an allosteric site located in the vestibule of the receptor offers an attractive target for receptor modulation. We introduced seven additional humanizing mutations in the vestibule-located binding site of AChBP to improve its suitability as a model for studying allosteric binding. Using a fragment-based screening approach, we uncovered an allosteric binding site located near the β8–β9 loop, which critically contributes to coupling ligand binding to channel opening in human α7 nAChR. This work expands our understanding of the topology of allosteric binding sites in AChBP and, by extrapolation, in the human α7 nAChR as determined by electrophysiology measurements. Our insights pave the way for drug design strategies targeting nAChRs involved in ion channel–mediated disorders. PMID:29237730

  6. Sarsasapogenin reverses depressive-like behaviors and nicotinic acetylcholine receptors induced by olfactory bulbectomy.

    PubMed

    Feng, Bo; Zhao, Xiao-Yang; Song, Yi-Zhou; Liang, Wen-Na; Liu, Ji-Liang

    2017-02-03

    Cholinergic signalling in the hippocampus may contribute to the aetiology of mood regulation. Antidepressants can reverse the increase in acetylcholinesterase (AChE) activity induced by olfactory bulbectomy. The activation of nicotinic acetylcholine receptors (nAChRs) also alleviates the symptoms of depression. This study advances the development of sarsasapogenin, which interacts with cholinergic signalling and has a favourable antidepressant profile in olfactory bulbectomised (OB) rats. We examined OB-induced changes in cholinergic signalling, as well as AChE, α4-nAChR, and α7-nAChR expression in the hippocampus. The results indicate that abnormal cholinergic signalling in the hippocampus contributes to the development of depression in the OB rat model. This depression may be alleviated following treatment with sarsasapogenin. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Exon-intron structure of the human neuronal nicotinic acetylcholine receptor {alpha}4 subunit (CHRNA4)

    SciTech Connect

    Steinlein, O.; Weiland, S.; Stoodt, J.

    1996-03-01

    The human neuronal nicotinic acetylcholine receptor {alpha}4 subunit gene (CHRNA4) is located in the candidate region for three different phenotypes: benign familial neonatal convulsions, autosomal dominant nocturnal frontal lobe epilepsy, and low-voltage EEG. Recently, a missense mutation in transmembrane domain 2 of CHRNA4 was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy in one extended pedigree. We have determined the genomic organization of CHRNA4, which consists of six exons distributed over approximately 17 kb of genomic DNA. The nucleotide sequence obtained from the genomic regions adjacent to the exon boundaries enabled us to develop a set ofmore » primer pairs for PCR amplification of the complete coding region. The sequence analysis provides the basis for a comprehensive mutation screening of CHRNA4 in the above-mentioned phenotypes and possibly in other types of idopathic epilepsies. 29 refs., 3 figs., 1 tab.« less

  8. Roles of agonist-binding sites in nicotinic acetylcholine receptor function.

    PubMed

    Dunn, S M; Raftery, M A

    2000-12-20

    Under equilibrium conditions, the nicotinic acetylcholine receptor from Torpedo electroplax carries two high affinity-binding sites for agonists. It is generally assumed that these are the only agonist sites on the receptor and that their occupancy results in rapid channel activation followed by slower conformational transitions that lead to the high affinity equilibrium state. These slow transitions are thought to reflect the physiological process of desensitization. Here we show that preequilibration of the high affinity sites with saturating concentrations of carbamylcholine does not diminish the ion flux response to subsequent exposure to higher (activating) concentrations of this agonist. This finding has profound implications with respect to receptor function: (1) occupancy of the high affinity sites per se does not desensitize the receptor and (2) these sites cannot be directly involved in receptor activation. It is thus necessary to invoke the presence of additional binding sites in channel opening. Copyright 2000 Academic Press.

  9. Nicotinic acetylcholine receptor β2 subunits in the medial prefrontal cortex control attention.

    PubMed

    Guillem, Karine; Bloem, Bernard; Poorthuis, Rogier B; Loos, Maarten; Smit, August B; Maskos, Uwe; Spijker, Sabine; Mansvelder, Huibert D

    2011-08-12

    More than one-third of all people are estimated to experience mild to severe cognitive impairment as they age. Acetylcholine (ACh) levels in the brain diminish with aging, and nicotinic ACh receptor (nAChR) stimulation is known to enhance cognitive performance. The prefrontal cortex (PFC) is involved in a range of cognitive functions and is thought to mediate attentional focus. We found that mice carrying nAChR β2-subunit deletions have impaired attention performance. Efficient lentiviral vector-mediated reexpression of functional β2-subunit-containing nAChRs in PFC neurons of the prelimbic area (PrL) completely restored the attentional deficit but did not affect impulsive and motivational behavior. Our findings show that β2-subunit expression in the PrL PFC is sufficient for endogenous nAChR-mediated cholinergic regulation of attentional performance.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  12. Peptides from puff adder Bitis arietans venom, novel inhibitors of nicotinic acetylcholine receptors.

    PubMed

    Vulfius, Catherine A; Spirova, Ekaterina N; Serebryakova, Marina V; Shelukhina, Irina V; Kudryavtsev, Denis S; Kryukova, Elena V; Starkov, Vladislav G; Kopylova, Nina V; Zhmak, Maxim N; Ivanov, Igor A; Kudryashova, Ksenia S; Andreeva, Tatyana V; Tsetlin, Victor I; Utkin, Yuri N

    2016-10-01

    Phospholipase A 2 (named bitanarin) possessing capability to block nicotinic acetylcholine receptors (nAChRs) was isolated earlier (Vulfius et al., 2011) from puff adder Bitis arietans venom. Further studies indicated that low molecular weight fractions of puff adder venom inhibit nAChRs as well. In this paper, we report on isolation from this venom and characterization of three novel peptides called baptides 1, 2 and 3 that reversibly block nAChRs. To isolate the peptides, the venom of B. arietans was fractionated by gel-filtration and reversed phase chromatography. The amino acid sequences of peptides were established by de novo sequencing using MALDI mass spectrometry. Baptide 1 comprised 7, baptides 2 and 3-10 amino acid residues, the latter being acetylated at the N-terminus. This is the first indication for the presence of such post-translational modification in snake venom proteins. None of the peptides contain cysteine residues. For biological activity studies the peptides were prepared by solid phase peptide synthesis. Baptide 3 and 2 blocked acetylcholine-elicited currents in isolated Lymnaea stagnalis neurons with IC 50 of about 50 μM and 250 μM, respectively. In addition baptide 2 blocked acetylcholine-induced currents in muscle nAChR heterologously expressed in Xenopus oocytes with IC 50 of about 3 μM. The peptides did not compete with radioactive α-bungarotoxin for binding to Torpedo and α7 nAChRs at concentration up to 200 μM that suggests non-competitive mode of inhibition. Calcium imaging studies on α7 and muscle nAChRs heterologously expressed in mouse neuroblastoma Neuro2a cells showed that on α7 receptor baptide 2 inhibited acetylcholine-induced increasing intracellular calcium concentration with IC 50 of 20.6 ± 3.93 μM. On both α7 and muscle nAChRs the suppression of maximal response to acetylcholine by about 50% was observed at baptide 2 concentration of 25 μM, the value being close to IC 50 on α7 nAChR. These data are

  13. Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 subunits.

    PubMed

    Person, Alexandra M; Bills, Kathy L; Liu, Hong; Botting, Shaleen K; Lindstrom, Jon; Wells, Gregg B

    2005-12-02

    Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.

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

    PubMed

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

    2015-05-29

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Dynamic structural investigations on the torpedo nicotinic acetylcholine receptor by time-resolved photoaffinity labeling.

    PubMed

    Mourot, Alexandre; Grutter, Thomas; Goeldner, Maurice; Kotzyba-Hibert, Florence

    2006-04-01

    An increasing number of high-resolution structures of membrane-embedded ion channels (or soluble homologues) have emerged during the last couple of years. The most pressing need now is to understand the complex mechanism underlying ion-channel function. Time-resolved photoaffinity labeling is a suitable tool for investigating the molecular function of membrane proteins, especially when high-resolution structures of related proteins are available. However until now this methodology has only been used on the Torpedo nicotinic acetylcholine receptor (nAChR). nAChRs are allosteric cation-selective receptor channels that are activated by the neurotransmitter acetylcholine (ACh) and implicated in numerous physiological and pathological processes. Time-resolved photoaffinity labeling has already enabled local motions of nAChR subdomains (i.e. agonist binding sites, ion channel, subunit interface) to be understood at the molecular level, and has helped to explain how small molecules can exert their physiological effect, an important step toward the development of drug design. Recent analytical and technical improvements should allow the application of this powerful methodology to other membrane proteins in the near future.

  17. Epibatidine Binds to Four Sites on the Torpedo Nicotinic Acetylcholine Receptor

    PubMed Central

    Kawai, Hideki; Dunn, Susan M. J.; Raftery, Michael A.

    2008-01-01

    The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabelled ligand binding techniques, we show that the binding of [3H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and 1 µM. These classes of sites exist in approximately equal numbers and all [3H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses. PMID:18083118

  18. Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor.

    PubMed

    Kawai, Hideki; Dunn, Susan M J; Raftery, Michael A

    2008-02-15

    The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for alpha-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [(3)H]-(+/-)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15nM and 1muM. These classes of sites exist in approximately equal numbers and all [(3)H]-(+/-)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.

  19. Whole-cell patch-clamp recording of nicotinic acetylcholine receptors in adult Brugia malayi muscle.

    PubMed

    Robertson, A P; Buxton, S K; Martin, R J

    2013-12-01

    Lymphatic filariasis is a debilitating disease caused by clade III parasites like Brugia malayi and Wuchereria bancrofti. Current recommended treatment regimen for this disease relies on albendazole, ivermectin and diethylcarbamazine, none of which targets the nicotinic acetylcholine receptors in these parasitic nematodes. Our aim therefore has been to develop adult B. malayi for electrophysiological recordings to aid in characterizing the ion channels in this parasite as anthelmintic target sites. In that regard, we recently demonstrated the amenability of adult B. malayi to patch-clamp recordings and presented results on the single-channel properties of nAChR in this nematode. We have built on this by recording whole-cell nAChR currents from adult B. malayi muscle. Acetylcholine, levamisole, pyrantel, bephenium and tribendimidine activated the receptors on B. malayi muscle, producing robust currents ranging from >200 pA to ~1.5 nA. Levamisole completely inhibited motility of the adult B. malayi within 10 min and after 60 min, motility had recovered back to control values. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  20. Exploring the Relationship between Nicotinic Acetylcholine Receptor Ligand Size, Efficiency, Efficacy, and C-Loop Opening.

    PubMed

    Ma, Qianyun; Tae, Han-Shen; Wu, Guanzhao; Jiang, Tao; Yu, Rilei

    2017-08-28

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mediating fundamental physiological activities in the nervous system and have become important targets for drug design. For a long time, the acetylcholine binding protein (AChBP) has been used as a surrogate to study the nAChR structure-function. Taking advantage of more than 100 AChBP crystal structures in the Protein DataBank (PDB), we explored the relationship between the size, efficiency, and efficacy of nAChR ligands and the C-loop movement. We found that the size of the ligand is correlated with the opening of the C-loop, which can be used in selecting AChBP crystal structures with appropriate C-loop opening to be used for nAChR ligand docking. Ligand size and C-loop opening are reversely correlated with the ligand efficiency rather than the binding affinity. Ligand efficiency could be accurately predicted using simple computational docking, giving a correlation coefficients (R 2 ) up to 0.73. The efficacy of nAChR ligands might be related to ligand size, C-loop opening, and ligand efficiency. Results from this study are useful for engineering the binding affinity and efficacy of nAChR ligands.

  1. Intersubunit Bridge Formation Governs Agonist Efficacy at Nicotinic Acetylcholine α4β2 Receptors

    PubMed Central

    Rohde, Line Aagot Hede; Ahring, Philip Kiær; Jensen, Marianne Lerbech; Nielsen, Elsebet Østergaard; Peters, Dan; Helgstrand, Charlotte; Krintel, Christian; Harpsøe, Kasper; Gajhede, Michael; Kastrup, Jette Sandholm; Balle, Thomas

    2012-01-01

    The α4β2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4β2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4β2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4β2 receptors. Crystal structures of five agonists with efficacies at α4β2 from 21–76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring. PMID:22170047

  2. Selective potentiation of (α4)3(β2)2 nicotinic acetylcholine receptors augments amplitudes of prefrontal acetylcholine- and nicotine-evoked glutamatergic transients in rats.

    PubMed

    Grupe, Morten; Paolone, Giovanna; Jensen, Anders A; Sandager-Nielsen, Karin; Sarter, Martin; Grunnet, Morten

    2013-11-15

    Prefrontal glutamate release evoked through activation of α4β2* nicotinic acetylcholine receptors (nAChRs) situated on thalamic glutamatergic afferents mediates cue detection processes and thus contributes to attentional performance. However, little is known about the respective contributions of the high sensitivity and low sensitivity (LS) stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, to these processes. In the present study we employed glutamate-sensitive microelectrodes and the (α4)3(β2)2-selective positive allosteric modulator (PAM) NS9283 to investigate the importance of the LS α4β2 nAChR for glutamate release in the rat medial prefrontal cortex (mPFC). Firstly, the signaling evoked by physiologically relevant ACh concentrations through the (α4)3(β2)2 nAChR in HEK293 cells was potentiated by NS9283, consistent with the classification of NS9283 as a PAM. In urethane-anesthetized rats, intra-prefrontal pressure ejections of NS9283 evoked glutamatergic transients. Importantly, this glutamate release was attenuated by removal of cholinergic projections to the recording area. This finding indicates that the effects of NS9283 depend on endogenous ACh, again consistent with effects of a PAM. We then conducted microdialysis to demonstrate the presence of extracellular ACh in urethane-anesthetized control rats. While detectable, those levels were significantly lower than in awake rats. Finally, the amplitudes of glutamatergic transients evoked by local pressure ejections of a low concentration of nicotine were significantly augmented following systemic administration of NS9283 (3.0mg/kg). In conclusion, our results indicate that a LS α4β2 nAChR PAM such as NS9283 may enhance the cholinergic modulation of glutamatergic neurotransmission in the cortex, thereby perhaps alleviating the attentional impairments common to a range of brain disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. 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.

  4. Acetylcholine Attenuates Hydrogen Peroxide-Induced Intracellular Calcium Dyshomeostasis Through Both Muscarinic and Nicotinic Receptors in Cardiomyocytes.

    PubMed

    Palee, Siripong; Apaijai, Nattayaporn; Shinlapawittayatorn, Krekwit; Chattipakorn, Siriporn C; Chattipakorn, Nipon

    2016-01-01

    Oxidative stress induced intracellular Ca2+ overload plays an important role in the pathophysiology of several heart diseases. Acetylcholine (ACh) has been shown to suppress reactive oxygen species generation during oxidative stress. However, there is little information regarding the effects of ACh on the intracellular Ca2+ regulation in the presence of oxidative stress. Therefore, we investigated the effects of ACh applied before or after hydrogen peroxide (H2O2) treatment on the intracellular Ca2+ regulation in isolated cardiomyocytes. Single ventricular myocytes were isolated from the male Wistar rats for the intracellular Ca2+ transient study by a fluorimetric ratio technique. H2O2 significantly decreased both of intracellular Ca2+ transient amplitude and decay rate. ACh applied before, but not after, H2O2 treatment attenuated the reduction of intracellular Ca2+ transient amplitude and decay rate. Both atropine (a muscarinic acetylcholine receptor blocker) and mecamylamine (a nicotinic acetylcholine receptor blocker) significantly decreased the protective effects of acetylcholine on the intracellular Ca2+ regulation. Moreover, the combination of atropine and mecamylamine completely abolished the protective effects of acetylcholine on intracellular Ca2+ transient amplitude and decay rate. ACh pretreatment attenuates H2O2-induced intracellular Ca2+ dyshomeostasis through both muscarinic and nicotinic receptors. © 2016 The Author(s) Published by S. Karger AG, Basel.

  5. Effects of nicotine, methamphetamine and cocaine on extracellular levels of acetylcholine in the interpeduncular nucleus of rats

    PubMed Central

    Hussain, Rifat J.; Taraschenko, Olga D.; Glick, Stanley D.

    2008-01-01

    There is increasing evidence that the cholinergic habenulo-interpeduncular pathway and the dopaminergic mesolimbic pathway may jointly mediate the reinforcing properties of addictive drugs. However, the effects of addictive drug on the functioning of the habenulo-interpeduncular pathway have not been well-characterized. Thus, several drugs of abuse (i.e., nicotine, cocaine, amphetamine) have been shown to alter the morphology of the habenulo-interpeduncular pathway, causing selective degeneration of the cholinergic neurons in this area. On the other hand, morphine was shown to alter the neurochemistry of the habenulo-interpeduncular pathway, inducing biphasic changes in acetylcholine release in the interpeduncular nucleus. In order to determine the effects of cocaine, amphetamine and nicotine on cholinergic neurotransmission in the habenulo-interpeduncular pathway, levels of acetylcholine were assessed during microdialysis in freely-moving rats. Nicotine (0.1 and 0.4 mg/kg s.c.) produced a dose-dependent decrease in extracellular levels of acetylcholine, while methamphetamine (1 and 4 mg/kg i.p.) produced an increase in acetylcholine release in the interpeduncular nucleus. Cocaine (5 and 20 mg/kg i.p.) produced a biphasic effect on extracellular acetylcholine release, i.e., a low dose enhanced the release of acetylcholine and a high dose decreased its release. These results suggest that the habenulo-intepeduncular pathway may be a common target for drugs of abuse and, by modulating the mesolimbic pathway, may mediate unique aspects of the rewarding effects of different drugs. PMID:18583043

  6. 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. Published by Elsevier Inc.

  7. Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors.

    PubMed

    Dowell, Cheryl; Olivera, Baldomero M; Garrett, James E; Staheli, Sarah T; Watkins, Maren; Kuryatov, Alexander; Yoshikami, Doju; Lindstrom, Jon M; McIntosh, J Michael

    2003-09-17

    Until now, there have been no antagonists to discriminate between heteromeric nicotinic acetylcholine receptors (nAChRs) containing the very closely related alpha6 and alpha3 subunits. nAChRs containing alpha3, alpha4, or alpha6 subunits in combination with beta2, occasionally beta4, and sometimes beta3 or alpha5 subunits, are thought to play important roles in cognitive function, pain perception, and the reinforcing properties of nicotine. We cloned a novel gene from the predatory marine snail Conus purpurascens. The predicted peptide, alpha-conotoxin PIA, potently blocks the chimeric alpha6/alpha3beta2beta3 subunit combination as expressed in oocytes but neither the muscle nor the major neuronal nAChR alpha4beta2. Additionally, this toxin is the first described ligand to discriminate between nAChRs containing alpha6 and alpha3 subunits. Exploiting the unusual intron conservation of conotoxin genes may represent a more general approach for defining conotoxin ligand scaffolds to discriminate among closely related receptor populations.

  8. Sofinicline: a novel nicotinic acetylcholine receptor agonist in the treatment of attention-deficit/hyperactivity disorder.

    PubMed

    Fleisher, Carl; McGough, James

    2014-08-01

    Psychostimulants are first-line treatments for attention-deficit/hyperactivity disorder (ADHD), but their tolerability profiles and individual response variability fuel a continuing search for alternative medications. The observation that nicotinic agents improve cognition has led pharmaceutical companies to explore the potential utility of agonists of the nicotinic acetylcholine receptor (nAChR) system for ADHD treatments. This article reviews Phase I and Phase II trials of sofinicline (ABT-894), an agonist of the nAChR α4β2 subtype, as a potential non-stimulant treatment for ADHD. This includes one Phase II trial that compared sofinicline with atomoxetine, a noradrenergic reuptake inhibitor currently approved as a non-stimulant ADHD treatment. This article also reviews the chemistry, pharmacodynamics and pharmacokinetics of sofinicline. Sofinicline appears to be well tolerated and showing efficacy similar to that of atomoxetine. Although the number of patients studied to date is small, further evaluation of sofinicline in Phase II, and possibly Phase III, trials appears to be warranted. Additional studies are needed to explore the efficacy and tolerability of sofinicline with respect to: i) optimal dosing; ii) its use in combination with other medications for ADHD; and iii) its use in children and adolescents, who more commonly experience adverse effects when taking psychostimulant medications.

  9. Nicotinic acetylcholine receptor variants are related to smoking habits, but not directly to COPD.

    PubMed

    Budulac, Simona E; Vonk, Judith M; Postma, Dirkje S; Siedlinski, Mateusz; Timens, Wim; Boezen, Marike H

    2012-01-01

    Genome-wide association studies identified single nucleotide polymorphisms (SNPs) in the nicotinic acetylcholine receptors (nAChRs) cluster as a risk factor for nicotine dependency and COPD. We investigated whether SNPs in the nAChR cluster are associated with smoking habits and lung function decline, and if these potential associations are independent of each other. The SNPs rs569207, rs1051730 and rs8034191 in the nAChR cluster were analyzed in the Vlagtwedde-Vlaardingen cohort (n = 1,390) that was followed for 25 years. We used GEE and LME models to analyze the associations of the SNPs with quitting or restarting smoking and with the annual FEV(1) decline respectively. Individuals homozygote (CC) for rs569207 were more likely to quit smoking (OR (95%CI) = 1.58 (1.05-2.38)) compared to wild-type (TT) individuals. Individuals homozygote (TT) for rs1051730 were less likely to quit smoking (0.64 (0.42; 0.97)) compared to wild-type (CC) individuals. None of the SNPs was significantly associated with the annual FEV(1) decline in smokers and ex-smokers. We show that SNPs in the nAChR region are associated with smoking habits such as quitting smoking, but have no significant effect on the annual FEV(1) decline in smokers and ex-smokers, suggesting a potential role of these SNPs in COPD development via smoking habits rather than via direct effects on lung function.

  10. Use of Monoclonal Antibodies to Study the Structural Basis of the Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle, and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons

    DTIC Science & Technology

    1987-03-16

    brains. We have also been studying nicotinic receptors from a human neuronal cell line which appear to be of the muscle type. Using synthetic...AND MUSCLE, AND TO DETERMINE THE STRUCTURE OF NICOTINIC ACETYLCHOLINE RECEPTORS ON NEURONS Annual Report Jon M. Lindstrom D T IC ELECTE March 16, 1987...AUTHOR(S) ON NEURONS . LINDSTROM. Jon M. 1 3a. TYPE OF REPORT I1 3b. TIME COVERED 114. DTOF REPORT (Year, Mlonth, Day) IS15 PAGE COUNT Annual IFROm2/15/8

  11. Nicotine induces hypoxia-inducible factor-1alpha expression in human lung cancer cells via nicotinic acetylcholine receptor-mediated signaling pathways.

    PubMed

    Zhang, Qunzhou; Tang, Xudong; Zhang, Zuo-Feng; Velikina, Rita; Shi, Shihong; Le, Anh D

    2007-08-15

    Nicotine, the major component in cigarette smoke, can promote tumor growth and angiogenesis in various cancers, including lung cancer. Hypoxia-inducible factor-1alpha (HIF-1alpha) is overexpressed in human lung cancers, particularly in non-small cell lung cancers (NSCLC), and is closely associated with an advanced tumor grade, increased angiogenesis, and resistance to chemotherapy and radiotherapy. The purpose of this study was to investigate the effects of nicotine on the expression of HIF-1alpha and its downstream target gene, vascular endothelial growth factor (VEGF), in human lung cancer cells. Human NSCLC cell lines A549 and H157 were treated with nicotine and examined for expression of HIF-1alpha and VEGF using Western blot or ELISA. Loss of HIF-1alpha function using specific small interfering RNA was used to determine whether HIF-1alpha is directly involved in nicotine-induced tumor angiogenic activities, including VEGF expression, cancer cell migration, and invasion. Nicotine increased HIF-1alpha and VEGF expression in NSCLC cells. Pharmacologically blocking nicotinic acetylcholine receptor-mediated signaling cascades, including the Ca2+/calmodulin, c-Src, protein kinase C, phosphatidylinositol 3-kinase, mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2, and the mammalian target of rapamycin pathways, significantly attenuated nicotine-induced up-regulation of HIF-1alpha protein. Functionally, nicotine potently stimulated in vitro tumor angiogenesis by promoting tumor cell migration and invasion. These proangiogenic and invasive effects were partially abrogated by treatment with small interfering RNA specific for HIF-1alpha. These findings identify novel mechanisms by which nicotine promotes tumor angiogenesis and metastasis and provide further evidences that HIF-1alpha is a potential anticancer target in nicotine-associated lung cancer.

  12. Modulation of ethanol reward sensitivity by nicotinic acetylcholine receptors containing the α6 subunit.

    PubMed

    Guildford, Melissa J; Sacino, Anthony V; Tapper, Andrew R

    2016-12-01

    The prevalent co-abuse of nicotine and alcohol suggests a common neural mechanism underlying the actions of the two drugs. Nicotine, the addictive component of tobacco, activates nicotinic acetylcholine receptors (nAChRs) containing the α6 subunit (α6* nAChRs) in dopaminergic (DAergic) neurons of the ventral tegmental area (VTA), a region known to be crucial for drug reward. Recent evidence suggests that ethanol may potentiate ACh activation of these receptors as well, although whether α6* nAChR expression is necessary for behavioral effects of acute ethanol exposure is unknown. We compared binge-like ethanol consumption and ethanol reward sensitivity between knockout (KO) mice that do not express chrna6 (the gene encoding the α6 nAChR subunit, the α6 KO line) and wild-type (WT) littermates using the Drinking-in-the-Dark (DID) and Conditioned Place Preference (CPP) assay, respectively. In the DID assay, α6 KO female and male mice consumed ethanol similarly to WT mice at all concentrations tested. In the CPP assay, 2.0-g/kg and 3.0-g/kg, but not 0.5-mg/kg, ethanol conditioned a place preference in WT female and male mice, whereas only 2.0-g/kg ethanol conditioned a place preference in α6 KO mice. Acute challenge with ethanol reduced locomotor activity, an effect that developed tolerance with repeated injections, similarly between genotypes in both female and male mice. Together, these data indicate that expression of α6* nAChRs is not required for binge-like ethanol consumption and reward, but modulate sensitivity to the rewarding properties of the drug. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Tracking the Molecular Evolution of Calcium Permeability in a Nicotinic Acetylcholine Receptor

    PubMed Central

    Lipovsek, Marcela; Fierro, Angélica; Pérez, Edwin G.; Boffi, Juan C.; Millar, Neil S.; Fuchs, Paul A.; Katz, Eleonora; Elgoyhen, Ana Belén

    2014-01-01

    Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels. PMID:25193338

  14. Bidirectional Regulation of Aggression in Mice by Hippocampal Alpha-7 Nicotinic Acetylcholine Receptors.

    PubMed

    Lewis, Alan S; Pittenger, Steven T; Mineur, Yann S; Stout, Dawson; Smith, Philip H; Picciotto, Marina R

    2018-05-01

    Humans with 15q13.3 microdeletion syndrome (15q13.3DS) are typically hemizygous for CHRNA7, the gene coding for the α7 nicotinic acetylcholine receptor (nAChR), and manifest a variable neuropsychiatric phenotype that frequently includes persistent aggression. In mice, nAChR activation by nicotine is anti-aggressive, or 'serenic,' an effect which requires α7 nAChRs and is recapitulated by GTS-21, an α7 nAChR partial agonist. Pharmacotherapies potentiating α7 nAChR signaling have also been shown to reduce aggression in human 15q13.3DS. These findings identify the α7 nAChR as an important regulator of aggressive behavior, but the underlying neurobiological substrates remain to be determined. We therefore investigated the brain regions and potential neural circuits in which α7 nAChRs regulate aggressive behavior in male mice. As in 15q13.3DS, mice heterozygous for Chrna7 were significantly more aggressive compared to wild-type controls in the resident-intruder test. We subsequently examined the hippocampus, where α7 nAChRs are highly expressed, particularly in GABAergic interneurons. Resident-intruder interactions strongly activated granule cells in the dentate gyrus (DG). In contrast, GTS-21, which reduces aggression in mice, reduced DG granule cell activity during resident-intruder interactions. Short hairpin RNA knockdown of Chrna7 in the DG enhanced baseline aggression and eliminated the serenic effects of both nicotine and GTS-21 on attack latency. These data further implicate α7 nAChRs in regulation of aggression, and demonstrate that hippocampal α7 nAChR signaling is necessary and sufficient to limit aggression. These findings suggest that nAChR-mediated regulation of hippocampal excitatory-inhibitory balance could be a promising therapeutic intervention for aggression arising in certain forms of neuropsychiatric disease.

  15. Tracking the molecular evolution of calcium permeability in a nicotinic acetylcholine receptor.

    PubMed

    Lipovsek, Marcela; Fierro, Angélica; Pérez, Edwin G; Boffi, Juan C; Millar, Neil S; Fuchs, Paul A; Katz, Eleonora; Elgoyhen, Ana Belén

    2014-12-01

    Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  16. The nicotinic acetylcholine receptors of zebrafish and an evaluation of pharmacological tools used for their study

    PubMed Central

    Papke, Roger L.; Ono, Fumihito; Stokes, Clare; Urban, Jason; Boyd, R. Thomas

    2012-01-01

    Zebrafish (Danio rerio) have been used to study multiple effects of nicotine, for example on cognition, locomotion, and stress responses, relying on the assumption that pharmacological tools will operate similarly upon molecular substrates in the fish and mammalian systems. We have cloned the zebrafish nicotinic acetylcholine receptor (nAChR) subunits and expressed key nAChR subtypes in Xenopus oocytes including neuronal (α4β2, α2β2, α3β4, and α7) and muscle (α1β1bεδ) nAChR. Consistent with studies of mammalian nAChR, nicotine was relatively inactive on muscle-type receptors, having both low potency and efficacy. It had high efficacy but low potency for α7 receptors, and the best potency and good efficacy for α4β2 receptors. Cytisine, a key lead compound for the development of smoking cessation agents, is a full agonist for both mammalian α7 and α3β4 receptors, but a full agonist only for the fish α7, with surprisingly low efficacy for α3β4. The efficacy of cytisine for α4β2 was somewhat greater than typically reported for mammalian α4β2. The ganglionic blocker mecamylamine was most potent for blocking α3β4 receptors, least potent for α7, and roughly equipotent for the muscle receptors and the β2-containing nAChR. However, the block of β2-containing receptors was slowly reversible, consistent with effective targeting of these CNS-type receptors in vivo. Three prototypical α7-selective agonists, choline, tropane, and 4OH-GTS-21, were tested, and these agents were observed to activate both fish α7 and α4β2 nAChR. Our data therefore indicate that while some pharmacological tools used in zebrafish may function as expected, others will not. PMID:22580045

  17. Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors.

    PubMed

    Iwamoto, K; Mata, D; Linn, D M; Linn, C L

    2013-05-01

    Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3 days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500 μM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3 days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10 μM ACh, 100 μM nicotine or 100 nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double

  18. Selective down-regulation of α4β2 neuronal nicotinic acetylcholine receptors in the brain of uremic rats with cognitive impairment.

    PubMed

    Ballesta, Juan J; del Pozo, Carlos; Castelló-Banyuls, Juan; Faura, Clara C

    2012-07-01

    Cognitive impairment is common in patients with chronic kidney disease. Brain nicotinic acetylcholine receptors modulate cognitive functions, such as learning and memory. Pharmacological cholinergic enhancement is useful in patients with cognitive dysfunction. The major nicotinic acetylcholine receptor subtypes in the brain are heteromeric α4β2 and homomeric α7 receptors. To study the involvement of neuronal acetylcholine receptors in cognitive impairment in uremic rats, bilateral nephrectomy was performed. 24 weeks after nephrectomy, memory was assessed using the one trial step-down inhibitory avoidance test. Neuronal nicotinic acetylcholine receptors in the brain were studied by radioligand binding, immunoprecipitation, Western blot and sucrose gradient experiments. We demonstrated that rats with severe renal failure show disorders of short term memory. Long term memory was not altered in these rats. The number of functional α4β2 heteromeric neuronal nicotinic receptors was decreased in the brains of rats with severe renal failure. There was a significant correlation between the degree of renal impairment and the number of heteromeric nicotinic acetylcholine receptors in the brain. The down-regulation of functional α4β2 receptors in the brains of rats with severe renal failure was not due to a reduction of α4 or β2 subunit proteins. The number of α7 homomeric neuronal nicotinic acetylcholine receptors was not altered. These findings may have important clinical significance for the management of cognitive impairment in patients with chronic kidney disease. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. PI3K/Akt-Independent NOS/HO Activation Accounts for the Facilitatory Effect of Nicotine on Acetylcholine Renal Vasodilations: Modulation by Ovarian Hormones

    PubMed Central

    Gohar, Eman Y.; El-gowilly, Sahar M.; El-Gowelli, Hanan M.; El-Demellawy, Maha A.; El-Mas, Mahmoud M.

    2014-01-01

    We investigated the effect of chronic nicotine on cholinergically-mediated renal vasodilations in female rats and its modulation by the nitric oxide synthase (NOS)/heme oxygenase (HO) pathways. Dose-vasodilatory response curves of acetylcholine (0.01–2.43 nmol) were established in isolated phenylephrine-preconstricted perfused kidneys obtained from rats treated with or without nicotine (0.5–4.0 mg/kg/day, 2 weeks). Acetylcholine vasodilations were potentiated by low nicotine doses (0.5 and 1 mg/kg/day) in contrast to no effect for higher doses (2 and 4 mg/kg/day). The facilitatory effect of nicotine was acetylcholine specific because it was not observed with other vasodilators such as 5′-N-ethylcarboxamidoadenosine (NECA, adenosine receptor agonist) or papaverine. Increases in NOS and HO-1 activities appear to mediate the nicotine-evoked enhancement of acetylcholine vasodilation because the latter was compromised after pharmacologic inhibition of NOS (L-NAME) or HO-1 (zinc protoporphyrin, ZnPP). The renal protein expression of phosphorylated Akt was not affected by nicotine. We also show that the presence of the two ovarian hormones is necessary for the nicotine augmentation of acetylcholine vasodilations to manifest because nicotine facilitation was lost in kidneys of ovariectomized (OVX) and restored after combined, but not individual, supplementation with medroxyprogesterone acetate (MPA) and estrogen (E2). Together, the data suggests that chronic nicotine potentiates acetylcholine renal vasodilation in female rats via, at least partly, Akt-independent HO-1 upregulation. The facilitatory effect of nicotine is dose dependent and requires the presence of the two ovarian hormones. PMID:24733557

  20. Maternal nicotine exposure effects on adolescent learning and memory are abolished in alpha(α)2* nicotinic acetylcholine receptor-null mutant mice.

    PubMed

    Mojica, Celina; Bai, Yu; Lotfipour, Shahrdad

    2018-04-09

    The objective of the current study is to test the hypothesis that the deletion of alpha(α)2* nicotinic acetylcholine receptors (nAChRs) (encoded by the Chrna2 gene) ablate maternal nicotine-induced learning and memory deficits in adolescent mice. We use a pre-exposure-dependent contextual fear conditioning behavioral paradigm that is highly hippocampus-dependent. Adolescent wild type and α2-null mutant offspring are exposed to vehicle or maternal nicotine exposure (200 μg/ml, expressed as base) in the drinking water throughout pregnancy until weaning. Adolescent male offspring mice are tested for alterations in growth and development characteristics as well as modifications in locomotion, anxiety, shock-reactivity and learning and memory. As expected, maternal nicotine exposure has no effects on pup number, weight gain and only modestly reduces fluid intake by 19%. Behaviorally, maternal nicotine exposure impedes extinction learning in adolescent wild type mice, a consequence that is abolished in α2-null mutant mice. The effects on learning and memory are not confounded by alternations in stereotypy, locomotion, anxiety or sensory shock reactivity. Overall, the findings highlight that the deletion of α2* nAChRs eliminate the effects of maternal nicotine exposure on learning and memory in adolescent mice. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. CHRNA4 and ANKK1 Polymorphisms Influence Smoking-Induced Nicotinic Acetylcholine Receptor Upregulation.

    PubMed

    Esterlis, Irina; Hillmer, Ansel T; Bois, Frederic; Pittman, Brian; McGovern, Erin; O'Malley, Stephanie S; Picciotto, Marina R; Yang, Bao-Zhu; Gelernter, Joel; Cosgrove, Kelly P

    2016-09-01

    Tobacco smoking leads to increased numbers of β2*-containing nicotinic acetylcholine receptors (β2*-nAChRs) throughout the brain, which return to nonsmoker levels over extended abstinence. The goal of the current study was to determine whether the degree of tobacco smoking-induced changes in β2*-nAChR availability is genetically influenced. In this study, 113 European Americans participated in one or two [(123)I]5-IA-85380 single photon emission computed tomography (SPECT) brain scans. Smokers (n = 58) participated in one scan at 7-9 days of abstinence and those who remained abstinent (n = 27) were imaged again at 6-8 weeks of abstinence. Age- and sex-matched nonsmokers (n = 55) participated in one scan. Blood samples were collected for DNA analysis and genotyped for single nucleotide polymorphisms (SNPs) in the CHRNA4 and ANKK1 gene loci. β2*-nAChR availability was measured in the thalamus, striatum, cortical regions, and cerebellum. The CHRNA4 SNP rs2236196 and ANKK1 SNP rs4938015 were associated with significantly higher cerebellar and cortical β2*-nAChR availability in smokers versus nonsmokers for specific genotypes. There were no significant differences by carrier status in the change in β2*-nAChR availability in smokers from 7-9 days to 6-8 weeks of abstinence. This study provides evidence for genetic regulation of tobacco smoking-induced changes in β2*-nAChR availability and suggests that β2*-nAChR availability could be an endophenotype mediating influences of CHRNA4 variants on nicotine dependence. These results highlight individual differences in the neurochemistry of nicotine dependence and may suggest the need for individualized programs for smoking cessation. This study demonstrates genetic regulation of smoking-induced changes in β2*-nAChRs throughout the brain and highlights the need for personalized programs for smoking cessation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Research on Nicotine and

  2. [Development of physical dependence on nicotine and endogenous opioid system--participation of α7 nicotinic acetylcholine receptor].

    PubMed

    Kishioka, Shiroh; Kiguchi, Norikazu; Kobayashi, Yuka; Saika, Fumihiro; Yamamoto, Chizuko

    2014-10-01

    Nicotine (NIC) regulates various cellular functions acting on the nicotinic acetylcholine receptor (nAChR). And nAChR consists of ligand-gated cation channels with pentameric structure and composed of α and β subunits. In the central nervous system, α 4 β 2 and α 7 nAChRs are the most abundantly expressed as nAChR subtypes. There are several lines of evidence indicating that systemic administration of NIC elicits the release of endogenous opioids, such as, endorphins, enkephalins and dynorphins, in the brain. NIC exerts numerous acute effects, for example, antinociceptive effects and the activating effects of the hypothalamic-pituitary-adrenal (HPA) axis. In these effects, NIC-induced antinociception, but not HPA axis activation, was inhibited by opioid receptor antagonist, naloxone (NLX), and was also suppressed in morphine tolerated mice, indicating the participation of the endogenous opioid system in NIC-induced antinociception, but not HPA axis activation. Moreover, NIC-induced antinociception was antagonized by both α 4 β 2 and α 7 nAChR antagonists, while NIC-induced HPA axis activation was antagonized by α 4 β 2 nAChR antagonist, but not by α 7 nAChR antagonist. These results suggest that the endogenous opioid system may not be located on the downstream of α 4 β 2 nAChR. On the other hand, NIC has substantial physical dependence liability. NLX elicits NIC withdrawal after repeated NIC administration evaluated by corticosterone increase as a withdrawal sign, and NLX-precipitated NIC withdrawal is inhibited by concomitant administration of other opioid receptor antagonist, naltrexone, indicating the participation of endogenous opioid system in the development of physical dependence on NIC. NLX-precipitated NIC withdrawal was also inhibited by concomitant administration of an α 7 nAChR antagonist, but not an α 4 β 2 nAChR antagonist. Taken together, these findings suggest that the endogenous opioid system may be located on the downstream of α 7

  3. Oseltamivir produces hypothermic and neuromuscular effects by inhibition of nicotinic acetylcholine receptor functions: comparison to procaine and bupropion.

    PubMed

    Fukushima, Akihiro; Chazono, Kaori; Hashimoto, Yuichi; Iwajima, Yui; Yamamoto, Shohei; Maeda, Yasuhiro; Ohsawa, Masahiro; Ono, Hideki

    2015-09-05

    Oseltamivir, an anti-influenza virus drug, induces marked hypothermia in normal mice. We have proposed that the hypothermic effect arises from inhibition of the nicotinic acetylcholine receptor function of sympathetic ganglion neurons which innervate the brown adipose tissue (a heat generator). It has been reported that local anesthetics inhibit nicotinic acetylcholine receptor function by acting on its ionic channels, and that bupropion, a nicotinic antagonist, induces hypothermia. In this study, we compared the effects of oseltamivir, procaine and bupropion on body temperature, cardiovascular function and neuromuscular transmission. Intraperitoneal administration of oseltamivir (100mg/kg), procaine (86.6mg/kg) and bupropion (86.7mg/kg) lowered the core body temperature of normal mice. At lower doses (10-30mg/kg oseltamivir, 8.7-26mg/kg procaine and bupropion), when administered subcutaneously, the three drugs antagonized the hypothermia induced by intraperitoneal injection of nicotine (1mg/kg). In anesthetized rats, intravenous oseltamivir (30-100mg/kg), procaine (10mg/kg) and bupropion (10mg/kg) induced hypotension and bradycardia. Oseltamivir alone (100mg/kg) did not inhibit neuromuscular twitch contraction of rats, but at 3-30mg/kg it augmented the muscle-relaxing effect of d-tubocurarine. Similar effects were observed when lower doses of procaine (10-30mg/kg) and bupropion (3-10mg/kg) were administered, suggesting that systemic administration of oseltamivir inhibits muscular nicotinic acetylcholine receptors. These results support the idea that the hypothermic effect of oseltamivir is due to its effects on sympathetic ganglia which innervate the brown adipose tissue, and suggest that oseltamivir may exert non-selective ion channel blocking effects like those of ester-type local anesthetics. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica.

    PubMed

    Mordvintsev, Dmitry Y; Polyak, Yakov L; Kuzmine, Dmitry A; Levtsova, Olga V; Tourleigh, Yegor V; Kasheverov, Igor E

    2006-01-01

    Short- and long-chain alpha-neurotoxins from snake venoms are potent blockers of nicotinic acetylcholine receptors (nAChRs). Short alpha-neurotoxins consist of 60-62 amino acid residues and include 4 disulfide bridges, whereas long alpha-neurotoxins have 66-75 residues and 5 disulfides. The spatial structure of these toxins is built by three loops, I-III "fingers," confined by four disulfide bridges; the fifth disulfide of long-chain alpha-neurotoxins is situated close to the tip of central loop II. An accurate knowledge of the mode of alpha-neurotoxin-nAChR interaction is important for rational design of new nAChR agonists and antagonists for medical purposes. Ideas on the topography of toxin-nAChR complexes were based until recently on nAChR interactions with selectively labeled alpha-neurotoxins, mutations in toxins, nAChR, or both. Recently, crystal structures have been solved for the Torpedo marmorata nAChR (4A[Unwin, 2005]) and for the acetylcholine-binding protein (AChBP) complexed with mollusk alpha-conotoxin (2.4 A[Celie et al., 2005]) or alpha-cobratoxin, long-chain alpha-neurotoxin (4 A [Bourne et al., 2005]). However, there were no angstrom-resolution models for complexes of short-chain alpha-neurotoxins. Here, we report the model of the Torpedo californica nAChR extracellular domain complexed to a short-chain alpha-neurotoxin II (NTII) from Naja oxiana cobra venom.

  5. Computational determination of the binding mode of α-conotoxin to nicotinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Tabassum, Nargis; Yu, Rilei; Jiang, Tao

    2016-12-01

    Conotoxins belong to the large families of disulfide-rich peptide toxins from cone snail venom, and can act on a broad spectrum of ion channels and receptors. They are classified into different subtypes based on their targets. The α-conotoxins selectively inhibit the current of the nicotinic acetylcholine receptors. Because of their unique selectivity towards distinct nAChR subtypes, α-conotoxins become valuable tools in nAChR study. In addition to the X-ray structures of α-conotoxins in complex with acetylcholine-binding protein, a homolog of the nAChR ligand-binding domain, the high-resolution crystal structures of the extracellular domain of the α1 and α9 subunits are also obtained. Such structures not only revealed the details of the configuration of nAChR, but also provided higher sequence identity templates for modeling the binding modes of α-conotoxins to nAChR. This mini-review summarizes recent modeling studies for the determination of the binding modes of α-conotoxins to nAChR. As there are not crystal structures of the nAChR in complex with conotoxins, computational modeling in combination of mutagenesis data is expected to reveal the molecular recognition mechanisms that govern the interactions between α-conotoxins and nAChR at molecular level. An accurate determination of the binding modes of α-conotoxins on AChRs allows rational design of α-conotoxin analogues with improved potency or selectivity to nAChRs.

  6. Mode of action of the positive modulator PNU-120596 on α7 nicotinic acetylcholine receptors.

    PubMed

    Szabo, Anett K; Pesti, Krisztina; Mike, Arpad; Vizi, E Sylvester

    2014-06-01

    We investigated the mode of action of PNU-120596, a type II positive allosteric modulator of the rat α7 nicotinic acetylcholine receptor expressed by GH4C1 cells, using patch-clamp and fast solution exchange. We made two important observations: first, while PNU-120596 rapidly associated to desensitized receptors, it had at least hundredfold lower affinity to resting conformation, therefore at 10 μM concentration it dissociated from resting receptors; and second, binding of PNU-120596 slowed down dissociation of choline molecules from the receptor radically. We propose that when agonist concentration is transiently elevated in the continuous presence of the modulator (as upon the neuronal release of acetylcholine in a modulator-treated animal) these two elements together cause occurrence of a cycle of events: Binding of the modulator is limited in the absence of the agonist. When the agonist is released, it binds to the receptor, and induces desensitization, thereby enabling modulator binding. Modulator binding in turn traps the agonist within its binding site for a prolonged period of time. Once the agonist finally dissociated, the modulator can also dissociate without re-binding, and the receptor assumes its original resting conformation. In kinetic simulations this "trapped agonist cycle" mechanism did not require that the orthosteric and allosteric ligands symmetrically modify each other's affinity, only the modulator must decrease agonist accessibility, and the agonist must induce a conformation that is accessible to the modulator. This mechanism effectively prolongs and amplifies the effect of the agonist. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Modes of Action, Resistance and Toxicity of Insecticides Targeting Nicotinic Acetylcholine Receptors.

    PubMed

    Ihara, Makoto; Buckingham, Steven D; Matsuda, Kazuhiko; Sattelle, David B

    2017-01-01

    Nicotinic acetylcholine receptors (nAChRs) of insects play a key role in fast excitatory neurotransmission. Several classes of insecticides target insect nAChRs, which are composed of subunit members of a family of multiple subunit encoding genes. Alternative splicing and RNA A-to-I editing can add further to receptor diversity. Native and recombinant receptors have been explored as sites of insecticide action using radioligands, electrophysiology and site-directed mutagenesis. We have reviewed the properties of native and recombinant insect nAChRs, the challenges of functional recombinant insect nAChR expression, nAChR interactions with ligands acting at orthosteric and allosteric sites and in particular their interactions with insecticides. Actions on insect nAChRs of cartap, neonicotinoids, spinosyns, sulfoxamines, butenolides and mesoionic insecticides are reviewed and current knowledge of their modes of action are addressed. Mutations that add to our understanding of insecticide action and those leading to resistance are discussed. Co-crystallisation of neonicotinoids with the acetylcholine binding protein (AChBP), a surrogate for the nAChR ligand binding domain, has proved instructive. Toxicity issues relating to insecticides targeting nAChRs are also considered. An overview of insecticide classes targeting insect nAChRs has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native nAChRs remains poorly understood and functional expression still presents difficulties. These topics together with improved understanding of the precise sites of insecticide actions on insect nAChRs will be the subject of future research. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors.

    PubMed

    Durek, Thomas; Shelukhina, Irina V; Tae, Han-Shen; Thongyoo, Panumart; Spirova, Ekaterina N; Kudryavtsev, Denis S; Kasheverov, Igor E; Faure, Grazyna; Corringer, Pierre-Jean; Craik, David J; Adams, David J; Tsetlin, Victor I

    2017-11-30

    Human SLURP-1 is a secreted protein of the Ly6/uPAR/three-finger neurotoxin family that co-localizes with nicotinic acetylcholine receptors (nAChRs) and modulates their functions. Conflicting biological activities of SLURP-1 at various nAChR subtypes have been based on heterologously produced SLURP-1 containing N- and/or C-terminal extensions. Here, we report the chemical synthesis of the 81 amino acid residue human SLURP-1 protein, characterization of its 3D structure by NMR, and its biological activity at nAChR subtypes. Radioligand assays indicated that synthetic SLURP-1 did not compete with [ 125 I]-α-bungarotoxin (α-Bgt) binding to human neuronal α7 and Torpedo californica muscle-type nAChRs, nor to mollusk acetylcholine binding proteins (AChBP). Inhibition of human α7-mediated currents only occurred in the presence of the allosteric modulator PNU120596. In contrast, we observed robust SLURP-1 mediated inhibition of human α3β4, α4β4, α3β2 nAChRs, as well as human and rat α9α10 nAChRs. SLURP-1 inhibition of α9α10 nAChRs was accentuated at higher ACh concentrations, indicating an allosteric binding mechanism. Our results are discussed in the context of recent studies on heterologously produced SLURP-1 and indicate that N-terminal extensions of SLURP-1 may affect its activity and selectivity on its targets. In this respect, synthetic SLURP-1 appears to be a better probe for structure-function studies.

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

    SciTech Connect

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

    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 andmore » 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.« less

  10. Modulation of TNF Release by Choline Requires α7 Subunit Nicotinic Acetylcholine Receptor-Mediated Signaling

    PubMed Central

    Parrish, William R; Rosas-Ballina, Mauricio; Gallowitsch-Puerta, Margot; Ochani, Mahendar; Ochani, Kanta; Yang, Li-Hong; Hudson, LaQueta; Lin, Xinchun; Patel, Nirav; Johnson, Sarah M; Chavan, Sangeeta; Goldstein, Richard S; Czura, Christopher J; Miller, Edmund J; Al-Abed, Yousef; Tracey, Kevin J; Pavlov, Valentin A

    2008-01-01

    The α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR) is an essential component in the vagus nerve-based cholinergic anti-inflammatory pathway that regulates the levels of TNF, high mobility group box 1 (HMGB1), and other cytokines during inflammation. Choline is an essential nutrient, a cell membrane constituent, a precursor in the biosynthesis of acetylcholine, and a selective natural α7nAChR agonist. Here, we studied the anti-inflammatory potential of choline in murine endotoxemia and sepsis, and the role of the α7nAChR in mediating the suppressive effect of choline on TNF release. Choline (0.1–50 mM) dose-dependently suppressed TNF release from endotoxin-activated RAW macrophage-like cells, and this effect was associated with significant inhibition of NF-κB activation. Choline (50 mg/kg, intraperitoneally [i.p.]) treatment prior to endotoxin administration in mice significantly reduced systemic TNF levels. In contrast to its TNF suppressive effect in wild type mice, choline (50 mg/kg, i.p.) failed to inhibit systemic TNF levels in α7nAChR knockout mice during endotoxemia. Choline also failed to suppress TNF release from endotoxin-activated peritoneal macrophages isolated from α7nAChR knockout mice. Choline treatment prior to endotoxin resulted in a significantly improved survival rate as compared with saline-treated endotoxemic controls. Choline also suppressed HMGB1 release in vitro and in vivo, and choline treatment initiated 24 h after cecal ligation and puncture (CLP)-induced polymicrobial sepsis significantly improved survival in mice. In addition, choline suppressed TNF release from endotoxin-activated human whole blood and macrophages. Collectively, these data characterize the anti-inflammatory efficacy of choline and demonstrate that the modulation of TNF release by choline requires α7nAChR-mediated signaling. PMID:18584048

  11. Baroreflex deficiency aggravates atherosclerosis via α7 nicotinic acetylcholine receptor in mice.

    PubMed

    Chen, Li; Liu, Dian-Hua; Zhang, Xin; Zhang, En-Hui; Liu, Chong; Su, Ding-Feng; Cai, Guo-Jun

    2016-12-01

    Inflammation and oxidative stress play a key role in the initiation, propagation, and development of atherosclerosis. Arterial baroreflex (ABR) dysfunction induced by sinoaortic denervation (SAD) promoted the development of atherosclerosis in ApoE -/- mice. The present work was designed to examine whether ABR deficiency affected inflammation and oxidative stress via α7 nicotinic acetylcholine receptor (α7nAChR) leading to the aggravation of atherosclerosis in mice. ApoE -/- mice were fed with a high-cholesterol diet for 6weeks and half of the mice received sinoaortic denervation that destroyed ABR. We studied the expression of vesicular acetylcholine transporter (VAChT), α7nAChR and levels of inflammatory response and oxidative stress. The results showed that baroreflex dysfunction could promote atherosclerosis, meanwhile, decrease the expression of VAChT and α7nAChR and significantly increase the levels of oxidative stress and inflammation in SAD mice. After treated with PNU-282987 (a selective α7nAChR agonist, 0.53mg/kg/day) for 6weeks in SAD and Sham mice, we found that PNU-282987 could attenuate atherosclerosis and significantly decreased oxidative stress and inflammation after SAD. In addition, α7nAChR +/+ and α7nAChR -/- mice fed with a high-cholesterol diet for 8weeks were co-treated with ketanserin (0.6mg/kg/day), a drug that can enhance baroreflex sensitivity (BRS). Ketanserin could alleviate atherosclerosis and markedly decrease oxidative stress and inflammation in α7nAChR +/+ mice. But there were no effects in α7nAChR knockout mice. Our results demonstrate that ABR dysfunction aggravates atherosclerosis in mice via the vagus-ACh-α7nAChR-inflammation and oxidative stress pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. A role for α4(non-α6)* nicotinic acetylcholine receptors in motor behavior

    PubMed Central

    Soll, Lindsey G.; Grady, Sharon R.; Salminen, Outi; Marks, Michael J.; Tapper, Andrew R.

    2013-01-01

    Nicotinic acetylcholine receptors (nAChRs) containing either the α4 and/or α6 subunit are robustly expressed in dopaminergic nerve terminals in dorsal striatum where they are hypothesized to modulate dopamine (DA) release via acetylcholine (ACh) stimulation from cholinergic interneurons. However, pharmacological blockade of nAChRs or genetic deletion of individual nAChR subunits, including α4 and α6, in mice, yields little effect on motor behavior. Based on the putative role of nAChRs containing the α4 subunit in modulation of DA in dorsal striatum, we hypothesized that mice expressing a single point mutation in the α4 nAChR subunit, Leu9′Ala, that renders nAChRs hypersensitive to agonist, would exhibit exaggerated differences in motor behavior compared to WT mice. To gain insight into these differences, we challenged WT and Leu9′Ala mice with the α4β2 nAChR antagonist dihydro-β-erythroidine (DHβE). Interestingly, in Leu9′Ala mice, DHβE elicited a robust, reversible motor impairment characterized by hypolocomotion, akinesia, catalepsy, clasping, and tremor; whereas the antagonist had little effect in WT mice at all doses tested. Pre-injection of nicotine (0.1 mg/kg) blocked DHβE-induced motor impairment in Leu9′Ala mice confirming that the phenotype was mediated by antagonism of nAChRs. In addition, SKF 82958 (1 mg/kg) and amphetamine (5 mg/kg) prevented the motor phenotype. DHβE significantly activated more neurons within striatum and substantia nigra pars reticulata in Leu9′Ala mice compared to WT animals, suggesting activation of the indirect motor pathway as the circuit underlying motor dysfunction. ACh evoked DA release from Leu9′Ala striatal synaptosomes revealed agonist hypersensitivity only at α4(non-α6)* nAChRs. Similarly, α6 nAChR subunit deletion in an α4 hypersensitive nAChR (Leu9′Ala/α6KO) background had little effect on the DHβE-induced phenotype, suggesting an α4(non-α6)* nAChR-dependent mechanism. Together, these

  13. Lobeline esters as novel ligands for neuronal nicotinic acetylcholine receptors and neurotransmitter transporters

    PubMed Central

    Hojahmat, Marhaba; Horton, David B.; Norrholm, Seth D.; Miller, Dennis K.; Grinevich, Vladimir P.; Deaciuc, Agripina Gabriela; Dwoskin, Linda P.; Crooks, Peter A.

    2013-01-01

    Vescular monoamine transporter-2 (VMAT2) is a viable target for development of pharmacotherapies for psychostimulant abuse. Lobeline (1) is a potent antagonist at α4β2* nicotinic acetylcholine receptors, has moderate affinity (Ki=5.46 μM) for VMAT2, and is being investigated currently as a clinical candidate for treatment of psychostimulant abuse. A series of carboxylic acid and sulfonic acid ester analogs 2–20 of lobeline were synthesized and evaluated for interaction with α4β2* and α7* neuronal nicotinic acetylcholine receptors (nAChRs), the dopamine transporter (DAT), serotonin transporter (SERT) and VMAT2. Both carboxylic acid and sulfonic acid esters had low affinity at α7* nAChRs. Similar to lobeline (Ki=4 nM), sulfonic acid esters had high affinity at α4β2* (Ki=5–17 nM). Aromatic carboxylic acid ester analogs of lobeline (2–4) were 100–1000-fold less potent than lobeline at α4β2* nAChRs, whereas aliphatic carboxylic acid ester analogs were 10–100-fold less potent than lobeline at α4β2*. Two representative lobeline esters, the 10-O-benzoate (2) and the 10-O-benzenesulfonate (10) were evaluated in the 36Rb+ efflux assay using rat thalamic synaptosomes, and were shown to be antagonists with IC50 values of 0.85 μM and 1.60 μM, respectively. Both carboxylic and sulfonic acid esters exhibited a range of potencies (equipotent to 13–45-fold greater potency compared to lobeline) for inhibiting DAT and SERT, respectively, and like lobeline, had moderate affinity (Ki=1.98–10.8 μM) for VMAT2. One of the more interesting analogs, p-methoxybenzoic acid ester 4, had low affinity at α4β2* nAChRs (Ki=19.3 μM) and was equipotent with lobeline, at VMAT2 (Ki=2.98 μM), exhibiting a 6.5-fold selectivity for VMAT2 over α4β2 nAChRs. Thus, esterification of the lobeline molecule may be a useful structural modification for the development of lobeline analogs with improved selectivity at VMAT2. PMID:20036131

  14. A role for α4(non-α6)* nicotinic acetylcholine receptors in motor behavior.

    PubMed

    Soll, Lindsey G; Grady, Sharon R; Salminen, Outi; Marks, Michael J; Tapper, Andrew R

    2013-10-01

    Nicotinic acetylcholine receptors (nAChRs) containing either the α4 and/or α6 subunit are robustly expressed in dopaminergic nerve terminals in dorsal striatum where they are hypothesized to modulate dopamine (DA) release via acetylcholine (ACh) stimulation from cholinergic interneurons. However, pharmacological blockade of nAChRs or genetic deletion of individual nAChR subunits, including α4 and α6, in mice, yields little effect on motor behavior. Based on the putative role of nAChRs containing the α4 subunit in modulation of DA in dorsal striatum, we hypothesized that mice expressing a single point mutation in the α4 nAChR subunit, Leu9'Ala, that renders nAChRs hypersensitive to agonist, would exhibit exaggerated differences in motor behavior compared to WT mice. To gain insight into these differences, we challenged WT and Leu9'Ala mice with the α4β2 nAChR antagonist dihydro-β-erythroidine (DHβE). Interestingly, in Leu9'Ala mice, DHβE elicited a robust, reversible motor impairment characterized by hypolocomotion, akinesia, catalepsy, clasping, and tremor; whereas the antagonist had little effect in WT mice at all doses tested. Pre-injection of nicotine (0.1 mg/kg) blocked DHβE-induced motor impairment in Leu9'Ala mice confirming that the phenotype was mediated by antagonism of nAChRs. In addition, SKF82958 (1 mg/kg) and amphetamine (5 mg/kg) prevented the motor phenotype. DHβE significantly activated more neurons within striatum and substantia nigra pars reticulata in Leu9'Ala mice compared to WT animals, suggesting activation of the indirect motor pathway as the circuit underlying motor dysfunction. ACh evoked DA release from Leu9'Ala striatal synaptosomes revealed agonist hypersensitivity only at α4(non-α6)* nAChRs. Similarly, α6 nAChR subunit deletion in an α4 hypersensitive nAChR (Leu9'Ala/α6 KO) background had little effect on the DHβE-induced phenotype, suggesting an α4(non-α6)* nAChR-dependent mechanism. Together, these data indicate

  15. Nicotine provokes impulsive-like action by stimulating alpha4beta2 nicotinic acetylcholine receptors in the infralimbic, but not in the prelimbic cortex.

    PubMed

    Tsutsui-Kimura, Iku; Ohmura, Yu; Izumi, Takeshi; Yamaguchi, Taku; Yoshida, Takayuki; Yoshioka, Mitsuhiro

    2010-05-01

    Nicotine, a major addictive component of tobacco, has been suggested to provoke impulsivity by activating central alpha4beta2 nicotinic acetylcholine receptors (nAChRs). Although lesion studies have demonstrated the involvement of the medial prefrontal cortex (mPFC) in impulsive action, the precise brain sites responsible for nicotine-induced impulsive action have not been identified. Our goal was to determine whether alpha4beta2 nAChRs in the prelimbic cortex (PL) and/or infralimbic cortex (IL), which are subregions of the mPFC, mediate nicotine-induced impulsive-like action in the three-choice serial reaction time task (3-CSRTT). The 3-CSRTT is a simple version of five-choice serial reaction time task and a rodent model of impulsive action in which the animal is required to inhibit the response until a light stimulus is presented randomly in one of three holes. Following the completion of the training, rats were bilaterally injected with dihydro-beta-erythroidine (DHbetaE; 6 and 18 microg/side), a selective alpha4beta2 nAChRs antagonist, into the PL or IL before systemic injection of nicotine (0.2 mg/kg, salt, s.c.). Intra-IL DHbetaE infusions dose-dependently blocked nicotine-induced impulsive-like action, while infusions of DHbetaE into the PL failed to block the effects of nicotine on impulsive-like action. The present results suggest a critical role for alpha4beta2 nAChRs in the IL in mediating the effects of nicotine on impulsive-like action in the 3-CSRTT.

  16. Blockade of central nicotine acetylcholine receptor signaling attenuate ghrelin-induced food intake in rodents.

    PubMed

    Dickson, S L; Hrabovszky, E; Hansson, C; Jerlhag, E; Alvarez-Crespo, M; Skibicka, K P; Molnar, C S; Liposits, Z; Engel, J A; Egecioglu, E

    2010-12-29

    Here we sought to determine whether ghrelin's central effects on food intake can be interrupted by nicotine acetylcholine receptor (nAChR) blockade. Ghrelin regulates mesolimbic dopamine neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens, partly via cholinergic VTA afferents originating in the laterodorsal tegmental area (LDTg). Given that these cholinergic projections to the VTA have been implicated in natural as well as drug-induced reinforcement, we sought to investigate the role of cholinergic signaling in ghrelin-induced food intake as well as fasting-induced food intake, for which endogenous ghrelin has been implicated. We found that i.p. treatment with the non-selective centrally active nAChR antagonist, mecamylamine decreased fasting-induced food intake in both mice and rats. Moreover, central administration of mecamylamine decreased fasting-induced food intake in rats. I.c.v. ghrelin-induced food intake was suppressed by mecamylamine i.p. but not by hexamethonium i.p., a peripheral nAChR antagonist. Furthermore, mecamylamine i.p. blocked food intake following ghrelin injection into the VTA. Expression of the ghrelin receptor, the growth hormone secretagogue receptor 1A, was found to co-localize with choline acetyltransferase, a marker of cholinergic neurons, in the LDTg. Finally, mecamylamine treatment i.p. decreased the ability of palatable food to condition a place preference. These data suggest that ghrelin-induced food intake is partly mediated via nAChRs and that nicotinic blockade decreases the rewarding properties of food. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  17. Nicotinic acetylcholine receptor availability in cigarette smokers: effect of heavy caffeine or marijuana use.

    PubMed

    Brody, Arthur L; Hubert, Robert; Mamoun, Michael S; Enoki, Ryutaro; Garcia, Lizette Y; Abraham, Paul; Young, Paulina; Mandelkern, Mark A

    2016-09-01

    Upregulation of α4β2* nicotinic acetylcholine receptors (nAChRs) is one of the most well-established effects of chronic cigarette smoking on the brain. Prior research by our group gave a preliminary indication that cigarette smokers with concomitant use of caffeine or marijuana have altered nAChR availability. We sought to determine if smokers with heavy caffeine or marijuana use have different levels of α4β2* nAChRs than smokers without these drug usages. One hundred and one positron emission tomography (PET) scans, using the radiotracer 2-FA (a ligand for β2*-containing nAChRs), were obtained from four groups of males: non-smokers without heavy caffeine or marijuana use, smokers without heavy caffeine or marijuana use, smokers with heavy caffeine use (mean four coffee cups per day), and smokers with heavy marijuana use (mean 22 days of use per month). Total distribution volume (Vt/fp) was determined for the brainstem, prefrontal cortex, and thalamus, as a measure of nAChR availability. A significant between-group effect was found, resulting from the heavy caffeine and marijuana groups having the highest Vt/fp values (especially for the brainstem and prefrontal cortex), followed by smokers without such use, followed by non-smokers. Direct between-group comparisons revealed significant differences for Vt/fp values between the smoker groups with and without heavy caffeine or marijuana use. Smokers with heavy caffeine or marijuana use have higher α4β2* nAChR availability than smokers without these drug usages. These findings are likely due to increased nicotine exposure but could also be due to an interaction on a cellular/molecular level.

  18. Partial blockade of nicotinic acetylcholine receptors improves the counterregulatory response to hypoglycemia in recurrently hypoglycemic rats.

    PubMed

    LaGamma, Edmund F; Kirtok, Necla; Chan, Owen; Nankova, Bistra B

    2014-10-01

    Recurrent exposure to hypoglycemia can impair the normal counterregulatory hormonal responses that guard against hypoglycemia, leading to hypoglycemia unawareness. This pathological condition known as hypoglycemia-associated autonomic failure (HAAF) is the main adverse consequence that prevents individuals with type 1 diabetes mellitus from attaining the long-term health benefits of tight glycemic control. The underlying molecular mechanisms responsible for the progressive loss of the epinephrine response to subsequent bouts of hypoglycemia, a hallmark sign of HAAF, are largely unknown. Normally, hypoglycemia triggers both the release and biosynthesis of epinephrine through activation of nicotinic acetylcholine receptors (nAChR) on the adrenal glands. We hypothesize that excessive cholinergic stimulation may contribute to impaired counterregulation. Here, we tested whether administration of the nAChR partial agonist cytisine to reduce postganglionic synaptic activity can preserve the counterregulatory hormone responses in an animal model of HAAF. Compared with nicotine, cytisine has limited efficacy to activate nAChRs and stimulate epinephrine release and synthesis. We evaluated adrenal catecholamine production and secretion in nondiabetic rats subjected to two daily episodes of hypoglycemia for 3 days, followed by a hyperinsulinemic hypoglycemic clamp on day 4. Recurrent hypoglycemia decreased epinephrine responses, and this was associated with suppressed TH mRNA induction (a measure of adrenal catecholamine synthetic capacity). Treatment with cytisine improved glucagon responses as well as epinephrine release and production in recurrently hypoglycemic animals. These data suggest that pharmacological manipulation of ganglionic nAChRs may be promising as a translational adjunctive therapy to avoid HAAF in type 1 diabetes mellitus. Copyright © 2014 the American Physiological Society.

  19. Partial blockade of nicotinic acetylcholine receptors improves the counterregulatory response to hypoglycemia in recurrently hypoglycemic rats

    PubMed Central

    LaGamma, Edmund F.; Kirtok, Necla; Chan, Owen

    2014-01-01

    Recurrent exposure to hypoglycemia can impair the normal counterregulatory hormonal responses that guard against hypoglycemia, leading to hypoglycemia unawareness. This pathological condition known as hypoglycemia-associated autonomic failure (HAAF) is the main adverse consequence that prevents individuals with type 1 diabetes mellitus from attaining the long-term health benefits of tight glycemic control. The underlying molecular mechanisms responsible for the progressive loss of the epinephrine response to subsequent bouts of hypoglycemia, a hallmark sign of HAAF, are largely unknown. Normally, hypoglycemia triggers both the release and biosynthesis of epinephrine through activation of nicotinic acetylcholine receptors (nAChR) on the adrenal glands. We hypothesize that excessive cholinergic stimulation may contribute to impaired counterregulation. Here, we tested whether administration of the nAChR partial agonist cytisine to reduce postganglionic synaptic activity can preserve the counterregulatory hormone responses in an animal model of HAAF. Compared with nicotine, cytisine has limited efficacy to activate nAChRs and stimulate epinephrine release and synthesis. We evaluated adrenal catecholamine production and secretion in nondiabetic rats subjected to two daily episodes of hypoglycemia for 3 days, followed by a hyperinsulinemic hypoglycemic clamp on day 4. Recurrent hypoglycemia decreased epinephrine responses, and this was associated with suppressed TH mRNA induction (a measure of adrenal catecholamine synthetic capacity). Treatment with cytisine improved glucagon responses as well as epinephrine release and production in recurrently hypoglycemic animals. These data suggest that pharmacological manipulation of ganglionic nAChRs may be promising as a translational adjunctive therapy to avoid HAAF in type 1 diabetes mellitus. PMID:25117409

  20. Discovery of peptide ligands through docking and virtual screening at nicotinic acetylcholine receptor homology models.

    PubMed

    Leffler, Abba E; Kuryatov, Alexander; Zebroski, Henry A; Powell, Susan R; Filipenko, Petr; Hussein, Adel K; Gorson, Juliette; Heizmann, Anna; Lyskov, Sergey; Tsien, Richard W; Poget, Sébastien F; Nicke, Annette; Lindstrom, Jon; Rudy, Bernardo; Bonneau, Richard; Holford, Mandë

    2017-09-19

    Venom peptide toxins such as conotoxins play a critical role in the characterization of nicotinic acetylcholine receptor (nAChR) structure and function and have potential as nervous system therapeutics as well. However, the lack of solved structures of conotoxins bound to nAChRs and the large size of these peptides are barriers to their computational docking and design. We addressed these challenges in the context of the α4β2 nAChR, a widespread ligand-gated ion channel in the brain and a target for nicotine addiction therapy, and the 19-residue conotoxin α-GID that antagonizes it. We developed a docking algorithm, ToxDock, which used ensemble-docking and extensive conformational sampling to dock α-GID and its analogs to an α4β2 nAChR homology model. Experimental testing demonstrated that a virtual screen with ToxDock correctly identified three bioactive α-GID mutants (α-GID[A10V], α-GID[V13I], and α-GID[V13Y]) and one inactive variant (α-GID[A10Q]). Two mutants, α-GID[A10V] and α-GID[V13Y], had substantially reduced potency at the human α7 nAChR relative to α-GID, a desirable feature for α-GID analogs. The general usefulness of the docking algorithm was highlighted by redocking of peptide toxins to two ion channels and a binding protein in which the peptide toxins successfully reverted back to near-native crystallographic poses after being perturbed. Our results demonstrate that ToxDock can overcome two fundamental challenges of docking large toxin peptides to ion channel homology models, as exemplified by the α-GID:α4β2 nAChR complex, and is extendable to other toxin peptides and ion channels. ToxDock is freely available at rosie.rosettacommons.org/tox_dock.

  1. Antagonist pharmacology of desensitizing and non-desensitizing nicotinic acetylcholine receptors in cockroach neurons.

    PubMed

    Salgado, Vincent L

    2016-09-01

    Two α-bungarotoxin-sensitive nicotinic acetylcholine (ACh) receptor subtypes in neurons of the American cockroach have been identified as desensitizing (nAChD) and selectively inhibitable with 100nM imidacloprid, and non-desensitizing (nAChN) and selectively inhibitable with 100pM methyllycaconitine. In this paper, the single-electrode voltage-clamp technique was used to measure concentration-response relations for the action of ACh and five antagonists on pharmacologically separated nAChD and nAChN receptors of acutely dissociated neurons from thoracic ganglia of the American cockroach. A dual bath and U-tube perfusion system was used to achieve rapid application of ACh in the continued presence of antagonists, which was essential to accurately measure inhibition by rapidly-reversible antagonists. ACh activated both receptors with an EC 50 of 7μM and the antagonist potencies were (nAChD/nAChN in nM): dihydro-β-erythroidine: 1.0/5.6, d-tubocurarine: 1000/34, condelphine: 0.39/0.65, phencyclidine: 74/980 and mecamylamine 47/1150. While each of these antagonists displayed some subtype selectivity, none are selective enough to be used as subtype-selective tools. These results bring to a total of 16 the number of nicotinic compounds that have been measured on nAChD and nAChN currents. Characterization of these receptors is important for understanding the role of nAChRs in the insect nervous system and the mechanism of action of insecticides. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Effects of cannabidiol on the function of α7-nicotinic acetylcholine receptors.

    PubMed

    Mahgoub, Mohamed; Keun-Hang, Susan Yang; Sydorenko, Vadym; Ashoor, Abrar; Kabbani, Nadine; Al Kury, Lina; Sadek, Bassem; Howarth, Christopher F; Isaev, Dmytro; Galadari, Sehamuddin; Oz, Murat

    2013-11-15

    The effects of cannabidiol (CBD), a non-psychoactive ingredient of cannabis plant, on the function of the cloned α7 subunit of the human nicotinic acetylcholine (α7 nACh) receptor expressed in Xenopus oocytes were tested using the two-electrode voltage-clamp technique. CBD reversibly inhibited ACh (100 μM)-induced currents with an IC50 value of 11.3 µM. Other phytocannabinoids such as cannabinol and Δ(9)-tetrahydrocannabinol did not affect ACh-induced currents. CBD inhibition was not altered by pertussis toxin treatment. In addition, CBD did not change GTP-γ-S binding to the membranes of oocytes injected with α7 nACh receptor cRNA. The effect of CBD was not dependent on the membrane potential. CBD (10 µM) did not affect the activity of endogenous Ca(2+)-dependent Cl(-) channels, since the extent of inhibition by CBD was unaltered by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing 2mM Ba(2+). Inhibition by CBD was not reversed by increasing ACh concentrations. Furthermore, specific binding of [(125)I] α-bungarotoxin was not inhibited by CBD (10 µM) in oocytes membranes. Using whole cell patch clamp technique in CA1 stratum radiatum interneurons of rat hippocampal slices, currents induced by choline, a selective-agonist of α7-receptor induced currents were also recoded. Bath application of CBD (10 µM) for 10 min caused a significant inhibition of choline induced currents. Finally, in hippocampal slices, [(3)H] norepinephrine release evoked by nicotine (30 µM) was also inhibited by 10 µM CBD. Our results indicate that CBD inhibits the function of the α7-nACh receptor. © 2013 Published by Elsevier B.V.

  3. Expression patterns for nicotinic acetylcholine receptor subunit genes in smoking-related lung cancers

    PubMed Central

    Bordas, Anna; Cedillo, José Luis; Arnalich, Francisco; Esteban-Rodriguez, Isabel; Guerra-Pastrián, Laura; de Castro, Javier; Martín-Sánchez, Carolina; Atienza, Gema; Fernández-Capitan, Carmen; Rios, Juan José; Montiel, Carmen

    2017-01-01

    Cigarette smoking is associated with increased risk for all histologic types of lung cancer, but why the strength of this association is stronger for squamous cell carcinoma than adenocarcinoma of the lung (SQC-L, ADC-L) is not fully understood. Because nicotine and tobacco-specific nitrosamines contribute to carcinogenesis by activating nicotinic acetylcholine receptors (nAChRs) on lung tumors and epithelial cells, we investigated whether differential expression of nAChR subtypes in these tumors could explain their different association with smoking. Expression of nAChR subunit genes in paired tumor and non-tumor lung specimens from 40 SQC-L and 38 ADC-L patients was analyzed by quantitative PCR. Compared to normal lung, both tumors share: i) transcriptional dysregulation of CHRNA3/CHRNA5/CHRNB4 (α3, α5, β4 subunits) at the chromosomal locus that predisposes to lung cancer; and ii) decreased expression of CHRFAM7A (dupα7 subunit); this last subunit negatively modulates α7-nAChR activity in oocytes. In contrast, CHRNA7 (α7 subunit) expression was increased in SQC-L, particularly in smokers and non-survivors, while CHRNA4 (α4 subunit) expression was decreased in ADC-L. Thus, over-representation of cancer-stimulating α7-nAChR in SQC-L, also potentiated by smoking, and under-representation of cancer-inhibiting α4β2-nAChR in ADC-L could explain the different tobacco influences on the tumorigenic process in each cancer type. PMID:28978081

  4. Expression patterns for nicotinic acetylcholine receptor subunit genes in smoking-related lung cancers.

    PubMed

    Bordas, Anna; Cedillo, José Luis; Arnalich, Francisco; Esteban-Rodriguez, Isabel; Guerra-Pastrián, Laura; de Castro, Javier; Martín-Sánchez, Carolina; Atienza, Gema; Fernández-Capitan, Carmen; Rios, Juan José; Montiel, Carmen

    2017-09-15

    Cigarette smoking is associated with increased risk for all histologic types of lung cancer, but why the strength of this association is stronger for squamous cell carcinoma than adenocarcinoma of the lung (SQC-L, ADC-L) is not fully understood. Because nicotine and tobacco-specific nitrosamines contribute to carcinogenesis by activating nicotinic acetylcholine receptors (nAChRs) on lung tumors and epithelial cells, we investigated whether differential expression of nAChR subtypes in these tumors could explain their different association with smoking. Expression of nAChR subunit genes in paired tumor and non-tumor lung specimens from 40 SQC-L and 38 ADC-L patients was analyzed by quantitative PCR. Compared to normal lung, both tumors share: i) transcriptional dysregulation of CHRNA3/CHRNA5/CHRNB4 (α3, α5, β4 subunits) at the chromosomal locus that predisposes to lung cancer; and ii) decreased expression of CHRFAM7A (dupα7 subunit); this last subunit negatively modulates α7-nAChR activity in oocytes. In contrast, CHRNA7 (α7 subunit) expression was increased in SQC-L, particularly in smokers and non-survivors, while CHRNA4 (α4 subunit) expression was decreased in ADC-L. Thus, over-representation of cancer-stimulating α7-nAChR in SQC-L, also potentiated by smoking, and under-representation of cancer-inhibiting α4β2-nAChR in ADC-L could explain the different tobacco influences on the tumorigenic process in each cancer type.

  5. Olfactory discrimination varies in mice with different levels of α7-nicotinic acetylcholine receptor expression

    PubMed Central

    Hellier, Jennifer L.; Arevalo, Nicole L.; Blatner, Megan J.; Dang, An K.; Clevenger, Amy C.; Adams, Catherine E.; Restrepo, Diego

    2010-01-01

    Previous studies have shown that schizophrenics have decreased expression of α7-nicotinic acetylcholine (α7) receptors in the hippocampus and other brain regions, paranoid delusions, disorganized speech, deficits in auditory gating (i.e., inability to inhibit neuronal responses to repetitive auditory stimuli), and difficulties in odor discrimination and detection. Here we use mice with decreased α7 expression that also show a deficit in auditory gating to determine if these mice have similar deficits in olfaction. In the adult mouse olfactory bulb (OB), α7 expression localizes in the glomerular layer; however, the functional role of α7 is unknown. We show that inbred mouse strains (i.e., C3H and C57) with varying α7 expression (e.g., α7 wild-type [α7+/+], α7 heterozygous knock-out [α7+/−] and α7 homozygous knockout mice [α7−/−]) significantly differ in odor discrimination and detection of chemically related odorant pairs. Using [125I] α-bungarotoxin (α-BGT) autoradiography, α7 expression was measured in the OB. As previously demonstrated, α-BGT binding was localized to the glomerular layer. Significantly more expression of α7 was observed in C57 α7+/+ mice compared to C3H α7+/+ mice. Furthermore, C57 α7+/+ mice were able to detect a significantly lower concentration of an odor in a mixture compared to C3H α7+/+ mice. Both C57 and C3H α7+/+ mice discriminated between chemically related odorants sooner than α7+/− or α7−/− mice. These data suggest that α7-nicotinic-receptors contribute strongly to olfactory discrimination and detection in mice and may be one of the mechanisms producing olfactory dysfunction in schizophrenics. PMID:20713028

  6. Nicotine effects on human endothelial intercellular communication via α4β2 and α3β2 nicotinic acetylcholine receptor subtypes.

    PubMed

    Duerrschmidt, Nicole; Hagen, Anja; Gaertner, Christiane; Wermke, Alice; Nowicki, Marcin; Spanel-Borowski, Katharina; Stepan, Holger; Mohr, Friedrich-Wilhelm; Dhein, Stefan

    2012-06-01

    Since previous in vitro experiments revealed that nicotine can impair endothelial intercellular communication via the downregulation of connexin43 (Cx43), we wanted to find out which nicotinic acetylcholine receptors are involved in the molecular mechanism of communication failure. Cultured human endothelial cells were exposed to 1 μM nicotine for 5 days. Intercellular communication was measured using dye transfer study with/without subtype-specific nicotinic acetylcholine receptor (nAChR) inhibitors. Reverse transcriptase (RT)-PCR was used to further investigate the regulation of nAChR subtypes. Electron microscopy together with MAP LC3-II western blot was used to investigate possible autophagy processes. In cultured human endothelial cells, nicotine decreased the Cx43 protein amount as shown by western blot and immunohistochemistry; however, together with an unaltered mRNA expression as shown by RT-PCR. The nicotine-induced Cx43 downregulation functionally impaired intercellular dye transfer, which could be prevented by mecamylamine, κ-bungarotoxin, lobeline, and dihydro-β-erythroidine but not α-bungarotoxin, indicating that the nAChR subtypes α4β2 and α3β2 but not α7 are involved in signal cascade. RT-PCR analysis revealed that nicotine exposure resulted in the upregulation of α3 and β4 and the downregulation of α4-nAChR, while α7- and β2-nAChR-mRNA expressions remained unaltered. Furthermore, nicotine increased total protein ubiquinylation and proteasome activity as was shown by immunohistochemistry and peptide degradation analysis. Evidence of enhanced autophagic processes was assured by the occurrence of autophagic vacuoles in transmission electron microscopy and enhanced formation of MAP LC3-II in western blot. Reduced intercellular endothelial communication together with programmed cell death helps to explain the toxic effect of nicotine leading to endothelial dysfunction. The nAChR involved in the impairment of intercellular communication

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

    PubMed

    Li, Ping; Ann, Jason; Akk, Gustav

    2011-08-01

    Neonicotinoids are synthetic, nicotine-derived insecticides used for agricultural and household pest control. Though highly effective at activating insect nicotinic receptors, many neonicotinoids are also capable of directly activating and/or modulating the activation of vertebrate nicotinic receptors. In this study, we have investigated the actions of the neonicotinoids clothianidin (CTD) and imidacloprid (IMI) on human neuronal α4β2 nicotinic acetylcholine receptors. The data demonstrate that the compounds are weak agonists of the human receptors with relative peak currents of 1-4% of the response to 1 mM acetylcholine (ACh). Coapplication of IMI strongly inhibited currents elicited by ACh. From Schild plot analysis, we estimate that the affinity of IMI for the human α4β2 receptor is 18 μM. The application of low concentrations of CTD potentiated responses to low concentrations of ACh, suggesting that receptors occupied by one ACh and one CTD molecule have a higher gating efficacy than receptors with one ACh bound. Interestingly, subunit stoichiometry affected inhibition by CTD, with (α4)(2) (β2)(3) receptors significantly more strongly inhibited than the (α4)(3) (β2)(2) receptors. Copyright © 2011 Wiley-Liss, Inc.

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

    PubMed Central

    Li, Ping; Ann, Jason; Akk, Gustav

    2013-01-01

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

  9. Novel and potent 6-chloro-3-pyridinyl ligands for the alpha4beta2 neuronal nicotinic acetylcholine receptor.

    PubMed

    Latli, B; D'Amour, K; Casida, J E

    1999-06-17

    1-[(6-Chloro-3-pyridinyl)methyl]-2-imidazolidine (1), the N-desnitro metabolite of the major insecticide imidacloprid, is known to have similar potency to that of (-)-nicotine as an inhibitor of [3H](-)-nicotine binding at the rat recombinant alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR); IC50 values in the present study are 3.8 nM for (-)-nicotine, 6.0 nM for 1, and 155 nM for imidacloprid. Synthesis of new analogues of 1, modified only in the heterocyclic moiety (five-, six-, or seven-membered rings with NH, S, O, and CH2 substituents), gave compounds varying from 4-fold higher potency (2-iminothiazole analogue 10) to >6000-fold less active than (-)-nicotine. Other potent N-[(6-chloro-3-pyridinyl)methyl] compounds are those in which the heterocyclic imine is replaced with pyrrolidine (19) (IC50 9 nM) or trimethylammonium (22) (IC50 18 nM). A novel conversion of (-)-nicotine to its 6-chloro analogue increased the potency 2-fold. These 6-chloro-3-pyridinyl compounds are of interest as novel nAChR probes and potential metabolites of candidate insecticides.

  10. Brain nicotinic acetylcholine receptor availability and response to smoking cessation treatment: a randomized trial.

    PubMed

    Brody, Arthur L; Mukhin, Alexey G; Mamoun, Michael S; Luu, Trinh; Neary, Meaghan; Liang, Lidia; Shieh, Jennifer; Sugar, Catherine A; Rose, Jed E; Mandelkern, Mark A

    2014-07-01

    Cigarette smoking leads to upregulation of nicotinic acetylcholine receptors (nAChRs) in the human brain, including the common α4β2* nAChR subtype. While subjective aspects of tobacco dependence have been extensively examined as predictors of quitting smoking with treatment, no studies to our knowledge have yet reported the relationship between the extent of pretreatment upregulation of nAChRs and smoking cessation. To determine whether the degree of nAChR upregulation in smokers predicts quitting with a standard course of treatment. Eighty-one tobacco-dependent cigarette smokers (volunteer sample) underwent positron emission tomographic (PET) scanning of the brain with the radiotracer 2-FA followed by 10 weeks of double-blind, placebo-controlled treatment with nicotine patch (random assignment). Pretreatment specific binding volume of distribution (VS/fP) on PET images (a value that is proportional to α4β2* nAChR availability) was determined for 8 brain regions of interest, and participant-reported ratings of nicotine dependence, craving, and self-efficacy were collected. Relationships between these pretreatment measures, treatment type, and outcome were then determined. The study took place at academic PET and clinical research centers. Posttreatment quit status after treatment, defined as a participant report of 7 or more days of continuous abstinence and an exhaled carbon monoxide level of 3 ppm or less. Smokers with lower pretreatment VS/fP values (a potential marker of less severe nAChR upregulation) across all brain regions studied were more likely to quit smoking (multivariate analysis of covariance, F8,69 = 4.5; P < .001), regardless of treatment group assignment. Furthermore, pretreatment average VS/fP values provided additional predictive power for likelihood of quitting beyond the self-report measures (stepwise binary logistic regression, likelihood ratio χ21 = 19.8; P < .001). Smokers with less upregulation of available α4β2* n

  11. Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system.

    PubMed

    Obaid, A L; Nelson, M E; Lindstrom, J; Salzberg, B M

    2005-08-01

    Nicotinic transmission in the enteric nervous system (ENS) is extensive, but the role of individual nicotinic acetylcholine receptor (nAChR) subtypes in the functional connectivity of its plexuses has been elusive. Using monoclonal antibodies (mAbs) against neuronal alpha3-, alpha4-, alpha3/alpha5-, beta2-, beta4- and alpha7-subunits, combined with radioimmunoassays and immunocytochemistry, we demonstrate that guinea-pig enteric ganglia contain all of these nAChR-subunits with the exception of alpha4, and so, differ from mammalian brain. This information alone, however, is insufficient to establish the functional role of the identified nAChR-subtypes within the enteric networks and, ultimately, their specific contributions to gastrointestinal physiology. We have used voltage-sensitive dyes and a high-speed CCD camera, in conjunction with specific antagonists to various nAChRs, to elucidate some of the distinct contributions of the individual subtypes to the behaviour of enteric networks. In the guinea-pig, the submucous plexus has the extraordinary advantage that it is virtually two-dimensional, permitting optical recording, with single cell resolution, of the electrical activity of all of its neurones. In this plexus, the block of alpha3beta2-, alpha3beta4- and/or alpha7-nAChRs always results in a decrease in the magnitude of the synaptic response. However, the magnitude of the fast excitatory post-synaptic potentials (epsps) evoked by electrical stimulation of a neighbouring ganglion varies from cell to cell, reflecting the differential expression of subunits already observed using mAbs, as well as the strengths of the activated synaptic inputs. At the same time, we observe that submucous neurones have a substantial mecamylamine (Mec)-insensitive (non-nicotinic) component to their fast epsps, which may point to the presence of purinergic or serotonergic fast epsps in this system. In the myenteric plexus, on the other hand, the antagonist-induced changes in the

  12. Effect of secondhand smoke on occupancy of nicotinic acetylcholine receptors in brain.

    PubMed

    Brody, Arthur L; Mandelkern, Mark A; London, Edythe D; Khan, Aliyah; Kozman, Daniel; Costello, Matthew R; Vellios, Evan E; Archie, Meena M; Bascom, Rebecca; Mukhin, Alexey G

    2011-09-01

    Despite progress in tobacco control, secondhand smoke (SHS) exposure remains prevalent worldwide and is implicated in the initiation and maintenance of cigarette smoking. To determine whether moderate SHS exposure results in brain α(4)β(2)* nicotinic acetylcholine receptor (nAChR) occupancy. Positron emission tomography scanning and the radiotracer 2-[18F]fluoro-3-(2(S)azetidinylmethoxy) pyridine (also known as 2-[(18)F]fluoro-A-85380, or 2-FA) were used to determine α(4)β(2)* nAChR occupancy from SHS exposure in 24 young adult participants (11 moderately dependent cigarette smokers and 13 nonsmokers). Participants underwent two bolus-plus-continuous-infusion 2-FA positron emission tomography scanning sessions during which they sat in the passenger's seat of a car for 1 hour and either were exposed to moderate SHS or had no SHS exposure. The study took place at an academic positron emission tomography center. Main Outcome Measure  Changes induced by SHS in 2-FA specific binding volume of distribution as a measure of α(4)β(2)* nAChR occupancy. An overall multivariate analysis of variance using specific binding volume of distribution values revealed a significant main effect of condition (SHS vs control) (F(1,22) = 42.5, P < .001) but no between-group (smoker vs nonsmoker) effect. Exposure to SHS led to a mean 19% occupancy of brain α(4)β(2)* nAChRs (1-sample t test, 2-tailed, P < .001). Smokers had both a mean 23% increase in craving with SHS exposure and a correlation between thalamic α(4)β(2)* nAChR occupancy and craving alleviation with subsequent cigarette smoking (Spearman ρ = -0.74, P = .01). Nicotine from SHS exposure results in substantial brain α(4)β(2)* nAChR occupancy in smokers and nonsmokers. Study findings suggest that such exposure delivers a priming dose of nicotine to the brain that contributes to continued cigarette use in smokers. This study has implications for both biological research into the link between SHS exposure

  13. CHRNA4 and ANKK1 Polymorphisms Influence Smoking-Induced Nicotinic Acetylcholine Receptor Upregulation

    PubMed Central

    Esterlis, Irina; Hillmer, Ansel T.; Bois, Frederic; Pittman, Brian; McGovern, Erin; O’Malley, Stephanie S.; Picciotto, Marina R.; Yang, Bao-zhu; Gelernter, Joel

    2016-01-01

    Introduction: Tobacco smoking leads to increased numbers of β2*-containing nicotinic acetylcholine receptors (β2*-nAChRs) throughout the brain, which return to nonsmoker levels over extended abstinence. The goal of the current study was to determine whether the degree of tobacco smoking-induced changes in β2*-nAChR availability is genetically influenced. Methods: In this study, 113 European Americans participated in one or two [123I]5-IA-85380 single photon emission computed tomography (SPECT) brain scans. Smokers (n = 58) participated in one scan at 7–9 days of abstinence and those who remained abstinent (n = 27) were imaged again at 6–8 weeks of abstinence. Age- and sex-matched nonsmokers (n = 55) participated in one scan. Blood samples were collected for DNA analysis and genotyped for single nucleotide polymorphisms (SNPs) in the CHRNA4 and ANKK1 gene loci. β2*-nAChR availability was measured in the thalamus, striatum, cortical regions, and cerebellum. Results: The CHRNA4 SNP rs2236196 and ANKK1 SNP rs4938015 were associated with significantly higher cerebellar and cortical β2*-nAChR availability in smokers versus nonsmokers for specific genotypes. There were no significant differences by carrier status in the change in β2*-nAChR availability in smokers from 7–9 days to 6–8 weeks of abstinence. Conclusion: This study provides evidence for genetic regulation of tobacco smoking-induced changes in β2*-nAChR availability and suggests that β2*-nAChR availability could be an endophenotype mediating influences of CHRNA4 variants on nicotine dependence. These results highlight individual differences in the neurochemistry of nicotine dependence and may suggest the need for individualized programs for smoking cessation. Implications: This study demonstrates genetic regulation of smoking-induced changes in β2*-nAChRs throughout the brain and highlights the need for personalized programs for smoking cessation. PMID:27611310

  14. Interaction of Bupropion with Muscle-Type Nicotinic Acetylcholine Receptors in Different Conformational States†

    PubMed Central

    Arias, Hugo R.; Gumilar, Fernanda; Rosenberg, Avraham; Targowska-Duda, Katarzyna M.; Feuerbach, Dominik; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W.; Bouzat, Cecilia

    2009-01-01

    To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion: (a) inhibits epibatidine-induced Ca2+ influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with ~100-fold higher potency compared to that in the open state, (c) increases desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits [3H]TCP and [3H]imipramine binding to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/α-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy–driven process, and (f) interacts with a binding domain located between the serine (position 6’) and valine (position 13’) rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process. PMID:19334677

  15. Spectroscopic investigation of the nicotinic acetylcholine receptor for application in medical diagnosis

    NASA Astrophysics Data System (ADS)

    Salzer, Reiner; Fischer, Wolfgang B.; Unverricht, Ines; Schwenke, Dirk; Steiner, Gerald; Schrattenholz, Andre; Maelicke, Alfred

    1998-04-01

    Native vesicles containing the nicotinic acetylcholine receptor (nAChR) prepared from the electric organ of the ray Torpedo marmorata were used to obtain fluorescence signal sin dependence of different concentrations of the local anesthetics procaine. Nonlinear concentration dependent spectral changes are found using ethidium bromide as a fluorescence marker. Structural changes are found for the proteins including the nAChR in the vesicles during immobilization onto surfaces such as IR transparent germanium (GE) crystal, Ge crystal coated with silver (Ag) cluster to use the SEIRA effect and/or crystals covered with a lipid subphase. A comparison between Ge and Ge coated with Ag (Ge/Ag) clusters reveals increased structural changes in the spectral regions around 1670 cm-1 upon adsorption of the vesicles on the latter surface. Is the Ge/Ag crystal precoated with a lipid subphase an almost similar spectral contour for the amide I band envelope as in the spectra recorded on a neat Ge crystal is found.

  16. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees.

    PubMed

    Moffat, Christopher; Buckland, Stephen T; Samson, Andrew J; McArthur, Robin; Chamosa Pino, Victor; Bollan, Karen A; Huang, Jeffrey T-J; Connolly, Christopher N

    2016-04-28

    There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species.

  17. Muscarinic and Nicotinic Acetylcholine Receptor Agonists and Allosteric Modulators for the Treatment of Schizophrenia

    PubMed Central

    Jones, Carrie K; Byun, Nellie; Bubser, Michael

    2012-01-01

    Muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs) are emerging as important targets for the development of novel treatments for the symptoms associated with schizophrenia. Preclinical and early proof-of-concept clinical studies have provided strong evidence that activators of specific mAChR (M1 and M4) and nAChR (α7 and α2β4) subtypes are effective in animal models of antipsychotic-like activity and/or cognitive enhancement, and in the treatment of positive and cognitive symptoms in patients with schizophrenia. While early attempts to develop selective mAChR and nAChR agonists provided important preliminary findings, these compounds have ultimately failed in clinical development due to a lack of true subtype selectivity and subsequent dose-limiting adverse effects. In recent years, there have been major advances in the discovery of highly selective activators for the different mAChR and nAChR subtypes with suitable properties for optimization as potential candidates for clinical trials. One novel strategy has been to identify ligands that activate a specific receptor subtype through actions at sites that are distinct from the highly conserved ACh-binding site, termed allosteric sites. These allosteric activators, both allosteric agonists and positive allosteric modulators, of mAChR and nAChR subtypes demonstrate unique mechanisms of action and high selectivity in vivo, and may provide innovative treatment strategies for schizophrenia. PMID:21956443

  18. 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.

  19. In vivo knockdown of nicotinic acetylcholine receptor α1 diminishes aortic atherosclerosis

    PubMed Central

    Zhang, Guoqiang; Marshall, Amanda L.; Thomas, Alison L.; Kernan, Kelly A.; Su, Yanyuan; LeBoeuf, Renee C.; Dong, Xiu Rong; Tchao, B.N. Angela

    2013-01-01

    Objective Nicotinic acetylcholine receptor α1 (nAChRα1) was recently identified as a functional cell receptor for urokinase, a potent atherogenic molecule. Here, we test the hypothesis that nAChRα1 plays a role in the pathogenesis of atherosclerosis. Methods Apolipoprotein E-deficient mice were initially fed a Western diet for 8 wks. Plasmid DNA encoding scramble RNA (pscr) or siRNA (psir2) for nAChRα1 was injected into the mice (n = 16) using an aortic hvdrodvnamic gene transfer protocol. Four mice from each group were sacrificed 7 days after the DNA injection to confirm the nAChRα1 gene silencing. The remaining mice continued on a Western diet for an additional 16 wks. Results The nAChRα1 was up-regulated in aortic atherosclerotic lesions. A 78% knockdown of the nAChRα1 gene resulted in remarkably less severe aortic plaque growth and neovascularization at 16 wks (both P< 0.05). In addition, significantly fewer macrophages (60% less) and myofibroblasts (80% less) presented in the atherosclerotic lesion of the psir2-treated mice. The protective mechanisms of the nAChRα1 knockdown may involve up-regulating interferon-γ/Y box protein-1 activity and down-regulating transforming growth factor-β expression. Conclusions The nAChRα1 gene plays a significant role at the artery wall, and reducing its expression decreases aortic plaque development. PMID:20810113

  20. Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor.

    PubMed

    Livett, Bruce G; Sandall, David W; Keays, David; Down, John; Gayler, Ken R; Satkunanathan, Narmatha; Khalil, Zeinab

    2006-12-01

    Pain therapeutics discovered by molecular mining of the expressed genome of Australian predatory cone snails are providing lead compounds for the treatment of neurological diseases such as multiple sclerosis, shingles, diabetic neuropathy and other painful neurological conditions. The high specificity exhibited by these novel compounds for neuronal receptors and ion channels in the brain and nervous system indicates the high degree of selectivity that this class of neuropeptides can be expected to show when used therapeutically in humans. A lead compound, ACV1 (conotoxin Vc1.1 from Conus victoriae), has entered Phase II clinical trials and is being developed for the treatment for neuropathic pain. ACV1 will be targeted initially for the treatment of sciatica, shingles and diabetic neuropathy. The compound is a 16 amino acid peptide [Sandall et al., 2003. A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo. Biochemistry 42, 6904-6911], an antagonist of neuronal nicotinic acetylcholine receptors. It has potent analgesic activity following subcutaneous or intramuscular administration in several preclinical animal models of human neuropathic pain [Satkunanathan et al., 2005. Alpha conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurons. Brain. Res. 1059, 149-158]. ACV1 may act as an analgesic by decreasing ectopic excitation in sensory nerves. In addition ACV1 appears to accelerate the recovery of injured nerves and tissues.

  1. Stimulation of nicotinic acetylcholine alpha7 receptors rescue schizophrenia-like cognitive impairments in rats.

    PubMed

    Potasiewicz, Agnieszka; Nikiforuk, Agnieszka; Hołuj, Małgorzata; Popik, Piotr

    2017-02-01

    Alpha7 nicotinic acetylcholine receptor (α7 nAChR) dysfunction plays an important role in schizophrenia. Positive allosteric modulators of α7 nAChR have emerged as a promising therapeutic approach to manage cognitive deficits that are inadequately treated in schizophrenic patients. The aim of the present study was to evaluate the ability of type I (CCMI) and type II (PNU120596) α7 nAChR positive allosteric modulators to counteract MK-801-induced cognitive and sensorimotor gating deficits. The activity of these compounds was compared with the action of the α7 nAChR agonist A582941. CCMI, PNU120596 and A582941 reversed the sensorimotor gating impairment evoked by MK-801 based on the prepulse inhibition of the startle response. Additionally, no MK-801-evoked working memory deficits were observed with α7 nAChR ligand pretreatment as assessed in a discrete paired-trial delayed alternation task. However, these compounds did not affect the rats' attentional performances in the five-choice serial reaction time test. The α7 nAChR agents demonstrated a beneficial effect on sensorimotor gating and some aspects of cognition tested in a rat model of schizophrenia. Therefore, these results support the use of α7 nAChR positive allosteric modulators as a potential treatment strategy in schizophrenia.

  2. Alpha7 Nicotinic Acetylcholine Receptor Is a Target in Pharmacology and Toxicology

    PubMed Central

    Pohanka, Miroslav

    2012-01-01

    Alpha7 nicotinic acetylcholine receptor (α7 nAChR) is an important part of the cholinergic nerve system in the brain. Moreover, it is associated with a cholinergic anti-inflammatory pathway in the termination of the parasympathetic nervous system. Antagonists of α7 nAChR are a wide group represented by conotoxin and bungarotoxin. Even Alzheimer’s disease drug memantine acting as an antagonist in its side pathway belongs in this group. Agonists of α7 nAChR are suitable for treatment of multiple cognitive dysfunctions such as Alzheimer’s disease or schizophrenia. Inflammation or even sepsis can be ameliorated by the agonistic acting compounds. Preparations RG3487, SEN34625/WYE-103914, SEN12333, ABT-107, Clozapine, GTS-21, CNI-1493, and AR-R17779 are representative examples of the novel compounds with affinity toward the α7 nAChR. Pharmacological, toxicological, and medicinal significance of α7 nAChR are discussed throughout this paper. PMID:22408449

  3. An essential role of acetylcholine-glutamate synergy at habenular synapses in nicotine dependence

    PubMed Central

    Frahm, Silke; Antolin-Fontes, Beatriz; Görlich, Andreas; Zander, Johannes-Friedrich; Ahnert-Hilger, Gudrun; Ibañez-Tallon, Ines

    2015-01-01

    A great deal of interest has been focused recently on the habenula and its critical role in aversion, negative-reward and drug dependence. Using a conditional mouse model of the ACh-synthesizing enzyme choline acetyltransferase (Chat), we report that local elimination of acetylcholine (ACh) in medial habenula (MHb) neurons alters glutamate corelease and presynaptic facilitation. Electron microscopy and immuno-isolation analyses revealed colocalization of ACh and glutamate vesicular transporters in synaptic vesicles (SVs) in the central IPN. Glutamate reuptake in SVs prepared from the IPN was increased by ACh, indicating vesicular synergy. Mice lacking CHAT in habenular neurons were insensitive to nicotine-conditioned reward and withdrawal. These data demonstrate that ACh controls the quantal size and release frequency of glutamate at habenular synapses, and suggest that the synergistic functions of ACh and glutamate may be generally important for modulation of cholinergic circuit function and behavior. DOI: http://dx.doi.org/10.7554/eLife.11396.001 PMID:26623516

  4. 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

    2015-03-06

    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.

  5. α7-Nicotinic Acetylcholine Receptor: Role in Early Odor Learning Preference in Mice

    PubMed Central

    Hellier, Jennifer L.; Arevalo, Nicole L.; Smith, Lynelle; Xiong, Ka-Na; Restrepo, Diego

    2012-01-01

    Recently, we have shown that mice with decreased expression of α7-nicotinic acetylcholine receptors (α7) in the olfactory bulb were associated with a deficit in odor discrimination compared to wild-type mice. However, it is unknown if mice with decreased α7-receptor expression also show a deficit in early odor learning preference (ELP), an enhanced behavioral response to odors with attractive value observed in rats. In this study, we modified ELP methods performed in rats and implemented similar conditions in mice. From post-natal days 5–18, wild-type mice were stroked simultaneously with an odor presentation (conditioned odor) for 90 s daily. Control mice were only stroked, exposed to odor, or neither. On the day of testing (P21), mice that were stroked in concert with a conditioned odor significantly investigated the conditioned odor compared to a novel odor, as observed similarly in rats. However, mice with a decrease in α7-receptor expression that were stroked during a conditioned odor did not show a behavioral response to that odorant. These results suggest that decreased α7-receptor expression has a role in associative learning, olfactory preference, and/or sensory processing deficits. PMID:22514723

  6. Mesoionic insecticides: a novel class of insecticides that modulate nicotinic acetylcholine receptors.

    PubMed

    Holyoke, Caleb W; Cordova, Daniel; Zhang, Wenming; Barry, James D; Leighty, Robert M; Dietrich, Robert F; Rauh, James J; Pahutski, Thomas F; Lahm, George P; Tong, My-Hanh Thi; Benner, Eric A; Andreassi, John L; Smith, Rejane M; Vincent, Daniel R; Christianson, Laurie A; Teixeira, Luis A; Singh, Vineet; Hughes, Kenneth A

    2017-04-01

    As the world population grows towards 9 billion by 2050, it is projected that food production will need to increase by 60%. A critical part of this growth includes the safe and effective use of insecticides to reduce the estimated 20-49% loss of global crop yields owing to pests. The development of new insecticides will help to sustain this protection and overcome insecticide resistance. A novel class of mesoionic compounds has been discovered, with exceptional insecticidal activity on a range of Hemiptera and Lepidoptera. These compounds bind to the orthosteric site of the nicotinic acetylcholine receptor and result in a highly potent inhibitory action at the receptor with minimal agonism. The synthesis, biological activity, optimization and mode of action will be discussed. Triflumezopyrim insect control will provide a powerful tool for control of hopper species in rice throughout Asia. Dicloromezotiaz can provide a useful control tool for lepidopteran pests, with an underexploited mode of action among these pests. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  7. Targeted drug delivery system to neural cells utilizes the nicotinic acetylcholine receptor.

    PubMed

    Huey, Rachel; O'Hagan, Barry; McCarron, Paul; Hawthorne, Susan

    2017-06-15

    Drug delivery to the brain is still a major challenge in the field of therapeutics, especially for large and hydrophilic compounds. In order to achieve drug delivery of therapeutic concentration in the central nervous system, the problematic blood brain barrier (BBB) must be overcome. This work presents the formulation of a targeted nanoparticle-based drug delivery system using a specific neural cell targeting ligand, rabies virus derived peptide (RDP). Characterization studies revealed that RDP could be conjugated to drug-loaded PLGA nanoparticles of average diameter 257.10±22.39nm and zeta potential of -5.51±0.73mV. In vitro studies showed that addition of RDP to nanoparticles enhanced drug accumulation in a neural cell line specifically as opposed to non-neural cell lines. It was revealed that this drug delivery system is reliant upon nicotinic acetylcholine receptor (nAChR) function for RDP-facilitated effects, supporting a cellular uptake mechanism of action. The specific neural cell targeting capabilities of RDP via the nAChR offers a non-toxic, non-invasive and promising approach to the delivery of therapeutics to the brain. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  8. Acetylcholine regulation of nicotinic receptor channels through a putative G protein in chick myotubes.

    PubMed Central

    Eusebi, F; Grassi, F; Molinaro, M; Zani, B M

    1987-01-01

    1. Single-channel currents induced by acetylcholine (ACh) were recorded from unstriated and non-innervated embryonic chick myotubes using the cell-attached patch-clamp technique. 2. ACh applied to the non-patched membrane decreased both channel opening probability and conductance. These ACh-induced effects occurred also when the non-patched membrane was exposed to nominally Ca2+-free extracellular medium, but were absent when it was treated with curare. 3. ACh-induced membrane current recorded under whole-cell patch-clamp conditions decreased in amplitude and time course when myotubes were intracellularly loaded with guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), but not with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) or cyclic adenosine-5'-monophosphate (cyclic AMP). Internal perfusion of GTP gamma S affected the ACh-induced openings in a similar manner to the non-patch ACh application. 4. These results suggest that ACh, in addition to its direct effect, acts indirectly on the nicotinic receptor channels by delivering an intracellular messenger and through the activation of a putative G protein. PMID:2451747

  9. NMR Structure and Dynamics of a Designed Water-soluble Transmembrane Domain of Nicotinic Acetylcholine Receptor

    PubMed Central

    Cui, Tanxing; Mowrey, David; Bondarenko, Vasyl; Tillman, Tommy; Ma, Dejian; Landrum, Elizabeth; Perez-Aguilar, Jose Manuel; He, Jing; Wang, Wei; Saven, Jeffery G.; Eckenhoff, Roderic G.; Tang, Pei; Xu, Yan

    2011-01-01

    The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ∼83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T2 dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites. PMID:22155685

  10. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees

    NASA Astrophysics Data System (ADS)

    Moffat, Christopher; Buckland, Stephen T.; Samson, Andrew J.; McArthur, Robin; Chamosa Pino, Victor; Bollan, Karen A.; Huang, Jeffrey T.-J.; Connolly, Christopher N.

    2016-04-01

    There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species.

  11. alpha7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not beta-amyloid-induced neurotoxicity.

    PubMed

    de Fiebre, Nancyellen C; de Fiebre, Christopher M

    2005-01-03

    The alpha7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, beta-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from alpha7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, "binge" ethanol and beta-amyloid. Knockout of the alpha7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to beta-amyloid induced toxicity, however, was unaffected by alpha7 nAChR gene null mutation. Further, beta-amyloid did not inhibit the binding of the highly alpha7-selective radioligand, [(125)I]alpha-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited alpha7 nAChR function. These data suggest that alpha7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by beta-amyloid. It is hypothesized that inhibition of alpha7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.

  12. 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

  13. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees

    PubMed Central

    Moffat, Christopher; Buckland, Stephen T.; Samson, Andrew J.; McArthur, Robin; Chamosa Pino, Victor; Bollan, Karen A.; Huang, Jeffrey T.-J.; Connolly, Christopher N.

    2016-01-01

    There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species. PMID:27124107

  14. Nicotine enhances hepatocyte growth factor-mediated lung cancer cell migration by activating the α7 nicotine acetylcholine receptor and phosphoinositide kinase-3-dependent pathway.

    PubMed

    Yoneyama, Remi; Aoshiba, Kazutetsu; Furukawa, Kinya; Saito, Makoto; Kataba, Hiroaki; Nakamura, Hiroyuki; Ikeda, Norihiko

    2016-01-01

    Cigarette smoking not only promotes lung carcinogenesis, but it has also been demonstrated to promote the progression of lung cancer. Despite nicotine being a major component of cigarette smoke, it is not carcinogenic when acting alone. Instead, it is believed to function as a tumor promoter. Due to the fatal consequences of lung cancer being primarily associated with the processes of invasion and metastasis, the present study aimed to determine the effect of nicotine on the migratory activity of lung cancer cells. The effect of nicotine on the migration of lung cancer A549 cells was evaluated by a wound healing assay. Hepatocyte growth factor (HGF) was used as a pro-migratory stimulus. During several of the experiments, specific inhibitors of α7-nicotine acetylcholine receptor (α7-nAchR), phosphoinositide kinase-3 (PI3K) and extracellular signal-related kinase (ERK)1/2 were included. The phosphorylation levels of Akt and ERK1/2 were examined using a cell-based protein phosphorylation assay. It was observed that nicotine did not induce cell migration by itself, but that it instead promoted HGF-induced cell migration. The effects of nicotine were inhibited by the pretreatment of the cells with the α7-nAchR inhibitor, methyllycaconitine, and the PI3K inhibitor, LY294002. The mitogen-activated protein kinase/ERK kinase kinase inhibitor exerted modest, but non-significant inhibitory activity on the effect of nicotine. Nicotine did not induce Akt phosphorylation by itself, but instead promoted the HGF-induced phosphorylation of Akt. It was also observed that nicotine had no effect on ERK1/2 phosphorylation. The results from the present study indicate that nicotine, when alone, does not have a pro-migratory function, but instead enhances responsiveness to the pro-migratory stimulus emitted by HGF. The current study provides an insight into the mechanism of tumor promotion by demonstrating that nicotine and α7-nAchRs act in synergy with the HGF-induced PI3K

  15. Molecular dissection of tropisetron, an alpha7 nicotinic acetylcholine receptor-selective partial agonist.

    PubMed

    Papke, Roger L; Schiff, Hillary C; Jack, Brian A; Horenstein, Nicole A

    2005-04-22

    The alpha7 nicotinic acetylcholine receptor (nAChR)-selective partial agonist tropisetron is a conjugate of an indole and a tropane group. We tested compounds structurally related to either the indole or tropane domains of tropisetron on oocytes expressing human alpha7. alpha4beta2, or alpha3beta4 nAChR or rat 5HT(3A) receptors. The simple compounds tropane and tropinone had alpha7-selective agonist activity comparable to that of tropisetron. Tropinone was more efficacious than tropisetron but 100-fold less potent. Some tropane compounds had antagonist activity on alpha3beta4 nAChR but no effect on alpha4beta2 nAChR. Some tropanes also affected the responses of 5HT3 receptors to serotonin. Tropisetron was more potent at inhibiting alpha3beta4 receptors (IC(50)=1.8+/-0.6) than was tropane or tropinone, suggesting that the presence of the indole group has a large impact on the potency of tropisetron, both as an alpha7 agonist and as an alpha3beta4 antagonist. The further reduced structures of dimethyl piperidinium and 1-methylpyrrolidine also had agonist activity on alpha7 receptors, suggesting that the minimal activating pharmacophore of these compounds, as with tetramethylammonium, may simply be the charged nitrogen, while additional structure elements impact subtype selectivity, potency, and efficacy. It has previously been reported that 5-hydroxyindole (5HI) can potentiate alpha7 receptor responses to acetylcholine (ACh). However, the site where 5HI binds to the receptor is not known. We tested the hypothesis that the tropisetron binding site might overlap the 5HI site and thereby produce a block of 5HI potentiation. Our results indicate that the indole portion of tropisetron is not likely to be binding to the same site where 5HI binds to potentiate alpha7 receptor responses since 5HI can greatly potentiate responses of tropisetron, tropinone, and other partial agonists such as 4OH-GTS-21.

  16. Nicotinic acetylcholine receptors in dorsal root ganglion neurons include the α6β4* subtype.

    PubMed

    Hone, Arik J; Meyer, Erin L; McIntyre, Melissa; McIntosh, J Michael

    2012-02-01

    The α6-containing nicotinic acetylcholine receptors (nAChRs) have recently been implicated in diseases of the central nervous system (CNS), including Parkinson's disease and substance abuse. In contrast, little is known about the role of α6* nAChRs in the peripheral nervous system (where the asterisk denotes the possible presence of additional subunits). Dorsal root ganglia (DRG) neurons are known to express nAChRs with a pharmacology consistent with an α7, α3β4*, and α4β2* composition. Here we present evidence that DRG neurons also express α6* nAChRs. We used RT-PCR to show the presence of α6 subunit transcripts and patch-clamp electrophysiology together with subtype-selective α-conotoxins to pharmacologically characterize the nAChRs in rat DRG neurons. α-Conotoxin BuIA (500 nM) blocked acetylcholine-gated currents (I(ACh)) by 90.3 ± 3.0%; the recovery from blockade was very slow, indicating a predominance of α(x)β4* nAChRs. Perfusion with either 300 nM BuIA[T5A;P6O] or 200 nM MII[E11A], α-conotoxins that target the α6β4* subtype, blocked I(ACh) by 49.3 ± 5 and 46.7 ± 8%, respectively. In these neurons, I(ACh) was relatively insensitive to 200 nM ArIB[V11L;V16D] (9.4±2.0% blockade) or 500 nM PnIA (23.0±4% blockade), α-conotoxins that target α7 and α3β2*/α6β2* nAChRs, respectively. We conclude that α6β4* nAChRs are among the subtypes expressed by DRG, and to our knowledge, this is the first demonstration of α6β4* in neurons outside the CNS.

  17. Pharmacological characterization of nicotine-induced acetylcholine release in the rat hippocampus in vivo: evidence for a permissive dopamine synapse

    PubMed Central

    Reid, Richard T; Lloyd, G Kenneth; Rao, Tadimeti S

    1999-01-01

    In this study, the mechanism of nicotine-induced hippocampal acetylcholine (ACh) release in awake, freely moving rats was examined using in vivo microdialysis.Systemic administration of nicotine (0.4 mg kg−1, s.c.) increased the levels of ACh in hippocampal dialysates.The nicotine-induced hippocampal ACh release was sensitive to the pretreatment of neuronal nicotinic acetylcholine receptor (nAChR) antagonists mecamylamine (3.0 mg kg−1, s.c.) and dihydro-β-erythrodine (DHβE; 4.0 mg kg−1, s.c.) as well as systemic administration of the dopamine (DA) D1 receptor antagonist SCH-23390 (R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-benzazepine; 0.3 mg kg−1, s.c.).Local perfusion of mecamylamine (100 μM), DHβE (100 μM) or SCH-23390 (10 μM) through microdialysis probe did not increase basal hippocampal ACh release.Hippocampal ACh release elicited by systemic administration of nicotine (0.4 mg kg−1, s.c.) was antagonized by local perfusion of SCH-23390 (10 μM), but not by MEC (100 μM) or DHβE (100 μM).Direct perfusion of nicotine (1 mM, but not 0.1 mM) increased hippocampal ACh levels; however, this effect was relatively insensitive to blockade by co-perfusion of either mecamylamine (100 μM) or SCH-23390 (10 μM).These results suggest that nicotine-induced hippocampal ACh release occurs by two distinct mechanisms: (1) activation of nAChRs outside the hippocampus leading to DA release and subsequent ACh release involving a permissive DA synapse, and (2) direct action of nicotine within the hippocampus leading to ACh release via non-DA-ergic mechanism. PMID:10455300

  18. Release of acetylcholine from murine embryonic stem cells: effect of nicotinic and muscarinic receptors and blockade of organic cation transporter.

    PubMed

    Wessler, Ignaz; Michel-Schmidt, Rosmarie; Dohle, Eva; Kirkpatrick, Charles James

    2012-11-27

    The non-neuronal cholinergic system is widely expressed in nature. The present experiments were performed to characterize the non-neuronal cholinergic system in murine embryonic stem cells (CGR8 cell line). CGR8 cells were cultured in gelatinized flasks with Glasgow's buffered minimal essential medium (Gibco, Germany). Acetylcholine was measured by HPLC combined with bioreactor and electrochemical detection. CGR8 cells contained 1.08±0.12 pmol acetylcholine/10(6) cells (n=7) which was reduced to 0.50±0.06 pmol/10(6) cells (n=6; p<0.05) in the presence (4h) of 30μM bromoacetylcholine to block choline acetyltransferase. A time-dependent release of acetylcholine into the incubation medium was demonstrated, when cholinesterase activity was blocked by 10 μM physostigmine, with 97±13, 180±15 and 216±14 pmol being released from 65×10(6) cells after incubation periods of 2, 4 and 6h, respectively. The cumulative release corresponds to a fractional release rate of 2%/min. Blockade of nicotine or muscarine receptors did not significantly modulate the release of acetylcholine which was substantially reduced by 300 μM quinine (inhibitor of organic cation transporters). This inhibition showed considerable fading over the incubation period, indicating additional release mechanisms activated upon inhibition of organic cation transporters. Murine embryonic stem cells contain and release significant amounts of acetylcholine. The high fractional release rate and the compensation for blocked organic cation transporters indicate that non-neuronal acetylcholine may play a functional role in the homeostasis of murine embryonic stem cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Potential of α7 nicotinic acetylcholine receptor PET imaging in atherosclerosis.

    PubMed

    Boswijk, Ellen; Bauwens, Matthias; Mottaghy, Felix M; Wildberger, Joachim E; Bucerius, Jan

    2017-11-01

    Atherosclerotic events are usually acute and often strike otherwise asymptomatic patients. Although multiple clinical risk factors have been associated with atherosclerosis, as of yet no further individual prediction can be made as to who will suffer from its consequences based on biomarker analysis or traditional imaging methods like CT, MRI or angiography. Previously, non-invasive imaging with 18 F-fluorodeoxyglucose ( 18 F-FDG) PET was shown to potentially fill this niche as it offers high sensitive detection of metabolic processes associated with inflammatory changes in atherosclerotic plaques. However, 18 F-FDG PET imaging of arterial vessels suffers from non-specificity and has still to be proven to reliably identify vulnerable plaques, carrying a high risk of rupture. Therefore, it may be regarded only as a secondary marker for monitoring treatment effects and it does not offer alternative treatment options or direct insight in treatment mechanisms. In this review, an overview is given of the current status and the potential of PET imaging of inflammation and angiogenesis in atherosclerosis in general and special emphasis is given to imaging of α7 nicotinic acetylcholine receptors (α7 nAChRs). Due to the gaps that still exist in our understanding of atherogenesis and the limitations of the available PET tracers, the search continues for a more specific radioligand, able to differentiate between stable atherosclerosis and plaques prone to rupture. The potential role of the α7 nAChR as imaging marker for plaque vulnerability is explored. Today, strong evidence exists that nAChRs are involved in the atherosclerotic disease process. They are suggested to mediate the deleterious effects of the major tobacco component, nicotine, a nAChR agonist. Mainly based on in vitro data, α7 nAChR stimulation might increase plaque burden via increased neovascularization. However, in animal studies, α7 nAChR manipulation appears to reduce plaque size due to its inhibitory

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

    PubMed Central

    Taillebois, Emiliane; Beloula, Abdelhamid; Quinchard, Sophie; Jaubert-Possamai, Stéphanie; Daguin, Antoine; Servent, Denis; Tagu, Denis

    2014-01-01

    Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16±0.04 nM and 41.7±5.9 nM) and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008±0.002 nM and 1.135±0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml) and TMX (LC50 = 0.034 µg/ml) were more toxic than CLT (LC50 = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies. PMID:24801634

  1. Role of neuronal nicotinic acetylcholine receptors (nAChRs) on learning and memory in zebrafish.

    PubMed

    Braida, Daniela; Ponzoni, Luisa; Martucci, Roberta; Sparatore, Fabio; Gotti, Cecilia; Sala, Mariaelvina

    2014-05-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) play a modulatory role in cognition, and zebrafish provide a preclinical model to study learning and memory. We investigated the effect of nicotine (NIC) and some new cytisine-derived partial agonists (CC4 and CC26) on spatial memory in zebrafish using a rapid assay on T-maze task. The role of α4/α6β2 and the α7 nAChRs in NIC-induced memory enhancement was evaluated using selective nAChR antagonists. Low and high doses of NIC, cytisine (CYT), CC4 and CC26 respectively improved and worsened the mean running time, showing an inverted U dose-response function. The effective dose (ED50) (×10⁻⁵ mg/kg) was 0.4 for CC4, 4.5 for CYT, 140 for NIC and 200 for CC26. NIC-induced cognitive enhancement was reduced by the selective nAChR subtype antagonists: methyllycaconitine (MLA) for α7, α-conotoxin (MII) for α6β2, dihydro-β-erythroidine (DhβE) for α4β2, the nonselective antagonist mecamylamine (MEC) and the muscarinic antagonist scopolamine (SCOP), with DhβE being more active than MLA or MII. All the partial agonists blocked the cognitive enhancement. The improvement with the maximal active dose of each partial agonist was blocked by low doses of DhβE (0.001 mg/kg) and MII (0.01 mg/kg). MLA reduced the effects of CC26 and CC4 at doses of 0.01 and 1 mg/kg, respectively, but did not antagonize CYT-induced memory improvement at any of the tested dose. No change in swimming activity was observed. Our findings demonstrate that zebrafish make a useful model for the rapid screening of the effect of new α4β2 nAChR compounds on spatial memory.

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

    PubMed

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

    2014-01-01

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

  3. Structure-activity relationships of acetylcholine derivatives with Lucilia cuprina nicotinic acetylcholine receptor α1 and α2 subunits in chicken β2 subunit hybrid receptors in comparison with chicken nicotinic acetylcholine receptor α4/β2.

    PubMed

    Dederer, H; Berger, M; Meyer, T; Werr, M; Ilg, T

    2013-04-01

    Insect nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several insecticide classes. In the present study, we report the gene identification and cloning of nAChR α1 and α2 subunits (Lcα1 and Lcα2) from the sheep blowfly Lucilia cuprina. Xenopus oocytes voltage clamp experiments as hybrids with the chicken β2 nAChR (Ggβ2) subunit resulted in ACh-gated ion channels with distinct dose-response curves for Lcα1/Ggβ2 (effective concentration 50% [EC50 ] = 80 nM; nH  = 1.05), and Lcα2/Ggβ2 (EC50  = 5.37 μM, nH  = 1.46). The neonicotinoid imidacloprid was a potent agonist for the α-bungarotoxin-sensitive Lcα1/Ggβ2 (EC50 ∼ 20 nM), while the α-bungarotoxin-resistant Lcα2/Ggβ2 showed a 30-fold lower sensitivity to this insecticide (EC50  = 0.62 μM). Thirteen close derivatives of ACh were analysed in EC50 , Hill coefficient and maximum current (relative to ACh) determinations for Lcα1/Ggβ2 and Lcα2/Ggβ2 and the chicken Ggα4/Ggβ2 nAChRs, and comparisons relative to ACh allowed the definition of novel structure-activity and structure-selectivity relationships. In the case of N-ethyl-acetylcholine, the EC50 of the chicken Ggα4/Ggβ2 rose by a factor of 1000, while for both Lcα1/Ggβ2 and Lcα2/Ggβ2, potency remained unchanged. Further derivatives with insect nAChR selectivity potential were acetyl-α-methylcholine and trimethyl-(3-methoxy-3-oxopropyl)ammonium, followed by acetylhomocholine and trimethyl-(4-oxopentyl) ammonium. Our results may provide guidance for the identification or design of insect-specific nAChR agonists using structure-based or in silico methods. © 2013 Royal Entomological Society.

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

    PubMed Central

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

    2013-01-01

    The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (±)-epibatidine-induced AChR Ca2+ influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [3H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [3H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6′) and valine (position 13′) rings, and (c) inhibits [3H]TCP, [3H] ibogaine, and [3H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization. PMID:20303928

  5. Site specificity of agonist-induced opening and desensitization of the Torpedo californica nicotinic acetylcholine receptor.

    PubMed

    Andreeva, Iraida E; Nirthanan, Selvanayagam; Cohen, Jonathan B; Pedersen, Steen E

    2006-01-10

    Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were measured using sequential-mixing stopped-flow fluorescence methods to determine the contribution of each individual site to agonist-induced opening and desensitization. Timed dansyl-C6-choline (DC6C) binding followed by its dissociation upon mixing with high, competing agonist concentrations revealed four kinetic components: an initial, fast fluorescence decay, followed by a transient increase, and then two characteristic decays that reflect dissociation from the desensitized agonist sites. The transient increase resulted from DC6C binding to the open-channel based on its prevention by proadifen, a noncompetitive antagonist. Further characterization of DC6C channel binding by the inhibition of [3H]phencyclidine binding and by equilibrium measurements of DC6C fluorescence yielded KD values of 2-4 microM for the desensitized AChR and approximately 600 microM for the closed state. At this site, DC6C displayed a strongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer from tryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned to DC6C dissociation from the alphadelta site of the AChR in its closed conformation, on the basis of inhibition with the site-selective antagonists d-tubocurarine and alpha-conotoxin MI. Fast decay amplitude data indicated an apparent affinity of 0.9 microM for the closed-state alphadelta site; the closed-state alphagamma-site affinity is inferred to be near 100 microM. These values and the known affinities for the desensitized conformation show that the alphagamma site drives AChR desensitization to a approximately 40-fold greater extent than the alphadelta site, undergoes energetically larger conformational changes, and is the primary determinant of agonist potency.

  6. Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor.

    PubMed

    Hamouda, Ayman K; Sanghvi, Mitesh; Sauls, Daniel; Machu, Tina K; Blanton, Michael P

    2006-04-04

    The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.

  7. Nicotinic acetylcholine receptor and the structural basis of neuromuscular transmission: insights from Torpedo postsynaptic membranes.

    PubMed

    Unwin, Nigel

    2013-11-01

    The nicotinic acetylcholine (ACh) receptor, at the neuromuscular junction, is a neurotransmitter-gated ion channel that has been fine-tuned through evolution to transduce a chemical signal into an electrical signal with maximum efficiency and speed. It is composed from three similar and two identical polypeptide chains, arranged in a ring around a narrow membrane pore. Central to the design of this assembly is a hydrophobic gate in the pore, more than 50 Å away from sites in the extracellular domain where ACh binds. Although the molecular properties of the receptor have been explored intensively over the last few decades, only recently have structures emerged revealing its complex architecture and illuminating how ACh entering the binding sites opens the distant gate. Postsynaptic membranes isolated from the (muscle-derived) electric organ of the Torpedo ray have underpinned most of the structural studies: the membranes form tubular vesicles having receptors arranged on a regular surface lattice, which can be imaged directly in frozen physiological solutions. Advances in electron crystallographic techniques have also been important, enabling analysis of the closed- and open-channel forms of the receptor in unreacted tubes or tubes reacted briefly with ACh. The structural differences between these two forms show that all five subunits participate in a concerted conformational change communicating the effect of ACh binding to the gate, but that three of them (αγ, β and δ) play a dominant role. Flexing of oppositely facing pore-lining α-helices is the principal motion determining the closed/open state of the gate. These results together with the findings of biochemical, biophysical and other structural studies allow an integrated description of the receptor and of its mode of action at the synapse.

  8. Evaluating Commercially Available Antibodies for Rat α7 Nicotinic Acetylcholine Receptors.

    PubMed

    Garg, Brijesh K; Loring, Ralph H

    2017-09-01

    Alpha7 nicotinic acetylcholine receptors (α7 nAChRs) are important drug targets in neurological disorders and inflammation, making their detection and localization by validated antibodies highly desirable. However, tests in knockout animals raised questions about specificity of antibodies to mouse α7 nAChRs. To date, methods for validating antibodies for rat or human α7 nAChR have not been reported. We developed a gel-shift assay for western blots using GH4C1 cells expressing either native rat receptors or α7 nAChR-green fluorescent protein (GFP) chimeras to evaluate seven commercially available α7 nAChR antibodies. Blots with anti-GFP antibody detected GFP or α7 nAChR-GFP expressed in GH4C1 cells, and 125 I-α-bungarotoxin binding and RNA analysis demonstrated α7 nAChR expression. Validated samples were used to evaluate α7 nAChR antibodies by western blot and immunofluorescence studies. These methods confirmed that two of seven α7 nAChR antibodies identify gel-shifts for α7 nAChR/nAChR-GFP but only one antibody demonstrated low background and significant immunofluorescence differences between wild-type and α7 nAChR expressing GH4C1 cells. However, that polyclonal antibody displayed lot-to-lot variability. Our findings suggest that careful validation methods are required for all α7 nAChR receptor species and antibody lots and that the gel-shift assay may allow for relatively rapid antibody screening.

  9. On the functional interaction between nicotinic acetylcholine receptor and Na+,K+-ATPase.

    PubMed

    Krivoi, Igor I; Drabkina, Tatiana M; Kravtsova, Violetta V; Vasiliev, Alexander N; Eaton, Misty J; Skatchkov, Serguei N; Mandel, Frederic

    2006-09-01

    Previous studies have shown that nanomolar acetylcholine (ACh) produces a 2 to 4-mV hyperpolarization of skeletal muscle fibers putatively due to Na(+),K(+)-ATPase activation. The present study elucidates the involvement of the nicotinic ACh receptor (nAChR) and of Na(+),K(+)-ATPase isoform(s) in ACh-induced hyperpolarization of rat diaphragm muscle fibers. A variety of ligands of specific binding sites of nAChR and Na(+),K(+)-ATPase were used. Dose-response curves for ouabain, a specific Na(+),K(+)-ATPase inhibitor, were obtained to ascertain which Na(+),K(+)-ATPase isoform(s) is involved. The ACh dose-response relationship for the hyperpolarization was also determined. The functional relationship between these two proteins was also studied in a less complex system, a membrane preparation from Torpedo electric organ. The possibility of a direct ACh effect on Na(+),K(+)-ATPase was studied in purified lamb kidney Na(+),K(+)-ATPase and in rat red blood cells, systems where no nAChR is present. The results indicate that binding of nAChR agonists to their specific sites results in modulation of ouabain-sensitive (most probably alpha2) isoform of Na(+),K(+)-ATPase, leading to muscle membrane hyperpolarization. In the Torpedo preparation, ouabain modulates dansyl-C6-choline binding to nAChR, and vice versa. These results provide the first evidence of a functional interaction between nAChR and Na(+),K(+)-ATPase. Possible interaction mechanisms are discussed.

  10. Resistance to cycloxaprid in Laodelphax striatellus is associated with altered expression of nicotinic acetylcholine receptor subunits.

    PubMed

    Zhang, Yueliang; Han, Yangchun; Yang, Qiong; Wang, Lihua; He, Peng; Liu, Zewen; Li, Zhong; Guo, Huifang; Fang, Jichao

    2018-04-01

    Cycloxaprid is a new oxabridged cis-configuration neonicotinoid insecticide, the resistance development potential and underlying resistance mechanism of which were investigated in the small brown planthopper, Laodelphax striatellus (Fallén), an important agricultural pest of rice. A cycloxaprid-resistant strain (YN-CPD) only achieved 10-fold higher resistance, in contrast to 106-fold higher resistance to buprofezin and 332-fold higher resistance to chlorpyrifos achieved after exposure to similar selection pressure, and the cycloxaprid selected line showed no cross-resistance to the buprofezin and chlorpyrifos-selected resistance strains. Moreover, we identified 10 nicotinic acetylcholine receptor (nAChR) subunits from the transcriptome of L. striatellus, and six segments had open reading frames (ORFs). While we did not find mutations in the nAChR genes of L. striatellus, subunits Lsα1 and Lsβ1 exhibited, respectively, 9.60-fold and 3.36-fold higher expression in the resistant strain, while Lsα8 exhibited 0.44-fold lower expression. Suppression of Lsα1 through ingestion of dsLsα1 led to an increase in susceptibility to cycloxaprid. The findings indicate that resistance to cycloxaprid develops slowly compared with resistance to other chemicals and without cross-resistance to chlorpyrifos or buprofezin; over-expressed Lsα1 is associated with low cycloxaprid resistance levels, but the importance of over-expressed Lsβ1 and reduced expression of Lsα8 could not be excluded. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  11. Inhibition of neuronal nicotinic acetylcholine receptor subtypes by alpha-Conotoxin GID and analogues.

    PubMed

    Millard, Emma L; Nevin, Simon T; Loughnan, Marion L; Nicke, Annette; Clark, Richard J; Alewood, Paul F; Lewis, Richard J; Adams, David J; Craik, David J; Daly, Norelle L

    2009-02-20

    alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

  12. α6β2 nicotinic acetylcholine receptors influence locomotor activity and ethanol consumption.

    PubMed

    Kamens, Helen M; Peck, Colette; Garrity, Caitlin; Gechlik, Alex; Jenkins, Brenita C; Rajan, Akshat

    2017-06-01

    Nicotinic acetylcholine receptors (nAChRs) in the mesolimbic dopamine system have been implicated in ethanol behaviors. In particular, work in genetically engineered mice has demonstrated that α6-containing nAChRs are involved in ethanol consumption and sedation. A limitation of these studies is that the alteration in the receptor was present throughout development. The recently described α6β2 antagonist, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), now makes it possible to test for the involvement of these receptors using a pharmacological approach. The aim of this study was to examine the role of α6β2 nAChRs in ethanol behaviors using a pharmacological approach. Adolescent C57BL/6J mice were treated with bPiDI 30 min prior to testing the mice for binge-like ethanol consumption in the drinking-in-the-dark (DID) test, ethanol-induced motor incoordination using the balance beam, and ethanol-induced sedation using the Loss of Righting Reflex (LORR) paradigm. Adolescent animals were chosen because they express a high amount of α6 mRNA relative to adult animals. Control studies were also performed to determine the effect of bPiDI on locomotor activity and ethanol metabolism. Female mice treated with 20 mg/kg bPiDI had reduced locomotor activity compared to saline-treated animals during the first 30 min following an acute injection. Pretreatment with the α6β2 antagonist reduced adolescent ethanol consumption but also reduced saccharin consumption. No significant effects were observed on ethanol-induced ataxia, sedation, or metabolism. This study provides evidence that α6β2 nAChRs are involved in locomotor activity as well as ethanol and saccharin consumption in adolescent animals. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Derivatives of dibenzothiophene for positron emission tomography imaging of α7-nicotinic acetylcholine receptors.

    PubMed

    Gao, Yongjun; Kellar, Kenneth J; Yasuda, Robert P; Tran, Thao; Xiao, Yingxian; Dannals, Robert F; Horti, Andrew G

    2013-10-10

    A new series of derivatives of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide with high binding affinities and selectivity for α7-nicotinic acetylcholine receptors (α7-nAChRs) (Ki = 0.4-20 nM) has been synthesized for positron emission tomography (PET) imaging of α7-nAChRs. Two radiolabeled members of the series [(18)F]7a (Ki = 0.4 nM) and [(18)F]7c (Ki = 1.3 nM) were synthesized. [(18)F]7a and [(18)F]7c readily entered the mouse brain and specifically labeled α7-nAChRs. The α7-nAChR selective ligand 1 (SSR180711) blocked the binding of [(18)F]7a in the mouse brain in a dose-dependent manner. The mouse blocking studies with non-α7-nAChR central nervous system drugs demonstrated that [(18)F]7a is highly α7-nAChR selective. In agreement with its binding affinity the binding potential of [(18)F]7a (BPND = 5.3-8.0) in control mice is superior to previous α7-nAChR PET radioligands. Thus, [(18)F]7a displays excellent imaging properties in mice and has been chosen for further evaluation as a potential PET radioligand for imaging of α7-nAChR in non-human primates.

  14. Intra-subunit flexibility underlies activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

    PubMed

    Chrisman, Paul A; Podair, Julie I; Jobe, Emily M; Levandoski, Mark M

    2014-04-01

    Allosteric modulation is a general feature of nicotinic acetylcholine receptors, yet the structural components and movements important for conversions among functional states are not well understood. In this study, we examine the communication between the binding sites for agonist and the modulator morantel (Mor) of neuronal α3β2 receptors, measuring evoked currents of receptors expressed in Xenopus oocytes with the two-electrode voltage-clamp method. We hypothesized that movement along an interface of β sheets connecting the agonist and modulator sites is necessary for allosteric modulation. To address this, we created pairs of substituted cysteines that span the cleft formed where the outer β sheet meets the β sheet constituting the (-)-face of the α3 subunit; the three pairs were L158C-A179C, L158C-G181C and L158C-K183C. Employing a disulfide trapping approach in which bonds are formed between neighboring cysteines under oxidation conditions, we found that oxidation treatments decreased the amplitude of currents evoked by either the agonist (ACh) or co-applied agonist and modulator (ACh + Mor), by as much as 51%, consistent with the introduced bond decreasing channel efficacy. Reduction treatment increased evoked currents up to 89%. The magnitude of the oxidation effects depended on whether agonists were present during oxidation and on the cysteine pair. Additionally, the cysteine mutations themselves decreased Mor potentiation, implicating these residues in modulation. Our findings suggest that these β sheets in the α3 subunit move with respect to each other during activation and modulation, and the residues studied highlight the contribution of this intramolecular allosteric pathway to receptor function. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Kinetic properties and open probability of α7 nicotinic acetylcholine receptors.

    PubMed

    Pesti, Krisztina; Szabo, Anett K; Mike, Arpad; Vizi, E Sylvester

    2014-06-01

    The alpha7 nicotinic acetylcholine receptor (nAChR) has some peculiar kinetic properties. From the literature of α7 nAChR-mediated currents we concluded that experimentally measured kinetic properties reflected properties of the solution exchange system, rather than genuine kinetic properties of the receptors. We also concluded that all experimentally measured EC50 values for agonists must inherently be inaccurate. The aim of this study was to assess the undistorted kinetic properties of α7 nAChRs, and to construct an improved kinetic model, which can also serve as a basis of modeling the effect of the positive allosteric modulator PNU-120596, as it is described in the accompanying paper. Agonist-evoked currents were recorded from GH4C1 cells stably transfected with pCEP4/rat α7 nAChR using patch-clamp and fast solution exchange. We used two approaches to circumvent the problem of insufficient solution exchange rate: extrapolation and kinetic modeling. First, using different solution exchange rates we recorded evoked currents, and extrapolated their amplitude and kinetics to instantaneous solution exchange. Second, we constructed a kinetic model that reproduced concentration-dependence and solution exchange rate-dependence of receptors, and then we simulated receptor behavior at experimentally unattainably fast solution exchange. We also determined open probabilities during choline-evoked unmodulated and modulated currents using nonstationary fluctuation analysis. The peak open probability of 10 mM choline-evoked currents was 0.033 ± 0.006, while in the presence of choline (10 mM) and PNU-120596 (10 μM), it was increased to 0.599 ± 0.058. Our kinetic model could adequately reproduce low open probability, fast kinetics, fast recovery and solution exchange rate-dependent kinetics. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Some properties of human neuronal alpha 7 nicotinic acetylcholine receptors fused to the green fluorescent protein.

    PubMed

    Palma, Eleonora; Mileo, Anna M; Martinez-Torres, Ataulfo; Eusebi, Fabrizio; Miledi, Ricardo

    2002-03-19

    The functional properties and cellular localization of the human neuronal alpha7 nicotinic acetylcholine (AcCho) receptor (alpha7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutalpha7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtalpha7 receptors decay much faster than those elicited by the mutalpha7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated alpha7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable "run-down" of the AcCho currents generated by mutalpha7-GFP receptors, whereas those of the wtalpha7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutalpha7-GFP oocytes was accompanied by a marked decrease of alpha-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtalpha7 and mutalpha7 receptors provides powerful tools to study the distribution and function of alpha7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins.

  17. Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

    PubMed Central

    Palma, Eleonora; Mileo, Anna M.; Martínez-Torres, Ataúlfo; Eusebi, Fabrizio; Miledi, Ricardo

    2002-01-01

    The functional properties and cellular localization of the human neuronal α7 nicotinic acetylcholine (AcCho) receptor (α7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutα7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtα7 receptors decay much faster than those elicited by the mutα7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated α7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable “run-down” of the AcCho currents generated by mutα7-GFP receptors, whereas those of the wtα7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutα7-GFP oocytes was accompanied by a marked decrease of α-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtα7 and mutα7 receptors provides powerful tools to study the distribution and function of α7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins. PMID:11891308

  18. 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

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

    PubMed

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

    2006-11-01

    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.

  20. Novel neonicotinoid-agarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors.

    PubMed

    Tomizawa, M; Latli, B; Casida, J E

    1996-10-01

    Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) to develop a neonicotinoid-agarose affinity column. The procedure-introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamicle gel electrophoresis-gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.

  1. The functional interaction between nicotinic acetylcholine receptors and Ly-6/neurotoxin proteins in Locusta migratoria.

    PubMed

    Bao, Haibo; Zhang, Yixi; Liu, Yang; Yu, Na; Liu, Zewen

    2017-09-01

    Failures in expressing insect nAChRs (nicotinic acetylcholine receptors) limit the understanding of their functional properties, although insect nAChRs have been developed as insecticide targets for decades. Some accessory proteins can modulate nAChRs, but only a few have been identified in insects, including lynx from Ly-6/neurotoxin superfamily. Until now, only one to three lynx were identified in one insect species. Here, 11 lynx were identified in Locusta migratoria with common features of Ly-6/neurotoxin members. The effects of these lynx on seven hybrid nAChRs functionally constructed in Xenopus oocytes were tested, in which each nAChR subtype contained one L. migratoria α subunit plus rat β2 subunit. One lynx could selectively modulate several nAChR subtypes to significantly increase the amplitude of epibatidine-evoked currents, while Loc-lynx10 acted on all test nAChRs. Among nine lynx with obvious modulation effects to increase inward currents, only four significantly increased the specific [ 3 H]epibatidine binding, and the binding increase existed in all modulated nAChRs by these lynx. The results indicated that the modulation mode was decided by the specific lynx and had nothing to do with nAChR subtypes. This study provides an approximate panorama for the functional interaction net between L. migratoria lynx and nAChRs, although some other nAChRs need further tests. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors.

    PubMed

    Arias, Hugo R; Bhumireddy, Pankaj; Spitzmaul, Guillermo; Trudell, James R; Bouzat, Cecilia

    2006-02-21

    We investigated the molecular mechanisms and the binding site location for the fluorophor crystal violet (CrV), a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR). To this end, radiolabeled competition binding, fluorescence spectroscopy, Schild-type analysis, patch-clamp recordings, and molecular dynamics approaches were used. The results indicate that (i) CrV interacts with the desensitized Torpedo AChR with higher affinity than with the resting state at several temperatures (5-37 degrees C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [(3)H]thienylcyclohexylpiperidine binding to the desensitized or resting AChR is mediated by a steric mechanism; (iii) tetracaine inhibits CrV binding to the resting AChR, probably by a steric mechanism; (iv) barbiturates modulate CrV binding to the resting AChR by an allosteric mechanism; (v) CrV itself induces AChR desensitization; (vi) CrV decreases the peak of macroscopic currents by acting on the resting AChR but without affecting the desensitization rate from the open state; and (vii) two tertiary amino groups from CrV may bind to the alpha1-Glu(262) residues (located at position 20') in the resting state. We conclude that the CrV binding site overlaps the PCP locus in the resting and desensitized state. The noncompetitive action of CrV may be explained by an allosteric mechanism in which the binding of CrV to the extracellular mouth of the resting receptor leads to an inhibition of channel opening. Binding of CrV probably increases desensitization of the resting channel and stabilizes the desensitized state.

  3. The Minimal Pharmacophore for Silent Agonism of the α7 Nicotinic Acetylcholine Receptor

    PubMed Central

    Chojnacka, Kinga; Horenstein, Nicole A.

    2014-01-01

    The minimum pharmacophore for activation of the human α7 nicotinic acetylcholine receptor (nAChR) is the tetramethylammonium cation. Previous work demonstrated that larger quaternary ammonium compounds, such as diethyldimethylammonium or 1-methyl quinuclidine, were α7-selective partial agonists, but additional increase in the size of the ammonium cation or the quinuclidine N-alkyl group by a single carbon to an N-ethyl group led to a loss of efficacy for ion channel activation. We report that although such compounds are ineffective at inducing the normal channel open state, they nonetheless regulate the induction of specific conformational states normally considered downstream of channel activation. We synthesized several panels of quaternary ammonium nAChR ligands that systematically varied the size of the substituents bonded to the central positively charged nitrogen atom. In these molecular series, we found a correlation between the molecular volume of the ligand and/or charge density, and the receptor’s preferred distribution among conformational states including the closed state, the active state, a nonconducting state that could be converted to an activated state by a positive allosteric modulator (PAM), and a PAM-insensitive nonconducting state. We hypothesize that the changes of molecular volume of an agonist’s cationic core subtly impact interactions at the subunit interface constituting the orthosteric binding site in such a way as to regulate the probability of conversions among the conformational states. We define a new minimal pharmacophore for the class of compounds we have termed “silent agonists,” which are able to induce allosteric modulator-dependent activation but not the normal activated state. PMID:24990939

  4. 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).

  5. Activation of the Torpedo nicotinic acetylcholine receptor. The contribution of residues alphaArg55 and gammaGlu93.

    PubMed

    Kapur, Ankur; Davies, Martin; Dryden, William F; Dunn, Susan M J

    2006-03-01

    The Torpedo nicotinic acetylcholine receptor is a heteropentamer (alpha2betagammadelta) in which structurally homologous subunits assemble to form a central ion pore. Viewed from the synaptic cleft, the likely arrangement of these subunits is alpha-gamma-alpha-delta-beta lying in an anticlockwise orientation. High affinity binding sites for agonists and competitive antagonists have been localized to the alpha-gamma and alpha-delta subunit interfaces. We investigated the involvement of amino acids lying at an adjacent interface (gamma-alpha) in receptor properties. Recombinant Torpedo receptors, expressed in Xenopus oocytes, were used to investigate the consequences of mutating alphaArg55 and gammaGlu93, residues that are conserved in most species of the peripheral nicotinic receptors. Based on homology modeling, these residues are predicted to lie in close proximity to one another and it has been suggested that they may form a salt bridge in the receptor's three-dimensional structure (Sine et al. 2002 J Biol Chem277, 29 210-29 223). Although substitution of alphaR55 by phenylalanine or tryptophan resulted in approximately a six-fold increase in the EC50 value for acetylcholine activation, the charge reversal mutation (alphaR55E) had no significant effect. In contrast, the replacement of gammaE93 by an arginine conferred an eight-fold increase in the potency for acetylcholine-induced receptor activation. In the receptor carrying the double mutations, alphaR55E-gammaE93R or alphaR55F-gammaE93R, the potency for acetylcholine activation was partially restored to that of the wild-type. The results suggest that, although individually these residues influence receptor activation, direct interactions between them are unlikely to play a major role in the stabilization of different conformational states of the receptor.

  6. Unorthodox Acetylcholine Binding Sites Formed by α5 and β3 Accessory Subunits in α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Jain, Akansha; Kuryatov, Alexander; Wang, Jingyi; Kamenecka, Theodore M.; Lindstrom, Jon

    2016-01-01

    All nicotinic acetylcholine receptors (nAChRs) evolved from homomeric nAChRs in which all five subunits are involved in forming acetylcholine (ACh) binding sites at their interfaces. Heteromeric α4β2* nAChRs typically have two ACh binding sites at α4/β2 interfaces and a fifth accessory subunit surrounding the central cation channel. β2 accessory subunits do not form ACh binding sites, but α4 accessory subunits do at the α4/α4 interface in (α4β2)2α4 nAChRs. α5 and β3 are closely related subunits that had been thought to act only as accessory subunits and not take part in forming ACh binding sites. The effect of agonists at various subunit interfaces was determined by blocking homologous sites at these interfaces using the thioreactive agent 2-((trimethylammonium)ethyl) methanethiosulfonate (MTSET). We found that α5/α4 and β3/α4 interfaces formed ACh binding sites in (α4β2)2α5 and (α4β2)2β3 nAChRs. The α4/α5 interface in (β2α4)2α5 nAChRs also formed an ACh binding site. Blocking of these sites with MTSET reduced the maximal ACh evoked responses of these nAChRs by 30–50%. However, site-selective agonists NS9283 (for the α4/α4 site) and sazetidine-A (for the α4/β2 site) did not act on the ACh sites formed by the α5/α4 or β3/α4 interfaces. This suggests that unorthodox sites formed by α5 and β3 subunits have unique ligand selectivity. Agonists or antagonists for these unorthodox sites might be selective and effective drugs for modulating nAChR function to treat nicotine addiction and other disorders. PMID:27645992

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

    PubMed

    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.

  8. 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

    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 ofmore » 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

  9. No involvement of nicotinic receptors in the facilitation of acetylcholine outflow in mouse cortex in the presence of neostigmine and atropine

    PubMed Central

    Iannazzo, Lydia; Majewski, Henryk

    2000-01-01

    The role of nicotinic and muscarinic receptors in the modulation of acetylcholine release was studied using field stimulated mouse cortex slices incubated with [3H]-choline.Both acetylcholine (100 μM) and the cholinesterase inhibitor neostigmine (100 μM) inhibited the stimulation-induced (S-I) outflow of radioactivity but in the presence of atropine (0.3 μM) an enhancement was seen, which may be indicative of facilitatory nicotinic receptors.Mecamylamine (100 μM) was unable to antagonize the enhancement seen in the presence of acetylcholine and atropine. The nicotinic agonist dimethylphenylpiperazinium (30 μM) did not facilitate S-I outflow of radioactivity.A range of nicotinic blockers had no effect on the enhancement seen in the presence of neostigmine and atropine, nor did indomethacin, the 5HT3 antagonist MDL 7222 nor the NMDA antagonist MK-801.The inability to block this effect suggests that nicotinic receptors are not involved. We postulate, at least for neostigmine, that the facilitation is an artefact because of the use of [3H]-choline as a radiotracer whereby the efflux of radioactivity is enhanced because the radiolabelled acetylcholine is not metabolized to choline and therefore flows out of the tissue more readily. PMID:10952694

  10. Spatial and intracellular relationships between the α7 nicotinic acetylcholine receptor and the vesicular acetylcholine transporter in the prefrontal cortex of rat and mouse

    PubMed Central

    Duffy, Aine M.; Zhou, Ping; Milner, Teresa A.; Pickel, Virginia M.

    2009-01-01

    The alpha-7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is expressed in the prefrontal cortex (PFC), a brain region where these receptors are implicated in cognitive function and in the pathophysiology of schizophrenia. Activation of this receptor is dependent on release of acetylcholine (ACh) from axon terminals that contain the vesicular acetylcholine transporter (VAChT). Since rat and mouse models are widely used for studies of specific abnormalities in schizophrenia, we sought to determine the subcellular location of the α7nAChR with respect to VAChT storage vesicles in axon terminals in the PFC in both species. For this, we used dual electron microscopic immunogold and immunoperoxidase labeling of antisera raised against the α7nAChR and VAChT. In both species, the α7nAChR-immunoreactivity (-ir) was principally identified within dendrites and dendritic spines, receptive to axon terminals forming asymmetric excitatory-type synapses, but lacking detectable α7nAChR or VAChT-ir. Quantitative analysis of the rat PFC revealed that of α7nAChR labeled neuronal profiles, 65% (299/463) were postsynaptic structures (dendrites and dendritic spine) and only 22% (104/463) were axon terminals or small unmyelinated axons. In contrast, VAChT was principally localized to varicose vesicle-filled axonal profiles, without recognized synaptic specializations (n = 240). Of the α7nAChR-labeled axons, 47% (37/79) also contained VAChT, suggesting that ACh release is autoregulated through the presynaptic α7nAChR. The VAChT-labeled terminals rarely formed synapses, but frequently apposed α7nAChR-containing neuronal profiles. These results suggest that in rodent PFC, the α7nAChR plays a major role in modulation of the postsynaptic excitation in spiny dendrites in contact with VAChT containing axons. PMID:19374941

  11. Thujone inhibits the function of α7-nicotinic acetylcholine receptors and impairs nicotine-induced memory enhancement in one-trial passive avoidance paradigm.

    PubMed

    Sultan, Ahmed; Yang, Keun-Hang Susan; Isaev, Dmitro; Nebrisi, Eslam El; Syed, Nurulain; Khan, Nadia; Howarth, Christopher F; Sadek, Bassem; Oz, Murat

    2017-06-01

    Effects of thujone, a major ingredient of absinthe, wormwood oil and some herbal medicines, were tested on the function of α 7 subunit of the human nicotinic acetylcholine (α 7 nACh) receptor expressed in Xenopus oocytes using the two-electrode voltage-clamp technique. Thujone reversibly inhibited ACh (100μM)-induced currents with an IC 50 value of 24.7μM. The effect of thujone was not dependent on the membrane potential and did not involve Ca 2+ -dependent Cl - channels expressed endogenously in oocytes. Inhibition by thujone was not reversed by increasing ACh concentrations. Moreover, specific binding of [ 125 I] α-bungarotoxin was not altered by thujone. Further experiments in SH-EP1 cells expressing human α 7 nACh receptor indicated that thujone suppressed choline induced Ca 2+ transients in a concentration-dependent manner. In rat hippocampal CA3-dentate gyrus synapses, nicotine-induced enhancement of long-term potentiation was also inhibited by thujone. Furthermore, the results observed in in-vivo one-trial passive avoidance paradigm show that thujone (1.25mg/kg, i.p.) significantly impaired nicotine-induced enhancement of learning and memory in Wistar rats. Collectively, our results indicate that thujone inhibits the function of the α7-nACh receptor and impairs cellular and behavioral correlates of cholinergic modulation of learning and memory. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization.

    PubMed

    Clause, Amanda; Lauer, Amanda M; Kandler, Karl

    2017-01-01

    Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

  14. 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

  15. Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor.

    PubMed

    Hamouda, Ayman K; Chiara, David C; Blanton, Michael P; Cohen, Jonathan B

    2008-12-02

    The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.

  16. Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog*

    PubMed Central

    Jayakar, Selwyn S.; Dailey, William P.; Eckenhoff, Roderic G.; Cohen, Jonathan B.

    2013-01-01

    Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [3H]AziPm, and labeled amino acids were identified by Edman degradation. [3H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18′ and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (−agonist) amino acids in the M2 helices (αM2-6′, βM2-6′ and -13′, and δM2-13′) that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10′. Propofol enhanced [3H]AziPm photolabeling at αM2-10′. Propofol inhibited [3H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC50 = 40 μm) than it inhibited ion channel photolabeling (IC50 = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site. PMID:23300078

  17. Probing the Structure of Affinity-Purified and Lipid-Reconstituted Torpedo Nicotinic Acetylcholine Receptor†

    PubMed Central

    Hamouda, Ayman K.; Chiara, David C.; Blanton, Michael P.; Cohen, Jonathan B.

    2010-01-01

    The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand gated ion channels (LGIC) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical/biophysical techniques, detergent-solubilization, purification, and lipid-reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to compare the state-dependent photolabeling of Torpedo nAChR before and after purification and reincorporation into lipid. For purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9 and M2-13, the agonist-enhanced labeling of δThr274 (δM2-18) within the δ subunit helix bundle, and the labeling at the lipid-protein interface (αM4) were the same as for nAChR in native membranes. However, addition of agonist did not enhance [125I]TID photolabeling of δIle288 within the δM2-M3 loop. These results indicate that after purification and reconstitution of Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the δM2-M3 loop. To further characterize the pharmacology of [125I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [125I]TID photolabeling. PCP inhibited [125I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6), while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the δ subunit helix bundle. PMID:18991407

  18. Strychnine, but not PMBA, inhibits neuronal nicotinic acetylcholine receptors expressed by rabbit retinal ganglion cells.

    PubMed

    Renna, J M; Strang, C E; Amthor, F R; Keyser, K T

    2007-01-01

    Strychnine is considered a selective competitive antagonist of glycine gated Cl- channels (Saitoh et al., 1994) and studies have used strychnine at low micromolar concentrations to study the role of glycine in rabbit retina (Linn, 1998; Protti et al., 2005). However, other studies have shown that strychnine, in the concentrations commonly used, is also a potent competitive antagonist of alpha7 nicotinic acetylcholine receptors (nAChRs; Matsubayashi et al., 1998). We tested the effects of low micromolar concentrations of strychnine and 3-[2'-phosphonomethyl[1,1'-biphenyl]-3-yl] alanine (PMBA), a specific glycine receptor blocker (Saitoh et al., 1994; Hosie et al., 1999) on the activation of both alpha7 nAChRs on retinal ganglion cells and on ganglion cell responses to a light flash. Extracellular recordings were obtained from ganglion cells in an isolated retina/choroid preparation and 500 microM choline was used as an alpha7 agonist (Alkondon et al., 1997). We recorded from brisk sustained and brisk transient OFF cells, many of which have been previously shown to have alpha7 receptors (Strang et al., 2005). Further, we tested the effect of strychnine, PMBA and alpha-bungarotoxin on the binding of tetramethylrhodamine alpha-bungarotoxin in the inner plexiform layer. Our data indicates that strychnine, at doses as low as 1.0 microM, can inhibit the alpha7 nAChR-mediated response to choline, but PMBA at concentrations as high as 0.4 microM does not. Binding studies show strychnine and alpha-bungarotoxin inhibit binding of labeled alpha-bungarotoxin in the IPL. Thus, the effects of strychnine application may be to inhibit glycine receptors expressed by ganglion cell or to inhibit amacrine cell alpha7 nAChRs, both of which would result in an increase in the ganglion cell responses. Further research will be required to disentangle the effects of strychnine previously believed to be caused by a single mechanism of glycine receptor inhibition.

  19. 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

  20. Roles of Amino Acids and Subunits in Determining the Inhibition of Nicotinic Acetylcholine Receptors by Competitive Antagonists

    PubMed Central

    Dilger, James P.; Vidal, Ana Maria; Liu, Man; Mettewie, Claire; Suzuki, Takahiro; Pham, Anh; Demazumder, Deeptankar

    2008-01-01

    Background Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the α–δ and α–ε subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the α–ε interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. Methods HEK293 cells were transfected with wild-type or mutant (αY198F, εD59A, εD59N, εD173A, εD173N, δD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcho-line in the absence and presence of antagonist. Concentration–response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. Results Effects of mutations were antagonist specific. αY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to εD173 mutations. εD59 mutations had large effects on metocurine and cisatracurium. δD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two δ subunits but no ε subunit. Differences in IC50 arose from differences in both dissociation and association rates. Conclusion Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the α–ε interface. Pancuronium, vecuronium, and cisatracurium were selective for the α–δ interface. This suggests the possibility of synergistic

  1. Corticosterone affects the differentiation of a neuronal cerebral cortex-derived cell line through modulation of the nicotinic acetylcholine receptor.

    PubMed

    Baier, C J; Franco, D L; Gallegos, C E; Mongiat, L A; Dionisio, L; Bouzat, C; Caviedes, P; Barrantes, F J

    2014-08-22

    Chronic exposure to stress hormones has an impact on brain structures relevant to cognition. Nicotinic acetylcholine receptors (AChRs) are involved in numerous cognitive processes including learning and memory formation. In order to better understand the molecular mechanisms of chronic stress-triggered mental disease, the effect of corticosterone (CORT) on the biology of AChRs was studied in the neuronal cell line CNh. We found that chronic treatment with CORT reduced the expression levels of the α7-type neuronal AChR and, to a lesser extent, of α4-AChR. CORT also delayed the acquisition of the mature cell phenotype in CNh cells. Chronic nicotine treatment affected the differentiation of CNh cells and exerted a synergistic effect with CORT, suggesting that AChR could participate in signaling pathways that control the cell cycle. Overexpression of α7-AChR-GFP abolished the CORT effects on the cell cycle and the specific α7-AChR inhibitor, methyllycaconitine, mimicked the proliferative action exerted by CORT. Whole-cell voltage-clamp recordings showed a significant decrease in nicotine-evoked currents in CORT-treated cells. Taken together, these observations indicate that AChRs, and the α7-AChR in particular, could act as modulators of the differentiation of CNh cells and that CORT could impair the acquisition of a mature phenotype by affecting the function of this AChR subtype. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. [Analysis of the interaction between nicotinic acetylcholine receptor and Na+,K(+)-ATPase in the rat skeletal muscle and the Torpedo electric organ membrane preparation].

    PubMed

    Krivoĭ, I I; Drabkina, T M; Vasil'ev, A N; Kravtsova, V V; Mandel, F

    2006-02-01

    The interaction between the nicotinic acetylcholine receptor and Na+,K(+)-ATPase described previously was further studied in isolated rat diaphragm and in a membrane preparation of Torpedo californica electric organ. Three specific agonists of the nicotinic receptor: acetylcholine, nicotine and carbamylcholine (100 nmol/L each), all hyperpolarized the non-synaptic membranes of muscle fibers by up to 4 mV. Competitive antagonists of nicotinic acetylcholine receptor, d-tubocurarine (2 mcmol/L) or alpha-bungarotoxin (5 nmol/L) completely blocked the acetylcholine-induced hyperpolarization indicating that the effect requires binding of the agonists to their specific sites. The noncompetitive antagonist, proadifen (5 mcmol/L), exerted no effect on the amplitude of hyperpolarized but decreased K0.5 for this effect from 28.3 +/- 3.6 nmol/L to 7.1 +/- 2.3 nmol/L. Involvement of the Na+,K(+)-ATPase was suggested by data demonstrating that three specific Na+,K(+)-ATPase inhibitors: ouabain, digoxin or marinobufagenin (100 nmol/L each), all inhibit the hyperpolarizing effect of acetylcholine. Acetylcholine did not affectation either the catalytic activity of the Na+,K(+)-ATPase purified from sheep kidney or the transport activity of the Na+,K(+)-ATPase in the rat erythrocytes, i. e. in preparations not containing acetylcholine receptors. Hence, acetylcholine does not directly affect the Na+,K(+)-ATPase. In a Torpedo membrane preparation, ouabain (< or = 100 nmol/L) increased the binding of the fluorescent ligand: Dansyl-C6-choline (DCC). No ouabain effect was observed either when the agonist binding sites of the receptor were occupied by 2 mmol/L carbamylcholine, or in the absence Mg2+, when the binding of ouabain to the Na+,K(+)-ATPase is negligible. These results indicate that ouabain only affects specific DCC binding and only when bound to the Na+,K(+)-ATPase. The data obtained suggest that, in two different systems, the interaction between the nicotinic acetylcholine

  3. Varenicline is a potent partial agonist at α6β2* nicotinic acetylcholine receptors in rat and monkey striatum.

    PubMed

    Bordia, Tanuja; Hrachova, Maya; Chin, Matthew; McIntosh, J Michael; Quik, Maryka

    2012-08-01

    Extensive evidence indicates that varenicline reduces nicotine craving and withdrawal symptoms by modulating dopaminergic function at α4β2* nicotinic acetylcholine receptors (nAChRs) (the asterisk indicates the possible presence of other nicotinic subunits in the receptor complex). More recent data suggest that α6β2* nAChRs also regulate dopamine release and mediate nicotine reinforcement. The present experiments were therefore done to test the effect of varenicline on α6β2* nAChRs and their function, because its interaction with this subtype is currently unclear. Receptor competition studies showed that varenicline inhibited α6β2* nAChR binding (K(i) = 0.12 nM) as potently as α4β2* nAChR binding (K(i) = 0.14 nM) in rat striatal sections and with ∼20-fold greater affinity than nicotine. Functionally, varenicline was more potent in stimulating α6β2* versus α4β2* nAChR-mediated [(3)H]dopamine release from rat striatal synaptosomes with EC(50) values of 0.007 and 0.086 μM, respectively. However, it acted as a partial agonist on α6β2* and α4β2* nAChR-mediated [(3)H]dopamine release with maximal efficacies of 49 and 24%, respectively, compared with nicotine. We also evaluated varenicline's action in striatum of monkeys, a useful animal model for comparison with humans. Varenicline again potently inhibited monkey striatal α6β2* (K(i) = 0.13 nM) and α4β2* (K(i) = 0.19 nM) nAChRs in competition studies. Functionally, it potently stimulated both α6β2* (EC(50) = 0.014 μM) and α4β2* (EC(50) = 0.029 μM) nAChR-mediated [(3)H]dopamine release from monkey striatal synaptosomes, again acting as a partial agonist relative to nicotine at both subtypes. These data suggest that the ability of varenicline to interact at α6β2* nAChRs may contribute to its efficacy as a smoking cessation aid.

  4. Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia, and airway mucus formation in vitro and in vivo.

    PubMed

    Gundavarapu, Sravanthi; Wilder, Julie A; Mishra, Neerad C; Rir-Sima-Ah, Jules; Langley, Raymond J; Singh, Shashi P; Saeed, Ali Imran; Jaramillo, Richard J; Gott, Katherine M; Peña-Philippides, Juan Carlos; Harrod, Kevin S; McIntosh, J Michael; Buch, Shilpa; Sopori, Mohan L

    2012-09-01

    Airway mucus hypersecretion is a key pathophysiologic feature in a number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. We sought to characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. IL-13 and γ-aminobutyric acid type A receptors (GABA(A)Rs) are implicated in airway mucus. We examined the role of IL-13 and GABA(A)Rs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells and secondhand cigarette smoke-induced, ovalbumin-induced, or both mucus formation in vivo. Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABA(A)R antagonist picrotoxin. Airway epithelial cells express α7-, α9-, and α10-nicotinic acetylcholine receptors (nAChRs), and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen- or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells, murine airways, or both. Nicotine-induced airway mucus formation is independent of IL-13, and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various promucoid agents, including IL-13. In the absence of nicotine, acetylcholine might be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABA(A)Rα2 in the MUC5AC pathway. Acetylcholine and α7-nAChRs might serve as therapeutic targets to control airway mucus. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  5. Activation of α7 nicotinic acetylcholine receptors persistently enhances hippocampal synaptic transmission and prevents Aß-mediated inhibition of LTP in the rat hippocampus.

    PubMed

    Ondrejcak, Tomas; Wang, Qinwen; Kew, James N C; Virley, David J; Upton, Neil; Anwyl, Roger; Rowan, Michael J

    2012-02-29

    Nicotinic acetylcholine receptors mediate fast cholinergic modulation of glutamatergic transmission and synaptic plasticity. Here we investigated the effects of subtype selective activation of the α7 nicotinic acetylcholine receptors on hippocampal transmission and the inhibition of synaptic long-term potentiation by the Alzheimer's disease associated amyloid ß-protein (Aß). The α7 nicotinic acetylcholine receptor agonist "compound A" ((R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl))thiophene-2-carboxamide) induced a rapid-onset persistent enhancement of synaptic transmission in the dentate gyrus in vitro. Consistent with a requirement for activation of α7 nicotinic acetylcholine receptors, the type II α7-selective positive allosteric modulator PheTQS ((3aR, 4S, 9bS)-4-(4-methylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) potentiated, and the antagonist methyllycaconitine (MLA) prevented the persistent enhancement. Systemic injection of the agonist also induced a similar MLA-sensitive persistent enhancement of synaptic transmission in the CA1 area in vivo. Remarkably, although compound A did not affect control long-term potentiation (LTP) in vitro, it prevented the inhibition of LTP by Aß1-42 and this effect was inhibited by MLA. These findings strongly indicate that activation of α7 nicotinic acetylcholine receptors is sufficient to persistently enhance hippocampal synaptic transmission and to overcome the inhibition of LTP by Aß. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Unorthodox Acetylcholine Binding Sites Formed by α5 and β3 Accessory Subunits in α4β2* Nicotinic Acetylcholine Receptors.

    PubMed

    Jain, Akansha; Kuryatov, Alexander; Wang, Jingyi; Kamenecka, Theodore M; Lindstrom, Jon

    2016-11-04

    All nicotinic acetylcholine receptors (nAChRs) evolved from homomeric nAChRs in which all five subunits are involved in forming acetylcholine (ACh) binding sites at their interfaces. Heteromeric α4β2* nAChRs typically have two ACh binding sites at α4/β2 interfaces and a fifth accessory subunit surrounding the central cation channel. β2 accessory subunits do not form ACh binding sites, but α4 accessory subunits do at the α4/α4 interface in (α4β2) 2 α4 nAChRs. α5 and β3 are closely related subunits that had been thought to act only as accessory subunits and not take part in forming ACh binding sites. The effect of agonists at various subunit interfaces was determined by blocking homologous sites at these interfaces using the thioreactive agent 2-((trimethylammonium)ethyl) methanethiosulfonate (MTSET). We found that α5/α4 and β3/α4 interfaces formed ACh binding sites in (α4β2) 2 α5 and (α4β2) 2 β3 nAChRs. The α4/α5 interface in (β2α4) 2 α5 nAChRs also formed an ACh binding site. Blocking of these sites with MTSET reduced the maximal ACh evoked responses of these nAChRs by 30-50%. However, site-selective agonists NS9283 (for the α4/α4 site) and sazetidine-A (for the α4/β2 site) did not act on the ACh sites formed by the α5/α4 or β3/α4 interfaces. This suggests that unorthodox sites formed by α5 and β3 subunits have unique ligand selectivity. Agonists or antagonists for these unorthodox sites might be selective and effective drugs for modulating nAChR function to treat nicotine addiction and other disorders. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Beta2-containing nicotinic acetylcholine receptors mediate calcium/calmodulin-dependent protein kinase-II and synapsin I protein levels in the nucleus accumbens after nicotine withdrawal in mice

    PubMed Central

    Jackson, Kia J.; Damaj, M. Imad

    2013-01-01

    Nicotinic acetylcholine receptors are calcium-permeable and the initial targets for nicotine. Studies suggest that calcium-dependent mechanisms mediate some behavioral responses to nicotine; however, the post-receptor calcium-dependent mechanisms associated with chronic nicotine and nicotine withdrawal remain unclear. The proteins calcium/calmodulin-dependent protein kinase II (CaMKII) and synapsin I are essential for neurotransmitter release and were shown to be involved in drug dependence. In the current study, using pharmacological techniques, we sought to (a) complement previously published behavioral findings from our lab indicating a role for calcium-dependent signaling in nicotine dependence and (b) expand on previously published acute biochemical and pharmacological findings indicating the relevance of calcium-dependent mechanisms in acute nicotine responses by evaluating the function of CaMKII and synapsin I after chronic nicotine and withdrawal in the nucleus accumbens, a brain region implicated in drug dependence. Male mice were chronically infused with nicotine for 14 days, and treated with the β2 -selective antagonist dihydro-β-erythroidine (DHβE), or the α7 antagonist, methyllycaconitine citrate (MLA) 20 minutes prior to dissection of the nucleus accumbens. Results show that phosphorylated and total CaMKII and synapsin I protein levels were significantly increased in the nucleus accumbens after chronic nicotine infusion, and reduced after treatment with DHβE, but not MLA. A spontaneous nicotine withdrawal assessment also revealed significant reductions in phosphorylated CaMKII and synapsin I levels 24 h after cessation of nicotine treatment. Our findings suggest that post-receptor calcium-dependent mechanisms associated with nicotine withdrawal are mediated through β2-containing nicotinic receptors. PMID:23313759

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

    PubMed Central

    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 α4α2* 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 cocaine

  9. 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

  10. Cognitive correlates of α4β2 nicotinic acetylcholine receptors in mild Alzheimer's dementia.

    PubMed

    Sabri, Osama; Meyer, Philipp M; Gräf, Susanne; Hesse, Swen; Wilke, Stephan; Becker, Georg-Alexander; Rullmann, Michael; Patt, Marianne; Luthardt, Julia; Wagenknecht, Gudrun; Hoepping, Alexander; Smits, Rene; Franke, Annegret; Sattler, Bernhard; Tiepolt, Solveig; Fischer, Steffen; Deuther-Conrad, Winnie; Hegerl, Ulrich; Barthel, Henryk; Schönknecht, Peter; Brust, Peter

    2018-04-17

    In early Alzheimer's dementia, there is a need for PET biomarkers of disease progression with close associations to cognitive dysfunction that may aid to predict further cognitive decline and neurodegeneration. Amyloid biomarkers are not suitable for that purpose. The α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) are widely abundant in the human brain. As neuromodulators they play an important role in cognitive functions such as attention, learning and memory. Post-mortem studies reported lower expression of α4β2-nAChRs in more advanced Alzheimer's dementia. However, there is ongoing controversy whether α4β2-nAChRs are reduced in early Alzheimer's dementia. Therefore, using the recently developed α4β2-nAChR-specific radioligand (-)-18F-flubatine and PET, we aimed to quantify the α4β2-nAChR availability and its relationship to specific cognitive dysfunction in mild Alzheimer's dementia. Fourteen non-smoking patients with mild Alzheimer's dementia, drug-naïve for cholinesterase therapy, were compared with 15 non-smoking healthy controls matched for age, sex and education by applying (-)-18F-flubatine PET together with a neuropsychological test battery. The one-tissue compartment model and Logan plot method with arterial input function were used for kinetic analysis to obtain the total distribution volume (VT) as the primary, and the specific binding part of the distribution volume (VS) as the secondary quantitative outcome measure of α4β2-nAChR availability. VS was determined by using a pseudo-reference region. Correlations between VT within relevant brain regions and Z-scores of five cognitive functions (episodic memory, executive function/working memory, attention, language, visuospatial function) were calculated. VT (and VS) were applied for between-group comparisons. Volume of interest and statistical parametric mapping analyses were carried out. Analyses revealed that in patients with mild Alzheimer's dementia compared to healthy controls

  11. Prenatal Exposure to Nicotine and Childhood Asthma: Role of Nicotine Acetylcholine Receptors, Neuropeptides and Fibronectin Expression in Lung

    DTIC Science & Technology

    2008-12-01

    of tissues, and this was associated with airway hyperreactivity to methacholine in physiological studies. These studies led to two published...when exposed to a bronchoconstrictor like methacholine (Fig 3). In general, we are adding more measurements to this study to strengthen the data...Legend to Figure 3. Eight-week old mice exposed to prenatal nicotine have higher minute ventilation in response to methacholine

  12. Activation of alpha 7 nicotinic acetylcholine receptor protects mice from radiation-induced intestinal injury and mortality.

    PubMed

    Chen, Ji-Kuai; Li, Zhang-Peng; Liu, Yun-Zi; Zhao, Ting; Zhao, Xin-Bin; Ni, Min; Jiang, Guo-Jun; Shen, Fu-Ming

    2014-06-01

    Radiation-induced gastrointestinal syndrome occurs when the body is exposed to a high dose of radiation. Currently, safe and effective radioprotectants are not available. Apoptosis was reported to play a primary role in radiation-induced injury. Recent evidence suggests that stimulation of α7 nicotinic acetylcholine receptor (α7nAChR) prevents cell death by inhibition of apoptosis. In this study, we demonstrated that a single dose of PNU282987 (100 μg/kg, i.p.), a selective α7nAChR agonist, protected mice from intestinal injury and significantly improved survival when administered prior to lethal 8 Gy total body irradiation. In vitro, PNU282987 protected against 8 Gy radiation-induced cell death in human umbilical venous endothelial cells by inhibiting apoptosis. We conclude that activation of α7nAChR may provide a new therapeutic pathway for the treatment of radiation-induced damage and mortality.

  13. Exploring the binding energy profiles of full agonists, partial agonists, and antagonists of the α7 nicotinic acetylcholine receptor.

    PubMed

    Tabassum, Nargis; Ma, Qianyun; Wu, Guanzhao; Jiang, Tao; Yu, Rilei

    2017-09-01

    Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor family and are important drug targets for the treatment of neurological diseases. However, the precise determinants of the binding efficacies of ligands for these receptors are unclear. Therefore, in this study, the binding energy profiles of various ligands (full agonists, partial agonists, and antagonists) were quantified by docking those ligands with structural ensembles of the α7 nAChR exhibiting different degrees of C-loop closure. This approximate treatment of interactions suggested that full agonists, partial agonists, and antagonists of the α7 nAChR possess distinctive binding energy profiles. Results from docking revealed that ligand binding efficacy may be related to the capacity of the ligand to stabilize conformational states with a closed C loop.

  14. Iptakalim inhibits nicotinic acetylcholine receptor-mediated currents in dopamine neurons acutely dissociated from rat substantia nigra pars compacta.

    PubMed

    Hu, J; DeChon, J; Yan, K C; Liu, Q; Hu, G; Wu, J

    2006-07-31

    Iptakalim hydrochloride, a novel cardiovascular ATP-sensitive K(+) (K(ATP)) channel opener, has shown remarkable antihypertensive and neuroprotective effects in a variety of studies using in vivo and in vitro preparations. We recently found that iptakalim blocked human alpha4-containing nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the human SH-EP1 cell line. In the present study, we examined the effects of iptakalim on several neurotransmitter-induced current responses in single DA neurons freshly dissociated from rat substantia nigra pars compacta (SNc), using perforated patch-clamp recordings combined with a U-tube rapid drug application. In identified DA neurons under voltage-clamp configuration, glutamate-, NMDA-, and GABA-induced currents were insensitive to co-application with iptakalim (100 microM), while whole-cell currents induced by ACh (1 mM+1 microM atropine) or an alpha4beta2 nicotinic acetylcholine receptors relatively selective agonist, RJR-2403 (300 microM), were eliminated by iptakalim. Iptakalim inhibited RJR-2403-induced current in a concentration-dependent manner, and reduced maximal RJR-2403-induced currents at the highest agonist concentration, suggesting a non-competitive block. In current-clamp mode, iptakalim failed to affect resting membrane potential and spontaneous action potential firing, but abolished RJR-2403-induced neuronal firing acceleration. Together, these results indicate that in dissociated SNc DA neurons, alpha4-containing nAChRs, rather than ionotropic glutamate receptors, GABA(A) receptors or perhaps K-ATP channels are the sensitive targets to mediate iptakalim's pharmacological roles.

  15. A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis

    PubMed Central

    Puinean, Alin M; Lansdell, Stuart J; Collins, Toby; Bielza, Pablo; Millar, Neil S

    2013-01-01

    High levels of resistance to spinosad, a macrocyclic lactone insecticide, have been reported previously in western flower thrips, Frankliniella occidentalis, an economically important insect pest of vegetables, fruit and ornamental crops. We have cloned the nicotinic acetylcholine receptor (nAChR) α6 subunit from F. occidentalis (Foα6) and compared the nucleotide sequence of Foα6 from susceptible and spinosad-resistant insect populations (MLFOM and R1S respectively). A single nucleotide change has been identified in Foα6, resulting in the replacement of a glycine (G) residue in susceptible insects with a glutamic acid (E) in resistant insects. The resistance-associated mutation (G275E) is predicted to lie at the top of the third α-helical transmembrane domain of Foα6. Although there is no direct evidence identifying the location of the spinosad binding site, the analogous amino acid in the C. elegans glutamate-gated chloride channel lies in close proximity (4.4 Å) to the known binding site of ivermectin, another macrocyclic lactone pesticide. The functional consequences of the resistance-associated mutation have been examined in the human nAChR α7 subunit. Introduction of an analogous (A272E) mutation in α7 abolishes the modulatory effects of spinosad whilst having no significant effect upon activation by acetylcholine, consistent with spinosad having an allosteric mechanism of action. PMID:23016960

  16. A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis.

    PubMed

    Puinean, Alin M; Lansdell, Stuart J; Collins, Toby; Bielza, Pablo; Millar, Neil S

    2013-03-01

    High levels of resistance to spinosad, a macrocyclic lactone insecticide, have been reported previously in western flower thrips, Frankliniella occidentalis, an economically important insect pest of vegetables, fruit and ornamental crops. We have cloned the nicotinic acetylcholine receptor (nAChR) α6 subunit from F. occidentalis (Foα6) and compared the nucleotide sequence of Foα6 from susceptible and spinosad-resistant insect populations (MLFOM and R1S respectively). A single nucleotide change has been identified in Foα6, resulting in the replacement of a glycine (G) residue in susceptible insects with a glutamic acid (E) in resistant insects. The resistance-associated mutation (G275E) is predicted to lie at the top of the third α-helical transmembrane domain of Foα6. Although there is no direct evidence identifying the location of the spinosad binding site, the analogous amino acid in the C. elegans glutamate-gated chloride channel lies in close proximity (4.4 Å) to the known binding site of ivermectin, another macrocyclic lactone pesticide. The functional consequences of the resistance-associated mutation have been examined in the human nAChR α7 subunit. Introduction of an analogous (A272E) mutation in α7 abolishes the modulatory effects of spinosad whilst having no significant effect upon activation by acetylcholine, consistent with spinosad having an allosteric mechanism of action. © 2012 International Society for Neurochemistry.

  17. Desensitization of neuronal nicotinic acetylcholine receptors conferred by N-terminal segments of the beta 2 subunit.

    PubMed

    Bohler, S; Gay, S; Bertrand, S; Corringer, P J; Edelstein, S J; Changeux, J P; Bertrand, D

    2001-02-20

    Desensitization is a general property of ligand-gated ion channels. Because of a wide array of available subunit combinations, it generates different time constants for channel closure, thereby modulating the processing of information in the brain. Within the family of neuronal nicotinic acetylcholine receptors (nAChRs), alpha 3 beta 2 and alpha 3 beta 4 receptors display contrasting properties of desensitization. When measured using two-electrode voltage-clamp in Xenopus oocytes, desensitization results in current decreases 2 s after initiation of acetylcholine application by 94% for alpha 3 beta 2 receptors, but only by 6% in the case of alpha 3 beta 4 receptors. Desensitization was analyzed by inserting different portions of the beta2 into the beta 4 subunit. Residues 1--212 of the beta2 subunit were able to confer 78% desensitization in 2 s, while smaller chimeras revealed desensitization in 2 s conferred by residues 1--42 alone to a level of 50%, by residues 72--89 to a level of 74%, and by residues 96--212 to a level of 77%. Some long-term (25 min) effects of desensitization driven by acetylcholine were found to rely partially on the same elements, including an enhancement mediated by residues 1--95 and 96--212 of the beta 2 subunit individually. Our results reveal that desensitization relies independently on diverse portions of the extracellular domain of the beta 2 subunit. Phenotype of alpha 3 beta 4 involves, in contrast, complex structural requirements involving residues dispersed throughout the entire N-terminal domain of the beta 4 subunit.

  18. Azemiopsin, a Selective Peptide Antagonist of Muscle Nicotinic Acetylcholine Receptor: Preclinical Evaluation as a Local Muscle Relaxant.

    PubMed

    Shelukhina, Irina V; Zhmak, Maxim N; Lobanov, Alexander V; Ivanov, Igor A; Garifulina, Alexandra I; Kravchenko, Irina N; Rasskazova, Ekaterina A; Salmova, Margarita A; Tukhovskaya, Elena A; Rykov, Vladimir A; Slashcheva, Gulsara A; Egorova, Natalya S; Muzyka, Inessa S; Tsetlin, Victor I; Utkin, Yuri N

    2018-01-07

    Azemiopsin (Az), a linear peptide from the Azemiops feae viper venom, contains no disulfide bonds, is a high-affinity and selective inhibitor of nicotinic acetylcholine receptor (nAChR) of muscle type and may be considered as potentially applicable nondepolarizing muscle relaxant. In this study, we investigated its preclinical profile in regard to in vitro and in vivo efficacy, acute and chronic toxicity, pharmacokinetics, allergenic capacity, immunotoxicity and mutagenic potency. The peptide effectively inhibited (IC 50 ~ 19 nM) calcium response of muscle nAChR evoked by 30 μM (EC 100 ) acetylcholine but was less potent (IC 50 ~ 3 μM) at α7 nAChR activated by 10 μM (EC 50 ) acetylcholine and had a low affinity to α4β2 and α3-containing nAChR, as well as to GABA A or 5HT₃ receptors. Its muscle relaxant effect was demonstrated at intramuscular injection to mice at doses of 30-300 µg/kg, 30 µg/kg being the initial effective dose and 90 µg/kg-the average effective dose. The maximal muscle relaxant effect of Az was achieved in 10 min after the administration and elimination half-life of Az in mice was calculated as 20-40 min. The longest period of Az action observed at a dose of 300 µg/kg was 55 min. The highest acute toxicity (LD 50 510 μg/kg) was observed at intravenous injection of Az, at intramuscular or intraperitoneal administration it was less toxic. The peptide showed practically no immunotoxic, allergenic or mutagenic capacity. Overall, the results demonstrate that Az has good drug-like properties for the application as local muscle relaxant and in its parameters, is not inferior to the relaxants currently used. However, some Az modification might be effective to extend its narrow therapeutic window, a typical characteristic and a weak point of all nondepolarizing myorelaxants.

  19. Azemiopsin, a Selective Peptide Antagonist of Muscle Nicotinic Acetylcholine Receptor: Preclinical Evaluation as a Local Muscle Relaxant

    PubMed Central

    Shelukhina, Irina V.; Zhmak, Maxim N.; Lobanov, Alexander V.; Ivanov, Igor A.; Garifulina, Alexandra I.; Kravchenko, Irina N.; Rasskazova, Ekaterina A.; Salmova, Margarita A.; Tukhovskaya, Elena A.; Rykov, Vladimir A.; Slashcheva, Gulsara A.; Egorova, Natalya S.; Muzyka, Inessa S.; Tsetlin, Victor I.

    2018-01-01

    Azemiopsin (Az), a linear peptide from the Azemiops feae viper venom, contains no disulfide bonds, is a high-affinity and selective inhibitor of nicotinic acetylcholine receptor (nAChR) of muscle type and may be considered as potentially applicable nondepolarizing muscle relaxant. In this study, we investigated its preclinical profile in regard to in vitro and in vivo efficacy, acute and chronic toxicity, pharmacokinetics, allergenic capacity, immunotoxicity and mutagenic potency. The peptide effectively inhibited (IC50 ~ 19 nM) calcium response of muscle nAChR evoked by 30 μM (EC100) acetylcholine but was less potent (IC50 ~ 3 μM) at α7 nAChR activated by 10 μM (EC50) acetylcholine and had a low affinity to α4β2 and α3-containing nAChR, as well as to GABAA or 5HT3 receptors. Its muscle relaxant effect was demonstrated at intramuscular injection to mice at doses of 30–300 µg/kg, 30 µg/kg being the initial effective dose and 90 µg/kg—the average effective dose. The maximal muscle relaxant effect of Az was achieved in 10 min after the administration and elimination half-life of Az in mice was calculated as 20–40 min. The longest period of Az action observed at a dose of 300 µg/kg was 55 min. The highest acute toxicity (LD50 510 μg/kg) was observed at intravenous injection of Az, at intramuscular or intraperitoneal administration it was less toxic. The peptide showed practically no immunotoxic, allergenic or mutagenic capacity. Overall, the results demonstrate that Az has good drug-like properties for the application as local muscle relaxant and in its parameters, is not inferior to the relaxants currently used. However, some Az modification might be effective to extend its narrow therapeutic window, a typical characteristic and a weak point of all nondepolarizing myorelaxants. PMID:29316656

  20. Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus.

    PubMed

    Vázquez-Gómez, Elizabeth; Arias, Hugo R; Feuerbach, Dominik; Miranda-Morales, Marcela; Mihailescu, Stefan; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof; García-Colunga, Jesús

    2014-10-05

    The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits.

    PubMed

    Criado, Manuel; Valor, Luis M; Mulet, José; Gerber, Susana; Sala, Salvador; Sala, Francisco

    2012-11-01

    Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α-bungarotoxin binding sites and acetylcholine-gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α-bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

  2. 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.

  3. Partial agonism at the α7 nicotinic acetylcholine receptor improves attention, impulsive action and vigilance in low attentive rats.

    PubMed

    Hayward, Andrew; Adamson, Lisa; Neill, Joanna C

    2017-04-01

    Inattention is a disabling symptom in conditions such as schizophrenia and attention deficit/hyperactivity disorder. Nicotine can improve attention and vigilance, but is unsuitable for clinical use due to abuse liability. Genetic knockout of the α7 nicotinic acetylcholine receptor (nAChR) induces attention deficits therefore selective agonism may improve attention, without the abuse liability associated with nicotine. The α7 nAChR partial agonist encenicline (formerly EVP-6124) enhances memory in rodents and humans. Here we investigate, for the first time, efficacy of encenicline to improve attention and vigilance in animals behaviourally grouped for low attentive traits in the 5 choice-continuous performance task (5C-CPT). Female Lister Hooded rats were trained to perform the 5C-CPT with a variable stimulus duration (SD). Animals were then grouped based on performance into upper and lower quartiles of d' (vigilance) and accuracy (selective attention), producing high-attentive (HA) and low-attentive (LA) groups. LA animals showed an increase in selective attention and vigilance at 0.3mg/kg encenicline, a reduction in impulsive action (probability of false alarms) and increase in vigilance following 1mg/kg at 0.75sSD. At 1mg/kg, HA animals had reduced selective attention at 0.75sSD and reduced vigilance at 0.75 and 1.25sSD. Improvement of attention, vigilance and impulsive action in LA animals demonstrates that encenicline has pro-attentive properties dependent on baseline levels of performance. Our work suggests that α7 nAChR partial agonism may improve attention particularly in conditions with low attention. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

  4. Effect of ispronicline, a neuronal nicotinic acetylcholine receptor partial agonist, in subjects with age associated memory impairment (AAMI).

    PubMed

    Dunbar, Geoffrey C; Inglis, Fraser; Kuchibhatla, Ramana; Sharma, Tonmoy; Tomlinson, Mark; Wamsley, James

    2007-03-01

    Cognitive decline seen in the normal elderly is associated with selective loss of neuronal nicotinic acetylcholine receptors (nAChRs). Nicotine given either by inhalation or transdermally helps cognition, but unacceptable side effects limit its utility. The present study assessed the safety, tolerability and effect on cognition of ispronicline, a highly selective partial agonist at the 4beta2 nAChR, in elderly subjects (n =76) with age associated memory impairment (AAMI). This double-blind, placebo-controlled cross-over study explored ascending oral doses of ispronicline in the range 50-150 mg given as a single morning dose for a period of 3 weeks. Pharmacokinetics (PK) were assessed, as well as cognitive function measured by means of the Cognitive Drug Research (CDR) computerized test battery. Ispronicline had a favourable safety profile and was well tolerated at doses below 150 mg. No effect of clinical importance was seen on biochemistry, haematology, urine analysis, vital signs, electrocardiogram (ECG) or Holter monitoring. The most frequent drug induced adverse event was light-headedness (dizziness). A beneficial effect was seen on cognition across the dose range. This was most marked at 50 mg on factors measuring attention and episodic memory. PK analysis indicated a plasma Cmax range of 5-25/35 ng/ml ispronicline was associated with the most beneficial effect. These early results demonstrate ispronicline was well tolerated and did not display the side effects typical of nicotine. Ispronicline also had a beneficial effect on cognition in subjects with AAMI. This was seen most strongly in a Cmax range that had been predicted from pre-clinical animal studies.

  5. Cross-reactivity of acid-sensing ion channel and Na+–H+ exchanger antagonists with nicotinic acetylcholine receptors

    PubMed Central

    Santos-Torres, Julio; Ślimak, Marta A; Auer, Sebastian; Ibañez-Tallon, Inés

    2011-01-01

    Abstract Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the mammalian central and peripheral nervous systems, where they contribute to neuronal excitability and synaptic communication. It has been reported that nAChRs are modulated by BK channels and that BK channels, in turn, are inhibited by acid-sensing ion channels (ASICs). Here we investigate the possible functional interaction between these channels in medial habenula (MHb) neurones. We report that selective antagonists of large-conductance calcium-activated potassium channels and ASIC1a channels, paxilline and psalmotoxin 1, respectively, did not induce detectable changes in nicotine-evoked currents. In contrast, the non-selective ASIC and Na+–H+ exchanger (NHE1) antagonists, amiloride and its analogues, suppressed nicotine-evoked responses in MHb neurones of wild-type and ASIC2 null mice, excluding a possible involvement of ASIC2 in the nAChR inhibition by amiloride. Zoniporide, a more selective inhibitor of NHE1, reversibly inhibited α3β4-, α7- and α4-containing (*) nAChRs in Xenopus oocytes and in brain slices, as well as in PS120 cells deficient in NHE1 and virally transduced with nAChRs, suggesting a generalized effect of zoniporide in most neuronal nAChR subtypes. Independently from nAChR antagonism, zoniporide profoundly blocked synaptic transmission onto MHb neurones without affecting glutamatergic and GABA receptors. Taken together, these results indicate that amiloride and zoniporide, which are clinically used to treat hypertension and cardiovascular disease, have an inhibitory effect on neuronal nAChRs when used experimentally at high doses. The possible cross-reactivity of these compounds with nAChRs in vivo will require further investigation. PMID:21911609

  6. 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. © FASEB.

  7. α-Conotoxins Identify the α3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells

    PubMed Central

    Hone, Arik J.; McIntosh, J. Michael; Azam, Layla; Lindstrom, Jon; Lucero, Linda; Whiteaker, Paul; Passas, Juan; Blázquez, Jesús

    2015-01-01

    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

  8. 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. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

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

    SciTech Connect

    Welch, Kevin D., E-mail: kevin.welch@ars.usda.gov; Pfister, James A.; Lima, Flavia G.

    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 neuromuscularmore » 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.« less

  10. Specific α4β2 Nicotinic Acetylcholine Receptor Binding of [F-18]Nifene in the Rhesus Monkey

    PubMed Central

    Hillmer, A.T.; Wooten, D.W.; Moirano, J.; Slesarev, M.; Barnhart, T.E.; Engle, J.W.; Nickles, R.J.; Murali, D.; Schneider, M.; Mukherjee, J.; Christian, B.T.

    2013-01-01

    Objective [F-18]Nifene is a PET radioligand developed to image α4β2* nicotinic acetylcholine receptors (nAChR) in the brain. This work assesses the in vivo binding and imaging characteristics of [F-18]nifene in rhesus monkeys for the development of PET experiments examining nAChR binding. Methods Dynamic PET imaging experiments with [F-18]nifene were acquired in 4 anesthetized macaca mulatta (rhesus) monkeys using a microPET P4 scanner. Data acquisition was initiated with a bolus injection of 109 ± 17 MBq [F-18]nifene and the time course of the radioligand in the brain was measured for up to 120 minutes. For two experiments, a displacement dose of (−)nicotine (0.03 mg/kg, i.v.) was given 45–60 minutes post injection and followed 30 minutes later with a second [F-18]nifene injection to measure radioligand nondisplaceable uptake. Time activity curves were extracted in the regions of the antereoventral thalamus (AVT), lateral geniculate nucleus region (LGN), frontal cortex, and the cerebellum (CB). Results The highest levels of [F-18]nifene uptake were observed in the AVT and LGN. Target-to-CB ratios reached maximum values of 3.3 ± 0.4 in the AVT and 3.2 ± 0.3 in the LG 30–45 minutes post-injection. Significant binding of [F-18]nifene was observed in the subiculum, insula cortex, temporal cortex, cingulate gyrus, frontal cortex, striatum, and midbrain areas. The (−)nicotine displaced bound [F-18]nifene to near background levels within 15 minutes post-drug injection. No discernable displacement was observed in the CB, suggesting its potential as a reference region. Logan graphical estimates using the CB as a reference region yielded binding potentials (BPND) of 1.6 ± 0.1 in the AVT, and 1.3 ± 0.1 in the LGN. The post-nicotine injection displayed uniform nondisplaceable uptake of [F-18]nifene throughout gray and white brain matter. Conclusions [F-18]Nifene exhibits rapid equilibration and a moderately high target to background binding profile in the α4

  11. The role of nicotinic acetylcholine and opioid systems of the ventral orbital cortex in modulation of formalin-induced orofacial pain in rats.

    PubMed

    Yousofizadeh, Shahnaz; Tamaddonfard, Esmaeal; Farshid, Amir Abbas

    2015-07-05

    Nicotinic acetylcholine and opioid receptors are involved in modulation of pain. In the present study, we investigated the effects of microinjection of nicotinic acetylcholine and opioid compounds into the ventral orbital cortex (VOC) on the formalin-induced orofacial pain in rats. For this purpose, two guide cannulas were placed into the left and right sides of the VOC of the brain. Orofacial pain was induced by subcutaneous injection of a diluted formalin solution (50μl, 1.5%) into the right vibrissa pad and face rubbing durations were recorded at 3-min blocks for 45min. Formalin produced a marked biphasic pain response (first phase: 0-3min and second phase: 15-33min). Epibatidine (a nicotinic receptor agonist) at doses of 0.05, 0.1 and 0.2μg/site, morphine (an opioid receptor agonist) at doses of 0.5, 1 and 2μg/site and their sub-analgesic doses (0.025μg/site epibatidine with 0.25μg/site morphine) combination treatment suppressed the second phase of pain. The antinociceptive effect induced by 0.2μg/site of epibatidine, but not morphine (2μg/site), was prevented by 2μg/site of mecamylamine (a nicotinic receptor antagonist). Naloxone (an opioid receptor antagonist) at a dose of 2μg/site prevented the antinociceptive effects induced by 2μg/site of morphine and 0.2μg/site of epibatidine. No above-mentioned chemical compounds affected locomotor activity. These results showed that at the VOC level, epibatidine and morphine produced antinociception. In addition, opioid receptor might be involved in epibatidine-induced antinociception, but the antinociception induced by morphine was not mediated through nicotinic acetylcholine receptor. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. 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 100 μM 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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor

    PubMed Central

    Absalom, Nathan L.; Quek, Gracia; Lewis, Trevor M.; Qudah, Taima; von Arenstorff, Ida; Ambrus, Joseph I.; Harpsøe, Kasper; Karim, Nasiara; Balle, Thomas; Mcleod, Malcolm D.; Chebib, Mary

    2013-01-01

    The α4β2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and α4β2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4β2 receptor containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(β2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(β2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(β2)2 nAChRs. PMID:23893416

  14. 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

  15. The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors.

    PubMed

    Chatzidaki, Anna; D'Oyley, Jarryl M; Gill-Thind, JasKiran K; Sheppard, Tom D; Millar, Neil S

    2015-10-01

    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. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Natural compounds interacting with nicotinic acetylcholine receptors: from low-molecular weight ones to peptides and proteins.

    PubMed

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

    2015-05-14

    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.

  17. 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

  18. In vivo evidence for β2 nicotinic acetylcholine receptor subunit upregulation in smokers as compared to nonsmokers with schizophrenia

    PubMed Central

    Esterlis, Irina; Bois, Frederic; Pittman, Brian; Picciotto, Marina R.; Shearer, Lara; Anticevic, Alan; Carlson, Jon; Niciu, Marc; Cosgrove, Kelly P.; D’Souza, D. Cyril

    2013-01-01

    Background: Schizophrenia is associated with very high rates of tobacco smoking. The latter may be related to an attempt to “self-medicate” symptoms and/or to alterations in function of high affinity nicotinic acetylcholine receptors (β2*-nAChRs). Methods: Smoking and nonsmoking subjects with schizophrenia (n=31) and age-, smoking- and sex-matched comparison subjects (n=31) participated in one [123I]5-IA-85380 single photon emission computed tomography (SPECT) scan to quantify β2*-nAChR availability. Psychiatric, cognitive, nicotine craving and mood assessments were obtained during active smoking as well as smoking abstinence. Results: There were no differences in smoking characteristics between smokers with and without schizophrenia. Subjects with schizophrenia had lower β2*-nAChR availability relative to comparison group, and nonsmokers had lower β2*-nAChR availability relative to smokers. However, there was no smoking by diagnosis interaction. Relative to nonsmokers with schizophrenia, smokers with schizophrenia had higher β2*-nAChR availability in limited brain regions. In smokers with schizophrenia, higher β2*-nAChR availability was associated with lower negative symptoms of schizophrenia and better performance on tests of executive control. Chronic exposure to antipsychotic drugs was not associated with changes in β2*-nAChR availability in schizophrenia. Conclusions: Although subjects with schizophrenia have lower β2*-nAChR availability as compared to comparison group, smokers with schizophrenia appear to upregulate in the cortical regions. Lower receptor availability in smokers with schizophrenia in the cortical regions is associated with a greater number of negative symptoms and worse performance on tests of executive function; suggesting smoking subjects with schizophrenia who upregulate to a lesser degree may be at risk for poorer outcomes. PMID:24360979

  19. Selective potentiation of gabapentin-mediated antinociception in the rat formalin test by the nicotinic acetylcholine receptor agonist ABT-594.

    PubMed

    Munro, Gordon; Dyhr, Helene; Grunnet, Morten

    2010-09-01

    The treatment of chronic pain is hampered by various issues including multiple underlying mechanisms contributing to disease pathology and treatment-related toxicity concerns. These can be partially circumvented by combining mechanistically distinct drugs with the aim of selectively potentiating analgesia as opposed to side-effects. This approach has been used to assess the antinociceptive efficacy of the nicotinic acetylcholine (nACh) receptor agonist ABT-594 when combined with the antiepileptic drug gabapentin, the mu-opioid receptor agonist morphine or the antidepressant drug duloxetine in the rat formalin test. Alone, ABT-594 (0.01-0.3 mg/kg) dose-dependently attenuated spontaneous flinching behaviour during first (P1) and second (P2) phase. Similarly, P1, interphase and P2 flinching were variously attenuated by gabapentin (25-200 mg/kg), morphine (0.3-3 mg/kg) and duloxetine (3-60 mg/kg). Remarkably, a completely inactive dose of ABT-594 reduced the dose of gabapentin required to produce antinociception during P1 by 4-8 fold and during P2 by 8-16 fold. This striking potentiation was blocked by mecamylamine and indicative of analgesic synergy. Similar, albeit less consistent results (3-10 fold potency increase) were obtained with morphine/ABT-594. Although a 3 fold increase in P2 antinociceptive potency was obtained with duloxetine in the presence of ABT-594, a corresponding increase in efficacy was lacking. Indeed, a mechanistically relevant reduction in antinociceptive efficacy and potency of duloxetine/ABT-594 occurred during interphase. Thus, activation of the nicotinic cholinergic system differentially modulates the antinociceptive actions of distinct mechanism of action compounds, and provides a novel framework for nACh receptor modulators mediating analgesia in the putative absence of adverse events associated with this mechanism of action. Copyright 2010 Elsevier Ltd. All rights reserved.

  20. Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.

    PubMed

    Hamouda, Ayman K; Stewart, Deirdre S; Husain, S Shaukat; Cohen, Jonathan B

    2011-06-10

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive

  1. Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor*

    PubMed Central

    Hamouda, Ayman K.; Stewart, Deirdre S.; Husain, S. Shaukat; Cohen, Jonathan B.

    2011-01-01

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC50 = 4 μm, whereas it inhibited the binding of [3H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC50 values of 2.5 and 0.7 mm, respectively. Similar to [3H]TDBzl-etomidate, [3H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [3H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl

  2. Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells.

    PubMed

    Singh, Nagendra S; Paul, Rajib K; Ramamoorthy, Anuradha; Torjman, Marc C; Moaddel, Ruin; Bernier, Michel; Wainer, Irving W

    2013-12-01

    Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cells primarily express the α7-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular d-Ser levels and SR protein expression. Incubation with (S)-nicotine increased d-Ser levels, which were attenuated by the α7-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of d-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α7-nAChR. The nAChR subtype selectivity was probed using α7-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α3β4-nAChR specific inhibitor AT-1001. The compounds reduced d-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca(2+) flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric

  3. Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells

    PubMed Central

    Singh, Nagendra S.; Paul, Rajib K.; Ramamoorthy, Anuradha; Torjman, Marc C.; Moaddel, Ruin; Bernier, Michel; Wainer, Irving W.

    2013-01-01

    Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cell primarily express the α7-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular D-Ser levels and SR protein expression. Incubation with (S)-nicotine increased D-Ser levels, which was attenuated by the α7-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of D-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α7-nAChR. The nAChR subtype selectivity was probed using α7-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α3β4-nAChR specific inhibitor AT-1001. The compounds reduced D-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca2+ flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric

  4. 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.

  5. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Implication of a multisubunit Ets-related transcription factor in synaptic expression of the nicotinic acetylcholine receptor.

    PubMed Central

    Schaeffer, L; Duclert, N; Huchet-Dymanus, M; Changeux, J P

    1998-01-01

    In adult muscle, transcription of the nicotinic acetylcholine receptor (AChR) is restricted to the nuclei located at the neuromuscular junction. The N-box, a new promoter element, was identified recently and shown to contribute to this compartmentalized synaptic expression of the AChR delta- and epsilon-subunits. We demonstrate that the N-box mediates transcriptional activation in cultured myotubes and identify the transcription factor that binds to the N-box as a heterooligomer in myotubes and adult muscle. The GABP (GA-binding protein) alpha-subunit belongs to the Ets family of transcription factors, whereas the beta-subunit shares homology with IkappaB and Drosophila Notch protein. GABP binding specificity to mutated N-box in vitro strictly parallels the sequence requirement for beta-galactosidase targeting to the endplate in vivo. In situ hybridization studies reveal that the mRNAs of both GABP subunits are abundant in mouse diaphragm, with preferential expression of the alpha-subunit at motor endplates. In addition, heregulin increases GABPalpha protein levels and regulates phosphorylation of both subunits in cultured chick myotubes. Finally, dominant-negative mutants of either GABPalpha or GABPbeta block heregulin-elicited transcriptional activation of the AChR delta and epsilon genes. These findings establish the expected connection with a presynaptic trophic factor whose release contributes to the accumulation of AChR subunit mRNAs at the motor endplate. PMID:9606190

  7. 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.

  8. 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

    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, amore » 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.« less

  9. Baroreflex deficiency hampers angiogenesis after myocardial infarction via acetylcholine-α7-nicotinic ACh receptor in rats.

    PubMed

    Yu, Jian-Guang; Song, Shu-Wei; Shu, He; Fan, Sai-Jun; Liu, Ai-Jun; Liu, Chong; Guo, Wei; Guo, Jin-Min; Miao, Chao-Yu; Su, Ding-Feng

    2013-08-01

    Angiogenesis is critical for re-establishing blood supply to ischaemic myocardium after myocardial infarction (MI). Human studies have associated arterial baroreflex (ABR) deficiency with higher rate of sudden death after MI. The present work was designed to examine whether ABR deficiency affects angiogenesis in MI rats. Baroreflex sensitivity (BRS) was determined in conscious rats at 1 month after occlusion of the left anterior descending coronary artery. The survival time was significantly shorter in Sprague-Dawley rats with BRS <0.60 ms/mmHg vs. those with BRS ≥0.60 ms/mmHg. Sinoaortic denervation destroyed ABR, and decreased capillary density, regional blood flow and vascular endothelial growth factor (VEGF) concentration after MI. Ketanserin (0.6 mg/kg/day) enhanced BRS, and increased capillary density, regional blood flow, and VEGF. Sinoaortic denervation also reduced the expression of vesicular acetylcholine (ACh) transporter and α7-nicotinic ACh receptor (α7-nAChR). Angiogenesis after MI was significantly attenuated in α7-nAChR knockout mice. In contrast, increase in endogenous ACh with cholinesterase inhibitor pyridostigmine (30 mg/kg/day) increased angiogenesis after MI. In cultured cardiac microvascular endothelial cells, ACh stimulated the expression of VEGF, phosphorylation of VEGF receptor 2, and tube formation in a manner dependent upon α7-nAChR. Our results demonstrated that ABR deficiency could attenuate angiogenesis in ischaemic myocardium. These findings provide further mechanistic basis for enhancing baroreflex function in the treatment of MI.

  10. Three-finger snake neurotoxins and Ly6 proteins targeting nicotinic acetylcholine receptors: pharmacological tools and endogenous modulators.

    PubMed

    Tsetlin, Victor I

    2015-02-01

    Snake venom neurotoxins and lymphocyte antigen 6 (Ly6) proteins, most of the latter being membrane tethered by a glycosylphosphatidylinositol (GPI) anchor, have a variety of biological activities, but their three-finger (3F) folding combines them in one Ly6/neurotoxin family. Subsets of two groups, represented by α-neurotoxins and Lynx1, respectively, interact with nicotinic acetylcholine receptors (nAChR) and, hence, are of therapeutic interest for the treatment of neurodegenerative diseases, pain, and cancer. Information on the mechanisms of action and 3D structure of the binding sites, which is required for drug design, is available from the 3D structure of α-neurotoxin complexes with nAChR models. Here, I compare the structural and functional features of α-neurotoxins versus Lynx1 and its homologs to get a clearer picture of Lynx1-nAChR interactions that is necessary for fundamental science and practical applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Requirement of Nicotinic Acetylcholine Receptor Subunit β2 in the Maintenance of Spiral Ganglion Neurons during Aging

    PubMed Central

    Bao, Jianxin; Lei, Debin; Du, Yafei; Ohlemiller, Kevin K.; Beaudet, Arthur L.; Role, Lorna W.

    2008-01-01

    Age-related hearing loss (presbycusis) is a major health concern for the elderly. Loss of spiral ganglion neurons (SGNs), the primary sensory relay of the auditory system, is associated consistently with presbycusis. The causative molecular events responsible for age-related loss of SGNs are unknown. Recent reports directly link age-related neuronal loss in cerebral cortex with the loss of high-affinity nicotine acetylcholine receptors (nAChRs). In cochlea, cholinergic synapses are made by olivocochlear efferent fibers on the outer hair cells that express α9 nAChR subunits and on the peripheral projections of SGNs that express α2, α4 –7, and β2–3 nAChR subunits. A significantly decreased expression of the β2 nAChR subunit in SGNs was found specifically in mice susceptible to presbycusis. Furthermore, mice lacking the β2 nAChR subunit (β2−/−), but not mice lacking the α5 nAChR subunit (α5−/−), have dramatic hearing loss and significant reduction in the number of SGNs. Our findings clearly established a requirement for β2 nAChR subunit in the maintenance of SGNs during aging. PMID:15788760

  12. 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.

    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 canmore » 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.« less

  13. 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.

    PubMed

    Shin, Vivian Yvonne; Jin, Hong Chuan; Ng, Enders K O; Yu, Jun; Leung, Wai Keung; Cho, Chi Hin; Sung, Joseph Jao Yiu

    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 (alpha7 nAChR) and beta-adrenergic receptors. Treatment of cells with alpha-bungarotoxin (alpha-BTX, alpha7nAChR antagonist) or propranolol (beta-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE(2) and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE(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.

  14. Nicotine promotes apoptosis resistance of breast cancer cells and enrichment of side population cells with cancer stem cell-like properties via a signaling cascade involving galectin-3, α9 nicotinic acetylcholine receptor and STAT3.

    PubMed

    Guha, Prasun; Bandyopadhyaya, Gargi; Polumuri, Swamy K; Chumsri, Saranya; Gade, Padmaja; Kalvakolanu, Dhananjaya V; Ahmed, Hafiz

    2014-05-01

    Nicotine, a main addictive compound in tobacco smoke, has been linked to promotion and progression of lung, head and neck, pancreatic, and breast cancers, but the detailed mechanisms of cancer progression remain elusive. Here, we show that nicotine induces the expression of galectin-3 (an anti-apoptotic β-galactoside-binding lectin) in breast cancer cell line and in primary tumors from breast cancer patients. Nicotine-induced up regulation of galectin-3 is due to an increased expression of α9 isoform of nicotinic acetylcholine receptor (α9nAChR), which activates transcription factor STAT3 that in turn, physically binds to galectin-3 (LGALS3) promoter and induces transcription of galectin-3. Intracellular galectin-3 increased mitochondrial integrity and suppressed chemotherapeutic-induced apoptosis of breast cancer cell. Moreover, nicotine-induced enrichment of side population cells with cancer stem cell-like properties was modulated by galectin-3 expression and could be significantly reduced by transient knock down of LGALS3 and its upstream signaling molecules STAT3 and α9nAChR. Thus, galectin-3 or its upstream signaling molecule STAT3 or α9nAChR could be a potential target to prevent nicotine-induced chemoresistance in breast cancer.

  15. Nicotine Promotes Apoptosis Resistance of Breast Cancer Cells and Enrichment of Side Population Cells with Cancer Stem Cell Like Properties via a Signaling Cascade Involving Galectin-3, α9 Nicotinic Acetylcholine Receptor and STAT3

    PubMed Central

    Guha, Prasun; Bandyopadhyaya, Gargi; Polumuri, Swamy K.; Chumsri, Saranya; Gade, Padmaja; Kalvakolanu, Dhananjaya V.; Ahmed, Hafiz

    2014-01-01

    Nicotine, a main addictive compound in tobacco smoke, has been linked to promotion and progression of lung, head and neck, pancreatic, and breast cancers, but the detailed mechanisms of cancer progression remain elusive. Here we show that nicotine induces the expression of galectin-3 (an anti-apoptotic β-galactoside-binding lectin) in breast cancer cell line and in primary tumors from breast cancer patients. Nicotine-induced up regulation of galectin-3 is due to an increased expression of α9 isoform of nicotinic acetylcholine receptor (α9nAChR), which activates transcription factor STAT3 that in turn, physically binds to galectin-3 (LGALS3) promoter and induces transcription of galectin-3. Intracellular galectin-3 increased mitochondrial integrity and suppressed chemotherapeutic-induced apoptosis of breast cancer cell. Moreover, nicotine induced enrichment of side population cells with cancer stem cell-like properties was modulated by galectin-3 expression and could be significantly reduced by transient knock down of LGALS3 and its upstream signaling molecules STAT3 and α9nAChR. Thus, galectin-3 or its upstream signaling molecule STAT3 or α9nAChR could be a potential target to prevent nicotine-induced chemoresistance in breast cancer. PMID:24668500

  16. Alpha 7 nicotinic acetylcholine receptor-mediated protection against ethanol-induced neurotoxicity.

    PubMed

    de Fiebre, NancyEllen C; de Fiebre, Christopher M

    2003-11-01

    The alpha(7)-selective nicotinic partial agonist 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB) was examined for its ability to modulate ethanol-induced neurotoxicity in primary cultures of rat neurons. Primary cultures of hippocampal neurons were established from Long-Evans, embryonic day (E)-18 rat fetuses and maintained for 7 days. Ethanol (0-150 mM), DMXB (0-56 microM), or both were subsequently co-applied to cultures. Ethanol was added two additional times to the cultures to compensate for evaporation. After 5 days, neuronal viability was assessed with the MTT cell proliferation assay. Results demonstrated that ethanol reduces neuronal viability in a concentration-dependent fashion and that DMXB protects against this ethanol-induced neurotoxicity, also in a concentration-dependent fashion. These results support the suggestion that nicotinic partial agonists may be useful in treating binge drinking-induced neurotoxicity and may provide clues as to why heavy drinkers are usually smokers.

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

    PubMed Central

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

    2013-01-01

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

  18. INHIBITION OF HUMAN A7 NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS BY THE VOLATILE ORGANIC SOLVENT TRICHLOROETHYLENE.

    EPA Science Inventory

    Volatile organic compounds such as toleune, trichloroethylene and perchloroethylene are potent and reversible blockers of voltage-gated calcium current in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells. It is hypothesized that effects of VOCs on ICa contri...

  19. Varenicline: a selective alpha4beta2 nicotinic acetylcholine receptor partial agonist approved for smoking cessation.

    PubMed

    Lam, Sum; Patel, Priti N

    2007-01-01

    Tobacco smoking remains a significant health problem in the United States. It has been associated with staggering morbidity and mortality, specifically due to malignancies and cardiovascular disease. Smoking cessation can be difficult and frequently requires pharmacologic interventions in addition to nonpharmacologic measures. Previously available agents are nicotine replacement products and bupropion, which increased quit rates by about 2-fold compared with placebo. Varenicline is the first drug in a new class known as the selective alpha4beta2 nicotinic receptor partial agonists. In several randomized, double-blind, 52-week clinical trials involving healthy chronic smokers, varenicline demonstrated superiority to placebo and bupropion in terms of efficacy measures. Additionally, it improved tobacco withdrawal symptoms and reinforcing effects of smoking in relapsed patients. Patients should start therapy in combination with tobacco cessation counseling 1 week before quit date and continue the regimen for 12 weeks. The dose of varenicline should be titrated to minimize nausea. The recommended dosage is 0.5 mg once daily (QD) on days 1-3; titrate to 0.5 mg twice daily (BID) on days 4-7; and 1 mg BID starting on day 8. An additional 12-week maintenance therapy may be considered for those who achieve abstinence. The most common side effects are nausea (30%), insomnia (18%), headache (15%), abnormal dreams (13%), constipation (8%), and abdominal pain (7%). Overall, varenicline is a breakthrough in the management of tobacco addiction and has demonstrated good efficacy in motivated quitters. It also provides an option for smokers who cannot tolerate other pharmacologic interventions.

  20. Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

    SciTech Connect

    Blanton, M.P.; Wang, H.H.

    1990-02-06

    A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchRmore » {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.« less

  1. PACAP Modulates Acetylcholine-Elicited Contractions at Nicotinic Neuromuscular Contacts of the Land Snail.

    PubMed

    Krajcs, Nóra; Hernádi, László; Pirger, Zsolt; Reglődi, Dóra; Tóth, Gábor; Kiss, Tibor

    2015-12-01

    In this study, we investigate the potentiating effect of PACAP27 on cholinergic neuromuscular transmission in the recently discovered flexor muscles of the land snail, Helix pomatia. Using immunohistochemistry, we show that PACAP and PAC1 receptors are present in nerve fibers innervating the flexor muscles but not in the muscle itself. We also observed that PACAP27 exerts both pre- and postsynaptic effects on the cholinergic synapse and performed tests using a broad spectrum of chemicals in order to explore the possible intracellular pathways through which PACAP mediates its stimulatory effect. Our pharmacological data demonstrate that PACAP27 presynaptically enhances the release of acetylcholine by activating the adenylate cyclase-cAMP-PKA pathway. Postsynaptically, PACAP27 was found to enhance muscle contractility by PKC-mediated signaling pathway resulting in an increased Ca(2+) release from intracellular stores. These findings suggest that regulation of Ca(2+) release may contribute to the stimulatory effect of PACAP. Our data are the first demonstration of the potentiating effect of PACAP27 at the molluscan excitatory neuromuscular contact.

  2. Subtype-Specific Mechanisms for Functional Interaction between α6β4* Nicotinic Acetylcholine Receptors and P2X Receptors

    PubMed Central

    Limapichat, Walrati; Dougherty, Dennis A.

    2014-01-01

    P2X receptors and nicotinic acetylcholine receptors (nAChRs) display functional and physical interactions in many cell types and heterologous expression systems, but interactions between α6β4-containing (α6β4*) nAChRs and P2X2 receptors and/or P2X3 receptors have not been fully characterized. We measured several types of crosstalk in oocytes coexpressing α6β4 nAChRs and P2X2, P2X3, or P2X2/3 receptors. A novel form of crosstalk occurs between α6β4 nAChRs and P2X2 receptors. P2X2 receptors were forced into a prolonged desensitized state upon activation by ATP through a mechanism that does not depend on the intracellular C terminus of the P2X2 receptors. Coexpression of α6β4 nAChRs with P2X3 receptors shifts the ATP dose-response relation to the right, even in the absence of acetylcholine (ACh). Moreover, currents become nonadditive when ACh and ATP are coapplied, as previously reported for other Cys-loop receptors interacting with P2X receptors, and this crosstalk is dependent on the presence of the P2X3 C-terminal domain. P2X2 receptors also functionally interact with α6β4β3 but through a different mechanism from α6β4. The interaction with P2X3 receptors is less pronounced for the α6β4β3 nAChR than the α6β4 nAChR. We also measured a functional interaction between the α6β4 nAChRs and the heteromeric P2X2/3 receptor. Experiments with the nAChR channel blocker mecamylamine on P2X2–α6β4 oocytes point to the loss of P2X2 channel activity during the crosstalk, whereas the ion channel pores of the P2X receptors were fully functional and unaltered by the receptor interaction for P2X2–α6β4β3, P2X2/3–α6β4, and P2X2/3–α6β4β3. These results may be relevant to dorsal root ganglion cells and to other neurons that coexpress these receptor subunits. PMID:24966348

  3. Subtype-specific mechanisms for functional interaction between α6β4* nicotinic acetylcholine receptors and P2X receptors.

    PubMed

    Limapichat, Walrati; Dougherty, Dennis A; Lester, Henry A

    2014-09-01

    P2X receptors and nicotinic acetylcholine receptors (nAChRs) display functional and physical interactions in many cell types and heterologous expression systems, but interactions between α6β4-containing (α6β4*) nAChRs and P2X2 receptors and/or P2X3 receptors have not been fully characterized. We measured several types of crosstalk in oocytes coexpressing α6β4 nAChRs and P2X2, P2X3, or P2X2/3 receptors. A novel form of crosstalk occurs between α6β4 nAChRs and P2X2 receptors. P2X2 receptors were forced into a prolonged desensitized state upon activation by ATP through a mechanism that does not depend on the intracellular C terminus of the P2X2 receptors. Coexpression of α6β4 nAChRs with P2X3 receptors shifts the ATP dose-response relation to the right, even in the absence of acetylcholine (ACh). Moreover, currents become nonadditive when ACh and ATP are coapplied, as previously reported for other Cys-loop receptors interacting with P2X receptors, and this crosstalk is dependent on the presence of the P2X3 C-terminal domain. P2X2 receptors also functionally interact with α6β4β3 but through a different mechanism from α6β4. The interaction with P2X3 receptors is less pronounced for the α6β4β3 nAChR than the α6β4 nAChR. We also measured a functional interaction between the α6β4 nAChRs and the heteromeric P2X2/3 receptor. Experiments with the nAChR channel blocker mecamylamine on P2X2-α6β4 oocytes point to the loss of P2X2 channel activity during the crosstalk, whereas the ion channel pores of the P2X receptors were fully functional and unaltered by the receptor interaction for P2X2-α6β4β3, P2X2/3-α6β4, and P2X2/3-α6β4β3. These results may be relevant to dorsal root ganglion cells and to other neurons that coexpress these receptor subunits. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  4. In vitro pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107.

    PubMed

    Malysz, John; Anderson, David J; Grønlien, Jens H; Ji, Jianguo; Bunnelle, William H; Håkerud, Monika; Thorin-Hagene, Kirten; Ween, Hilde; Helfrich, Rosalind; Hu, Min; Gubbins, Earl; Gopalakrishnan, Sujatha; Puttfarcken, Pamela S; Briggs, Clark A; Li, Jinhe; Meyer, Michael D; Dyhring, Tino; Ahring, Philip K; Nielsen, Elsebet Ø; Peters, Dan; Timmermann, Daniel B; Gopalakrishnan, Murali

    2010-09-01

    Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat

  5. The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

    SciTech Connect

    Grandič, Marjana; Aráoz, Romulo; Molgó, Jordi

    2012-12-01

    APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve–hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC{sub 50} = 0.74 μM), without affecting directly-elicited twitch tension up to 2.72 μM. The compound (0.007–3.40 μM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68 μM, without affecting their frequency. Full size endplate potentials,more » recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC{sub 50} = 0.36 μM) without significant change in the resting membrane potential of the muscle fibers up to 3.40 μM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1{sub 2}β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC{sub 50} = 0.0005 μM), indicating a higher affinity of the compound for Torpedo (α1{sub 2}β1γδ) than for the mouse (α1{sub 2}β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction. -- Highlights: ► APS12-2 produces concentration-dependent inhibition of nerve-evoked muscle contraction in vitro. ► APS12-2 blocks MEPPs and EPPs at the neuromuscular junction. APS12-2 blocks ACh-activated current in Xenopus oocytes incorporated with Torpedo nAChRs.« less

  6. A novel nicotinic acetylcholine receptor subtype in basal forebrain cholinergic neurons with high sensitivity to amyloid peptides

    PubMed Central

    Liu, Qiang; Huang, Yao; Xue, Fenqin; Simard, Alain; DeChon, Jamie; Li, Guohui; Zhang, Jianliang; Lucero, Linda; Wang, Min; Sierks, Michael; Hu, Gang; Chang, Yongchang; Lukas, Ronald J.; Wu, Jie

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) containing α7 subunits are thought to assemble as homomers. α7-nAChR function has been implicated in learning and memory, and alterations of α7-nAChR have been found in patients with Alzheimer’s disease (AD). Here we report findings consistent with a novel, naturally-occurring nAChR subtype in rodent, basal forebrain cholinergic neurons. In these cells, α7 subunits are co-expressed, co-localize and co-assemble with β2 subunit(s). Compared to homomeric α7-nAChRs from ventral tegmental area neurons, functional, presumably heteromeric α7β2-nAChRs on cholinergic neurons freshly dissociated from medial septum/diagonal band (MS/DB) exhibit relatively slow kinetics of whole-cell current responses to nicotinic agonists and are more sensitive to the β2 subunit-containing nAChR-selective antagonist, dihydro-β-erythroidine (DHβE). Interestingly, presumed, heteromeric α7β2-nAChRs are highly sensitive to functional inhibition by pathologically-relevant concentrations of oligomeric, but not monomeric or fibrillar forms of amyloid β1-42 (Aβ1-42). Slow whole-cell current kinetics, sensitivity to DHβE, and specific antagonism by oligomeric Aβ1-42 also are characteristic of heteromeric α7β2-nAChRs, but not of homomeric α7-nAChRs, heterologously expressed in Xenopus oocytes. Moreover, choline-induced currents have faster kinetics and less sensitivity to Aβ when elicited from MS/DB neurons derived from nAChR β2 subunit knockout mice rather than from wild-type mice. The presence of novel, functional, heteromeric α7β2-nAChRs on basal forebrain cholinergic neurons and their high sensitivity to blockade by low concentrations of oligomeric Aβ1-42 suggests possible mechanisms for deficits in cholinergic signaling that could occur early in the etiopathogenesis of AD and might be targeted by disease therapies. PMID:19176801

  7. Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

    SciTech Connect

    Gu, Y.

    1989-01-01

    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively,more » of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.« less

  8. Pharmacological characterisation of strychnine and brucine analogues at glycine and alpha7 nicotinic acetylcholine receptors.

    PubMed

    Jensen, Anders A; Gharagozloo, Parviz; Birdsall, Nigel J M; Zlotos, Darius P

    2006-06-06

    Strychnine and brucine from the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors, including some members of the superfamily of ligand-gated ion channels. In this study, we have characterised the pharmacological properties of tertiary and quaternary analogues as well as bisquaternary dimers of strychnine and brucine at human alpha1 and alpha1beta glycine receptors and at a chimera consisting of the amino-terminal domain of the alpha7 nicotinic receptor (containing the orthosteric ligand binding site) and the ion channel domain of the 5-HT3A serotonin receptor. Although the majority of the analogues displayed significantly increased Ki values at the glycine receptors compared to strychnine and brucine, a few retained the high antagonist potencies of the parent compounds. However, mirroring the pharmacological profiles of strychnine and brucine, none of the analogues displayed significant selectivity between the alpha1 and alpha1beta subtypes. The structure-activity relationships for the compounds at the alpha7/5-HT3 chimera were significantly different from those at the glycine receptors. Most strikingly, quaternization of strychnine and brucine with substituents possessing different steric and electronic properties completely eliminated the activity at the glycine receptors, whereas binding affinity to the alpha7/5-HT3 chimera was retained for the majority of the quaternary analogues. This study provides an insight into the structure-activity relationships for strychnine and brucine analogues at these ligand-gated ion channels.

  9. Protective effect of an alpha 7 nicotinic acetylcholine receptor agonist against enterovirus 71 infection in neuronal cells.

    PubMed

    Song, Feng Xia; Zhao, Lin Qing; Zhu, Ru Nan; Song, Qin Wei; Deng, Jie; Tian, Run; Wang, Fang; Qian, Yuan

    2018-01-01

    Enterovirus 71, as one of the dominant pathogens associated with severe hand, foot, and mouth disease, has been well reported to trigger severe neurological symptoms among young children over the last decade, particularly among children in the Asia-Pacific region. To date, no effective antiviral agent has been developed for the treatment of severe enterovirus 71 infection. PNU-282987, a selective alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonist, has been reported to have a neuroprotective effect by participating in inflammatory regulation in previous studies. Therefore, in the present study, we aimed to assess the cell-protective effect of PNU-282987 against enterovirus 71 infection in neuronal cells, and to discuss potential mechanisms underlying this cell-protective effect in order to elucidate the potential impact of such agonists in the treatment of neurotropic viral infection. We observed that treatment with PNU-282987 improved cell viability and inhibited viral replication in enterovirus 71-infected SH-SY5Y cells. Further investigation revealed that inhibition of enterovirus 71 production by PNU-282987 is likely associated with events of RNA replication, and that increased levels of INF mRNA and its downstream antiviral proteins stimulated by the JAK-STAT2 pathway may contribute to the antiviral effect of PNU-282987. Moreover, our findings suggest that both the antiviral and anti-inflammatory effects of PNU-282987 may contribute to the neural protective effect of the drug in enterovirus 71-infected cells. Taken together, the results suggest that selective α7nAChR agonists may represent viable candidates for future therapeutic treatment of severe enterovirus 71 infection, and for other cases of neurotropic viral infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments.

    PubMed

    Asmar-Rovira, Guillermo A; Asseo-García, Aloysha M; Quesada, Orestes; Hanson, Michael A; Cheng, Anchi; Nogueras, Carlos; Lasalde-Dominicci, José A; Stevens, Raymond C

    2008-05-01

    The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, high-resolution structural studies have been hampered by the lack of mechanistic molecular models that describe how detergents influence membrane protein stability and function. Furthermore, elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examines the effects of nine detergents on: (1) nAChR-lipid composition (gas chromatography with flame ionization; GC-FID and/or mass selective detectors; GC-MSD), (2) stability and aggregation state (analytical size exclusion chromatography; A-SEC and electron microscopy; EM) and (3) ion channel function (planar lipid bilayers). Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analogue detergents CHAPS {(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate}, FC-12 (n-dodecylphosphocholine) and sodium cholate (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid) maintaining stability and supporting ion channel function, and non-lipid-analogue detergents Cymal-6 (6-cyclohexyl-1-hexyl-beta-D-maltoside), DDM (n-dodecyl-beta-D-maltopyranoside), LDAO (lauryldimethylamine-N-oxide) and OG (n-octyl-beta-d-glucopyranoside) decreasing stability and significantly reducing or completely suppressing ion channel function. Anapoe-C(12)E(9 )(polyoxyethylene-[9]-dodecyl ether) and BigCHAP (N,N'-bis-[3-d-gluconamidopropyl] cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function compared to lipid-analogue detergents. Therefore, the nAChR can be stable and functional in lipid-analogue detergents or in detergents that retain moderate amounts of residual native lipids, but not in non

  11. Erysodine, a competitive antagonist at neuronal nicotinic acetylcholine receptors, decreases ethanol consumption in alcohol-preferring UChB rats.

    PubMed

    Quiroz, Gabriel; Guerra-Díaz, Nicolás; Iturriaga-Vásquez, Patricio; Rivera-Meza, Mario; Quintanilla, María Elena; Sotomayor-Zárate, Ramón

    2018-04-25

    Alcohol abuse is a worldwide health problem with high economic costs to health systems. Emerging evidence suggests that modulation of brain nicotinic acetylcholine receptors (nAChRs) may be a therapeutic target for alcohol dependence. In this work, we assess the effectiveness of four doses of erysodine (1.5, 2.0, 4.0 or 8.0 mg/kg/day, i.p.), a competitive antagonist of nAChRs, on voluntary ethanol consumption behavior in alcohol-preferring UChB rats, administered during three consecutive days. Results show that erysodine administration produces a dose-dependent reduction in ethanol consumption respect to saline injection (control group). The highest doses of erysodine (4 and 8 mg/kg) reduce (45 and 66%, respectively) the ethanol intake during treatment period and first day of post-treatment compared to control group. While, the lowest doses of erysodine (1.5 and 2 mg/kg) only reduce ethanol intake during one day of treatment period. These effective reductions in ethanol intake were 23 and 29% for 1.5 and 2 mg/kg erysodine, respectively. Locomotor activity induced by a high dose of erysodine (10 mg/kg) was similar to those observed with saline injection in control rats, showing that the reduction in ethanol intake was not produced by hypolocomotor effect induced by erysodine. This is the first report showing that erysodine reduces ethanol intake in UChB rats in a dose-dependent manner. Our results highlight the role of nAChRs in the reward effects of ethanol and its modulation as a potentially effective pharmacological alternative for alcohol dependence treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae).

    PubMed

    Xu, Gang; Wu, Shun-Fan; Teng, Zi-Wen; Yao, Hong-Wei; Fang, Qi; Huang, Jia; Ye, Gong-Yin

    2017-06-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop ligand-gated ion channel (cysLGIC) superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressalis, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs (cDNAs) via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine α-type and three β-type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species. ©2016 The Authors Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.

  13. The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors

    PubMed Central

    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 [125I]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

  14. 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

    2016-08-24

    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.

  15. HIV gp120 induces mucus formation in human bronchial epithelial cells through CXCR4/α7-nicotinic acetylcholine receptors.

    PubMed

    Gundavarapu, Sravanthi; Mishra, Neerad C; Singh, Shashi P; Langley, Raymond J; Saeed, Ali Imran; Feghali-Bostwick, Carol A; McIntosh, J Michael; Hutt, Julie; Hegde, Ramakrishna; Buch, Shilpa; Sopori, Mohan L

    2013-01-01

    Lung diseases such as chronic obstructive pulmonary disease (COPD), asthma, and lung infections are major causes of morbidity and mortality among HIV-infected patients even in the era of antiretroviral therapy (ART). Many of these diseases are strongly associated with smoking and smoking is more common among HIV-infected than uninfected people; however, HIV is an independent risk factor for chronic bronchitis, COPD, and asthma. The mechanism by which HIV promotes these diseases is unclear. Excessive airway mucus formation is a characteristic of these diseases and contributes to airway obstruction and lung infections. HIV gp120 plays a critical role in several HIV-related pathologies and we investigated whether HIV gp120 promoted airway mucus formation in normal human bronchial epithelial (NHBE) cells. We found that NHBE cells expressed the HIV-coreceptor CXCR4 but not CCR5 and produced mucus in response to CXCR4-tropic gp120. The gp120-induced mucus formation was blocked by the inhibitors of CXCR4, α7-nicotinic acetylcholine receptor (α7-nAChR), and γ-aminobutyric acid (GABA)AR but not the antagonists of CCR5 and epithelial growth factor receptor (EGFR). These results identify two distinct pathways (α7-nAChR-GABAAR and EGFR) for airway mucus formation and demonstrate for the first time that HIV-gp120 induces and regulates mucus formation in the airway epithelial cells through the CXCR4-α7-nAChR-GABAAR pathway. Interestingly, lung sections from HIV ± ART and simian immunodeficiency virus (SIV) ± ART have significantly more mucus and gp120-immunoreactivity than control lung sections from humans and macaques, respectively. Thus, even after ART, lungs from HIV-infected patients contain significant amounts of gp120 and mucus that may contribute to the higher incidence of obstructive pulmonary diseases in this population.

  16. 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.

  17. Prorocentrolide-A from Cultured Prorocentrum lima Dinoflagellates Collected in Japan Blocks Sub-Types of Nicotinic Acetylcholine Receptors.

    PubMed

    Amar, Muriel; Aráoz, Rómulo; Iorga, Bogdan I; Yasumoto, Takeshi; Servent, Denis; Molgó, Jordi

    2018-02-28

    Prorocentrolides are members of the cyclic imine phycotoxins family. Their chemical structure includes a 26-membered carbo-macrocycle and a 28-membered macrocyclic lactone arranged around a hexahydroisoquinoline that incorporates the characteristic cyclic imine group. Six prorocentrolides are already known. However, their mode of action remains undetermined. The aim of the present work was to explore whether prorocentrolide A acts on nicotinic acetylcholine receptors (nAChRs), using competition-binding assays and electrophysiological techniques. Prorocentrolide-A displaced [ 125 I]α-bungarotoxin binding to Torpedo membranes, expressing the muscle-type (α1₂β1γδ) nAChR, and in HEK-293 cells, expressing the chimeric chick neuronal α7-5HT₃ nAChR. Functional studies revealed that prorocentrolide-A had no agonist action on nAChRs, but inhibited ACh-induced currents in Xenopus oocytes that had incorporated the muscle-type α1₂β1γδ nAChR to their membranes, or that expressed the human α7 nAChR, as revealed by voltage-clamp recordings. Molecular docking calculations showed the absence of the characteristic hydrogen bond between the iminium group of prorocentrolide-A and the backbone carbonyl group of Trp147 in the receptor, explaining its weaker affinity as compared to all other cyclic imine toxins. In conclusion, this is the first study to show that prorocentrolide-A acts on both muscle and neuronal nAChRs, but with higher affinity on the muscle-type nAChR.

  18. Prorocentrolide-A from Cultured Prorocentrum lima Dinoflagellates Collected in Japan Blocks Sub-Types of Nicotinic Acetylcholine Receptors

    PubMed Central

    Amar, Muriel; Aráoz, Rómulo; Yasumoto, Takeshi; Servent, Denis

    2018-01-01

    Prorocentrolides are members of the cyclic imine phycotoxins family. Their chemical structure includes a 26-membered carbo-macrocycle and a 28-membered macrocyclic lactone arranged around a hexahydroisoquinoline that incorporates the characteristic cyclic imine group. Six prorocentrolides are already known. However, their mode of action remains undetermined. The aim of the present work was to explore whether prorocentrolide-A acts on nicotinic acetylcholine receptors (nAChRs), using competition-binding assays and electrophysiological techniques. Prorocentrolide-A displaced [125I]α-bungarotoxin binding to Torpedo membranes, expressing the muscle-type (α12β1γδ) nAChR, and in HEK-293 cells, expressing the chimeric chick neuronal α7-5HT3 nAChR. Functional studies revealed that prorocentrolide-A had no agonist action on nAChRs, but inhibited ACh-induced currents in Xenopus oocytes that had incorporated the muscle-type α12β1γδ nAChR to their membranes, or that expressed the human α7 nAChR, as revealed by voltage-clamp recordings. Molecular docking calculations showed the absence of the characteristic hydrogen bond between the iminium group of prorocentrolide-A and the backbone carbonyl group of Trp147 in the receptor, explaining its weaker affinity as compared to all other cyclic imine toxins. In conclusion, this is the first study to show that prorocentrolide-A acts on both muscle and neuronal nAChRs, but with higher affinity on the muscle-type nAChR. PMID:29495549

  19. The Alpha 7 Nicotinic Acetylcholine Receptor of Deciduous Dental Pulp Stem Cells Regulates Osteoclastogenesis During Physiological Root Resorption.

    PubMed

    Wang, Lulu; Zhou, Zhifei; Chen, Yujiang; Yuan, Shuai; Du, Yang; Ju, Xinke; Wu, Lizheng; Wang, Xiaojing

    2017-08-15

    The physiological root resorption of deciduous teeth is a normal phenomenon, but the mechanisms underlying this process are still unclear. In this study, deciduous dental pulp stem cells (DDPSCs) and permanent dental pulp stem cells (DPSCs) were derived from deciduous teeth and normal permanent teeth at different stages of resorption. In the middle stage of root resorption, DDPSCs exhibited an increase in the ability to induce osteoclast differentiation. Activation of the alpha 7 nicotinic acetylcholine receptor (α7 nAChR) by secretory mammalian Ly-6 urokinase-type plasminogen activator receptor-associated protein 1 (SLURP-1) caused a significant increase in the expression levels of NF-κB, receptor activator of nuclear factor-kappa B ligand (RANKL), and the ratio of RANKL/osteoprotegerin (OPG). These effects were inhibited by alpha-bungarotoxin (α-BTX). Furthermore, the expression levels of RANKL/OPG were significantly reduced following inhibition of NF-κB. High-strength, dynamic positive pressure increased the expression of SLURP-1 and α7 nAChR in DDPSCs in the stable stage. These data indicated that mechanical stress stimulated the expression of SLURP-1 and α7 nAChR in DDPSCs. Additionally, SLURP-1 activated α7 nAChR, thereby upregulating the expression of NF-κB and enhancing its activity, thus regulating RANKL/OPG expression and affecting the ability of DDPSCs to influence osteoclastogenesis, which likely enhances root resorption and leads to the physiological loss of deciduous teeth.

  20. Varenicline and cytisine: two nicotinic acetylcholine receptor ligands reduce ethanol intake in University of Chile bibulous rats.

    PubMed

    Sotomayor-Zárate, Ramón; Gysling, Katia; Busto, Usoa E; Cassels, Bruce K; Tampier, Lutske; Quintanilla, María Elena

    2013-05-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are pharmacological targets that have recently been implicated in the reinforcing effects of many drugs of abuse, including ethanol. Varenicline and cytisine are nAChR partial agonists in clinical use as smoking cessation aids. However, their efficacies to reduce alcohol consumption have not been fully studied. This study aims to compare the effects of varenicline and cytisine on ethanol consumption by rats bred for many generations as high ethanol drinkers (UChB). Repeated dosing (0.5 or 1.0 mg/kg/day i.p.) of varenicline or cytisine, for three consecutive days, to male UChB rats pre-exposed to 10 % (v/v) ethanol and water 24 h/day for 4 weeks, significantly reduced alcohol intake and preference of ethanol over water during 1- and 24-h ethanol access periods. This effect was specific for ethanol intake and was not observed for 0.2 % saccharin or water consumption. Varenicline appears to be more effective than cytisine, probably due to its more favorable pharmacokinetic and pharmacodynamic properties. Long-term use of both nAChRs ligands for more than 8-10 days induced tolerance to their effects on ethanol consumption. This preclinical study in UChB rats demonstrated that both varenicline and cytisine reduce alcohol intake, with varenicline producing a greater and longer-lasting reduction than cytisine. However, dose adjustment will have to be considered as a possible way to counter tolerance arising after continued use.

  1. Molecular basis of reactive oxygen species-induced inactivation of α4β2 nicotinic acetylcholine receptors.

    PubMed

    Zhao, Junjun; Zheng, Yan; Xue, Fenqin; Chang, Yongchang; Yang, Hui; Zhang, Jianliang

    2016-08-01

    The α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) are the most widespread heteromeric nAChR subtype in the brain, mediating fast synaptic transmission. Previous studies showed that α4β2 nAChRs could be inactivated by reactive oxygen species (ROS), but the underlying mechanism is still obscure. We found that H2O2 induced the rundown of ACh-evoked currents in human α4β2 nAChRs and the replacement of the conserved cysteine in the M1-M2 linker of either α4 Cys245 or β2 Cys237 with an alanine residue could prevent the current rundown. Structurally, α4 Cys245 and β2 Cys237 are hypothesized to be in close proximity when the receptor is activated. Western blotting results showed that α4 and β2 subunits were cross-linked when the agonist-bound receptor encountered H2O2, which could be prevented by the substitution of the conserved cysteine in the M1-M2 linker to an alanine. Thus, when agonist bound to the receptor, α4 Cys245 and β2 Cys237 came close to each other and ROS oxidized these conserved cysteines, leading subunits to be cross-linked and trapping α4β2 nAChRs into the inactivation state. In addition, we mimicked an experimental Parkinson's disease (PD) model in PC12 cells and found that ROS, generated by 6-hydroxydopamine (6-OHDA), could cause the current rundown in α4β2 nAChRs, which may play a role in PD. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Development of Automated Patch Clamp Assay for Evaluation of α7 Nicotinic Acetylcholine Receptor Agonists in Automated QPatch-16.

    PubMed

    Hao, Yuchen; Tang, Jingshu; Wang, KeWei

    2015-04-01

    The α7 nicotinic acetylcholine receptor (α7 nAChR) is an important and challenging target for drug discovery in the area of neuropsychiatric disorders. The current screening for chemicals targeting α7 nAChRs is primarily achieved by the use of low-throughput assay two-electrode voltage clamp (TEVC) in nonmammalian Xenopus oocytes. Automated patch clamp system has emerged as an attractive approach compared to conventional electrophysiology. To develop a mammalian cell-based functional assay in an automated electrophysiology system, we in this study generated a stable expression of α7 nAChRs in GH3 cells that originated from a rat pituitary tumor cell line and utilized automated QPatch-16 to test a set of tool compounds and chemicals identified as α7 agonists by TEVC. For the improvement of evaluating weak or partial α7 nAChRs agonists, we achieved enhancement of the signal-to-noise ratio by the addition of a positive allosteric modulator PNU-120596, which only activates α7 current in the presence of agonist. This improved assay was further validated by using known α7 partial agonists, such as RG3487, EVP-6124, and A-P90. Using this validated assay, we were able to identify a novel agonist 140507C that partially activates α7 nAChRs. Taken together, our results validate the use of QPatch-16 for evaluation α7 partial agonists, demonstrating its utility as an effective tool for α7 ion channel drug discovery.

  3. A novel inhibitor of α9α10 nicotinic acetylcholine receptors from Conus vexillum delineates a new conotoxin superfamily.

    PubMed

    Luo, Sulan; Christensen, Sean; Zhangsun, Dongting; Wu, Yong; Hu, Yuanyan; Zhu, Xiaopeng; Chhabra, Sandeep; Norton, Raymond S; McIntosh, J Michael

    2013-01-01

    Conotoxins (CTxs) selectively target a range of ion channels and receptors, making them widely used tools for probing nervous system function. Conotoxins have been previously grouped into superfamilies according to signal sequence and into families based on their cysteine framework and biological target. Here we describe the cloning and characterization of a new conotoxin, from Conus vexillum, named αB-conotoxin VxXXIVA. The peptide does not belong to any previously described conotoxin superfamily and its arrangement of Cys residues is unique among conopeptides. Moreover, in contrast to previously characterized conopeptide toxins, which are expressed initially as prepropeptide precursors with a signal sequence, a ''pro'' region, and the toxin-encoding region, the precursor sequence of αB-VxXXIVA lacks a ''pro'' region. The predicted 40-residue mature peptide, which contains four Cys, was synthesized in each of the three possible disulfide arrangements. Investigation of the mechanism of action of αB-VxXXIVA revealed that the peptide is a nicotinic acetylcholine receptor (nAChR) antagonist with greatest potency against the α9α10 subtype. (1)H nuclear magnetic resonance (NMR) spectra indicated that all three αB-VxXXIVA isomers were poorly structured in aqueous solution. This was consistent with circular dichroism (CD) results which showed that the peptides were unstructured in buffer, but adopted partially helical conformations in aqueous trifluoroethanol (TFE) solution. The α9α10 nAChR is an important target for the development of analgesics and cancer chemotherapeutics, and αB-VxXXIVA represents a novel ligand with which to probe the structure and function of this protein.

  4. 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

    2015-03-12

    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.

  5. 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

  6. Mechanisms Involved in Nicotinic Acetylcholine Receptor-Induced Neurotransmitter Release from Sympathetic Nerve Terminals in the Mouse Vas Deferens

    PubMed Central

    Williams, Damian J.; Sidaway, Peter; Cunnane, Thomas C.; Brain, Keith L.

    2011-01-01

    Prejunctional nicotinic acetylcholine receptors (nAChRs) amplify postganglionic sympathetic neurotransmission, and there are indications that intraterminal Ca2+ stores might be involved. However, the mechanisms by which nAChR activation stimulates neurotransmitter release at such junctions is unknown. Rapid local delivery (picospritzing) of the nAChR agonist epibatidine was combined with intracellular sharp microelectrode recording to monitor spontaneous and field-stimulation-evoked neurotransmitter release from sympathetic nerve terminals in the mouse isolated vas deferens. Locally applied epibatidine (1 µM) produced ‘epibatidine-induced depolarisations’ (EIDs) that were similar in shape to spontaneous excitatory junction potentials (SEJPs) and were abolished by nonselective nAChR antagonists and the purinergic desensitizing agonist α,β-methylene ATP. The amplitude distribution of EIDs was only slightly shifted towards lower amplitudes by the selective α7 nAChR antagonists α-bungarotoxin and methyllcaconitine, the voltage-gated Na+ channel blocker tetrodotoxin or by blocking voltage-gated Ca2+ channels with Cd2+. Lowering the extracellular Ca2+ concentration reduced the frequency of EIDs by 69%, but more surprisingly, the Ca2+-induced Ca2+ release blocker ryanodine greatly decreased the amplitude (by 41%) and the frequency of EIDs by 36%. Ryanodine had no effect on electrically-evoked neurotransmitter release, paired-pulse facilitation, SEJP frequency, SEJP amplitude or SEJP amplitude distribution. These results show that activation of non-α7 nAChRs on sympathetic postganglionic nerve terminals induces high-amplitude junctional potentials that are argued to represent multipacketed neurotransmitter release synchronized by intraterminal Ca2+-induced Ca2+ release, triggered by Ca2+ influx directly through the nAChR. This nAChR-induced neurotransmitter release can be targeted pharmacologically without affecting spontaneous or electrically

  7. Putative nicotinic acetylcholine receptor subunits express differentially through the life cycle of codling moth, Cydia pomonella (Lepidoptera: Tortricidae).

    PubMed

    Martin, Jessica A; Garczynski, Stephen F

    2016-04-01

    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). Orchardists in Washington State are concerned about the possibility of codling moth field populations developing resistance to these two insecticides. In an effort to help mitigate this issue, we initiated a project to identify and characterize codling moth nAChR subunits expressed in heads. This study had two main goals; (i) identify transcripts from a codling moth head transcriptome that encode for nAChR subunits, and (ii) determine nAChR subunit expression profiles in various life stages of codling moth. From a codling moth head transcriptome, 24 transcripts encoding for 12 putative nAChR subunit classes were identified and verified by PCR amplification, cloning, and sequence determination. Characterization of the deduced protein sequences encoded by putative nAChR transcripts revealed that they share the distinguishing features of the cys-loop ligand-gated ion channel superfamily with 9 α-type subunits and 3 β-type subunits identified. Phylogenetic analysis comparing these protein sequences to those of other insect nAChR subunits supports the identification of these proteins as nAChR subunits. Stage expression studies determined that there is clear differential expression of many of these subunits throughout the codling moth life cycle. The information from this study will be used in the future to monitor for potential target-site resistance mechanisms to neonicotinoids and spinosads in tolerant codling moth populations. © 2014 Institute of Zoology, Chinese Academy of Sciences.

  8. Attenuation of Compulsive-Like Behavior Through Positive Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors in Non-Induced Compulsive-Like Mice

    PubMed Central

    Mitra, Swarup; Mucha, Mckenzie; Khatri, Shailesh N.; Glenon, Richard; Schulte, Marvin K.; Bult-Ito, Abel

    2017-01-01

    Nicotinic α4β2 receptors are the most abundant subtypes of nicotinic acetylcholine receptors (nAChRs) expressed in brain regions implicated in obsessive compulsive disorder (OCD). These receptors are known to modify normal and addictive behaviors by modulating neuronal excitability. Desformylflustrabromine (dFBr) is a novel, positive allosteric modulator (PAM) of high acetylcholine sensitivity (HS) and low acetylcholine sensitivity (LS) α4β2 nAChRs. The present study tested the hypothesis that positive allosteric modulation of α4β2 receptors by dFBr will attenuate compulsive-like behavior in a non-induced compulsive-like mouse model. Male mice (Mus musculus) selected for compulsive-like nesting behavior (NB; 48 animals; 12 per group) received acute (once) and chronic (every day for 32 days) subcutaneous injection of dFBr at 2, 4 and 6 mg/kg doses. Saline was used as a control (0 mg/kg). Compulsive-like NB was assessed after 1, 2, 3, 4, 5 and 24 h, while compulsive-like marble burying (MB) and anxiety-like open field (OF) behaviors were performed 2 h after dFBr administration. In the acute administration protocol, dFBr dose dependently attenuated NB and MB. Rapid effects (1–2 h after drug administration) of dFBr on MB and NB were observed for the chronic administration which was in congruence with the acute study. Chronic administration also revealed sustained suppression of NB by dFBr following 5 weeks of treatment. In both the acute and chronic regimen dFBr did not modulate OF behaviors. This research demonstrates the novel role of positive allosteric modulation of α4β2 nicotinic receptors by dFBr as a translational potential for OCD. PMID:28105008

  9. Attenuation of Compulsive-Like Behavior Through Positive Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors in Non-Induced Compulsive-Like Mice.

    PubMed

    Mitra, Swarup; Mucha, Mckenzie; Khatri, Shailesh N; Glenon, Richard; Schulte, Marvin K; Bult-Ito, Abel

    2016-01-01

    Nicotinic α4β2 receptors are the most abundant subtypes of nicotinic acetylcholine receptors (nAChRs) expressed in brain regions implicated in obsessive compulsive disorder (OCD). These receptors are known to modify normal and addictive behaviors by modulating neuronal excitability. Desformylflustrabromine (dFBr) is a novel, positive allosteric modulator (PAM) of high acetylcholine sensitivity (HS) and low acetylcholine sensitivity (LS) α4β2 nAChRs. The present study tested the hypothesis that positive allosteric modulation of α4β2 receptors by dFBr will attenuate compulsive-like behavior in a non-induced compulsive-like mouse model. Male mice ( Mus musculus ) selected for compulsive-like nesting behavior (NB; 48 animals; 12 per group) received acute (once) and chronic (every day for 32 days) subcutaneous injection of dFBr at 2, 4 and 6 mg/kg doses. Saline was used as a control (0 mg/kg). Compulsive-like NB was assessed after 1, 2, 3, 4, 5 and 24 h, while compulsive-like marble burying (MB) and anxiety-like open field (OF) behaviors were performed 2 h after dFBr administration. In the acute administration protocol, dFBr dose dependently attenuated NB and MB. Rapid effects (1-2 h after drug administration) of dFBr on MB and NB were observed for the chronic administration which was in congruence with the acute study. Chronic administration also revealed sustained suppression of NB by dFBr following 5 weeks of treatment. In both the acute and chronic regimen dFBr did not modulate OF behaviors. This research demonstrates the novel role of positive allosteric modulation of α4β2 nicotinic receptors by dFBr as a translational potential for OCD.

  10. Effects of nicotine in combination with drugs described as positive allosteric nicotinic acetylcholine receptor modulators in vitro: discriminative stimulus and hypothermic effects in mice

    PubMed Central

    Moerke, Megan J.; de Moura, Fernando B.; Koek, Wouter; McMahon, Lance R.

    2016-01-01

    Some drugs that are positive allosteric nAChR modulators in vitro, desformylflustrabromine (dFBr), PNU-120596 and LY 2087101, have not been fully characterized in vivo. These drugs were examined for their capacity to share or modify the hypothermic and discriminative stimulus effects of nicotine (1 mg/kg s.c.) in male C57Bl/6J mice. Nicotine, dFBr, and PNU-120596 produced significant hypothermia, whereas LY 2087101 (up to 100 mg/kg) did not. Nicotine dose-dependently increased nicotine-appropriate responding and decreased response rate; the respective ED50 values were 0.56 mg/kg and 0.91 mg/kg. The modulators produced no more than 38% nicotine-appropriate responding up to doses that disrupted operant responding. Rank order potency was the same for hypothermia and rate-decreasing effects: nicotine>dFBr>PNU-120596=LY 2087101. Mecamylamine and the α4β2 nAChR antagonist dihydro-β-erythroidine, but not the α7 antagonist methyllycaconitine, antagonized the hypothermic effects of nicotine. In contrast, mecamylamine did not antagonize the hypothermic effects of the modulators. The combined discriminative stimulus effects of DFBr and nicotine were synergistic, whereas the combined hypothermic effects of nicotine with either dFBr or PNU-120596 were infra-additive. PNU-120596 did not modify the nicotine discriminative stimulus, and LY 2087101 did not significantly modify either effect of nicotine. Positive modulation of nicotine at nAChRs by PNU-120596 and LY 2087101 in vitro does not appear to confer enhancement of the nAChR-mediated hypothermic or discriminative stimulus effects of nicotine. However, dFBr appears to be a positive allosteric modulator of some behavioral effects of nicotine at doses of dFBr smaller than the doses producing unwanted effects (e.g. hypothermia) through non-nAChR mechanisms. PMID:27238974

  11. Impact of human D398N single nucleotide polymorphism on intracellular calcium response mediated by α3β4α5 nicotinic acetylcholine receptors

    PubMed Central

    Tammimäki, Anne; Herder, Penelope; Li, Ping; Esch, Caroline; Laughlin, James R.; Akk, Gustav; Stitzel, Jerry A.

    2013-01-01

    The human CHRNA5 D398N polymorphism (rs16969968) causes an aspartic acid to asparagine change in the nicotinic acetylcholine receptor (nAChR) α5 subunit gene. The N398 variant of CHRNA5 is linked to increased risk for nicotine dependence. In this study, we explored the effect of the CHRNA5 D398N polymorphism on the properties of human α3β4* nicotinic acetylcholine receptors in human embryonic kidney (HEK) cells. Addition of either D398 or N398 variant of α5 subunit in the α3β4* receptor did not affect total [125I]-epibatidine binding or surface expression of the receptor. However, addition of α5D398 into α3β4* receptor decreased the maximal response to agonist without significantly affecting EC50 in aequorin intracellular calcium assay. α3β4α5N398 nAChRs showed further decreased maximal response. The differences in agonist efficacy between the receptor subtypes were found to be dependent upon the concentration of external calcium but independent of external sodium. Moreover, activation of α3β4α5 nAChRs led to significantly greater intracellular calcium release from IP3 stores relative to α3β4 nAChRs although no effect of the α5 polymorphism was observed. Finally, inclusion of the α5 variant caused a small shift to the left in IC50 for some of the antagonists tested, depending upon α5 variant but did not affect sensitivity of α3β4* receptors to desensitization in response to incubation with nicotine. In conclusion, addition of either variant of a5 into an α3β4α5 receptor similarly effects receptor pharmacology and function. However, the N398 variant exhibits a reduced response to agonists when extracellular calcium is high and it may lead to distinct downstream cellular signaling. PMID:22820273

  12. A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: comparison with long-chain alpha-neurotoxins and alpha-conotoxins.

    PubMed

    Mordvintsev, D Yu; Polyak, Ya L; Levtsova, O V; Tourleigh, Ye V; Kasheverov, I E; Shaitan, K V; Utkin, Yu N; Tsetlin, V I

    2005-12-01

    Short-chain alpha-neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short alpha-neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using 1H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346, 967-989] and X-ray structures of acetylcholine-binding protein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907-914] and antagonists: alpha-cobratoxin, a long-chain alpha-neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512-1522] and alpha-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582-588]. In complex with the receptor, NTII was located at about 30 A from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the alpha/gamma or alpha/delta nAChR subunits, while the loops I and III

  13. Genetic Background Influences the Effects of Withdrawal from Chronic Nicotine on Learning and High-Affinity Nicotinic Acetylcholine Receptor Binding in the Dorsal and Ventral Hippocampus

    PubMed Central

    Wilkinson, Derek S.; Turner, Jill R.; Blendy, Julie A.; Gould, Thomas J.

    2013-01-01

    Rationale The effects of nicotine on cognitive processes may play an important role in nicotine addiction. Nicotine withdrawal impairs hippocampus-dependent learning and genetic factors influence this effect. However, the neural changes that contribute to these impairments are unknown. Chronic nicotine upregulates hippocampal nicotinic acetycholine receptors (nAChRs), which may contribute to cognitive deficits when nicotine administration ceases. If nAChR upregulation underlies withdrawal-deficits in learning, then strains of mice exhibiting withdrawal-deficits in hippocampus-dependent learning should also show upregulation of hippocampal nAChRs. Objectives Here, we examined the effects of nicotine withdrawal on fear conditioning and [3H]epibatidine binding in the dorsal and ventral hippocampus in two inbred mouse strains and their F1 hybrids. Methods Male C57BL/6NTac, 129S6/SvEvTac, and B6129SF1/Tac mice were administered chronic nicotine (18 mg/kg/d) for 12 days through osmotic pumps and then were trained and tested in fear conditioning 24 hours after cessation of nicotine treatment. Results Nicotine withdrawal impaired hippocampus-dependent contextual conditioning in C57BL/6NTac mice but not 129S6/SvEvTac or B6129SF1/Tac mice; no changes were observed in hippocampus-independent cued fear conditioning. Upregulated [3H]epibatidine binding was found in the dorsal, but not ventral, hippocampus of C57BL/6NTac mice and in the ventral hippocampus of B6129SF1/Tac mice after chronic nicotine. Conclusions Upregulation of high-affinity binding sites in the dorsal hippocampus of C57BL/6NTac mice, the only strain that exhibited nAChR upregulation in this region and withdrawal-deficits in contextual conditioning, suggests that upregulation of high-affinity binding sites in the dorsal hippocampus mediates, in part, nicotine withdrawal-deficits in contextual conditioning and genetic background modulates these effects. PMID:22836371

  14. Evaluation of the sensitivity of the novel α4β2* nicotinic acetylcholine receptor PET radioligand 18F-(-)-NCFHEB to increases in synaptic acetylcholine levels in rhesus monkeys.

    PubMed

    Gallezot, Jean-Dominique; Esterlis, Irina; Bois, Frederic; Zheng, Ming-Qiang; Lin, Shu-Fei; Kloczynski, Tracy; Krystal, John H; Huang, Yiyun; Sabri, Osama; Carson, Richard E; Cosgrove, Kelly P

    2014-11-01

    18F-(-)-NCFHEB (also known as 18F-(-)-Flubatine) is a new radioligand to image α4β2* nicotinic acetylcholine receptors in vivo with positron emission tomography (PET), with faster kinetics than previous radioligands such as 18F-2-F-A85380. The goal of this study was to assess the sensitivity of 18F-(-)-NCFHEB-PET to increases in synaptic acetylcholine concentration induced by acetylcholinesterase inhibitors. Two rhesus monkeys were scanned four times each on a Focus 220 scanner: first at baseline, then during two bolus plus infusions of physostigmine (0.06-0.28 mg/kg), and finally following a bolus injection of donepezil (0.25 mg/kg). The arterial input function and the plasma free fraction fP were measured. 18F-(-)-NCFHEB volume of distribution VT was estimated using the multilinear analysis MA1 and then normalized by plasma free fraction fP . 18F-(-)-NCFHEB fP was 0.89±0.04. At baseline, 18F-(-)-NCFHEB VT /fP ranged from 7.9±1.3 mL plasma/cm3 tissue in the cerebellum to 34.3±8.4 mL plasma/cm3 tissue in the thalamus. Physostigmine induced a dose-dependent reduction of 18F-(-)-NCFHEB VT /fP of 34±9% in the putamen, 32±8% in the thalamus, 25±8% in the cortex, and 23±10% in the hippocampus. With donepezil, 18F-(-)-NCFHEB VT /fP was reduced by 24±2%, 14+3% and 14±5%, 10±6% in the same regions. 18F-(-)-NCFHEB can be used to detect changes in synaptic acetylcholine concentration and is a promising tracer to study acetylcholine dynamics with shorter scan durations than previous radioligands. © 2014 Wiley Periodicals, Inc.

  15. Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments

    PubMed Central

    Asmar-Rovira, Guillermo A.; Asseo-García, Aloysha M.; Quesada, Orestes; Hanson, Michael A.; Nogueras, Carlos; Lasalde-Dominicci, José A.; Stevens, Raymond C.

    2009-01-01

    The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, the molecular mechanisms by which detergents influence membrane protein stability and function remain poorly understood, and elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examined nine detergents upon nAChR solubilization and purification, to assess receptor lipid composition using GC (Gas Chromatography)-FID (Flame Ionization) and/or GC-MSD (Mass Selective Detectors), stability and aggregation state using A-SEC (Analytical Size-Exclusion Chromatography) and EM (Electron Microscopy), and planar lipid bilayers to measure ion channel function. Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analog detergents [CHAPS (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate), FC-12 (n-Dodecylphosphocholine) and sodium cholate (3α,7α,12α-Trihydroxy-5β-cholan-24-oic acid)] maintaining stability and supporting ion channel function, while non-lipid analog detergents [Cymal-6 (6-Cyclohexyl-1-hexyl-β-d-maltoside), DDM (n-Dodecyl-β-d-maltopyranoside), LDAO (Lauryldimethylamine-N-oxide) and OG (n-Octyl-β-d-glucopyranoside)] showed decreased stability and significant reduction or loss of ion channel function. Anapoe-C12E9 (Polyoxyethylene-(9)-dodecyl ether) and BigCHAP (N,N′-bis-(3-d-Gluconamidopropyl) cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function when compared to lipid-analog detergents. Overall, these results show that the nAChR can be stable and functional in lipid-analog detergents or in detergents that retain moderate amounts of residual native lipids, while the opposite is true about non-lipid analog

  16. The role of the α7 subunit of the nicotinic acetylcholine receptor in the acute toxicosis of methyllycaconitine in mice.

    PubMed

    Welch, K D; Green, B T; Panter, K E; Pfister, J A; Gardner, D R

    2013-09-01

    The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research suggested a correlation between the lethal dose (LD50 ) of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. The objective of this study was to characterize the role of the α7 subunit in the acute toxicosis of MLA by evaluating the acute toxicity of MLA in mice lacking the α7 subunit. The LD50 values for MLA were 4.2 ± 0.9, 3.7 ± 1.1 and 3.3 ± 0.9 mg kg(-1) body weight (BW) for wild-type, heterozygous knockout and homozygous knockout mice, respectively. We also evaluated the response of anabasine in these mice. The LD50 values for anabasine were 1.6 ± 0.3, 2.0 ± 0.4 and 1.8 ± 0.3 mg kg(-1) BW for wild-type, heterozygous knockout and homozygous knockout mice, respectively. The protein expresson of various nAChR subunits was compared to determine if mice lacking the α7 subunit compensate by over expressing other nAChR subunits. There were no significant differences in the protein expression of the α3 , α4 , α5 , β2 and β4 subunits amongst the three genotypes of mice in brain or skeletal muscle. The results of this study suggest that α7 nAChR does not play an integral role in the acute toxicosis of MLA or anabasine. Consequently other nAChR subunits of nAChRs found in the neuromuscular junction are probably the primary target for MLA and anabasine resulting in acute toxicosis. Published 2013. This article is a US Government work and is in the public domain in the USA.

  17. A Distinct Functional Site in Ω-Neurotoxins: Novel Antagonists of Nicotinic Acetylcholine Receptors from Snake Venom.

    PubMed

    Hassan-Puttaswamy, Varuna; Adams, David J; Kini, R Manjunatha

    2015-12-18

    Snake venom α-neurotoxins from the three-finger toxin (3FTx) family are competitive antagonists with nanomolar affinity and high selectivity for nicotinic acetylcholine receptors (nAChR). Here, we report the characterization of a new group of competitive nAChR antagonists: Ω-neurotoxins. Although they belong to the 3FTx family, the characteristic functional residues of α-neurotoxins are not conserved. We evaluated the subtype specificity and structure-function relationships of Oh9-1, an Ω-neurotoxin from Ophiophagus hannah venom. Recombinant Oh9-1 showed reversible postsynaptic neurotoxicity in the micromolar range. Experiments with different nAChR subtypes expressed in Xenopus oocytes indicated Oh9-1 is selective for rat muscle type α1β1εδ (adult) and α1β1γδ (fetal) and rat neuronal α3β2 subtypes. However, Oh9-1 showed low or no affinity for other human and rat neuronal subtypes. Twelve individual alanine-scan mutants encompassing all three loops of Oh9-1 were evaluated for binding to α1β1εδ and α3β2 subtypes. Oh9-1's loop-II residues (M25, F27) were the most critical for interactions and formed the common binding core. Mutations at T23 and F26 caused a significant loss in activity at α1β1εδ receptors but had no effect on the interaction with the α3β2 subtype. Similarly, mutations at loop-II (H7, K22, H30) and -III (K45) of Oh9-1 had a distinctly different impact on its activity with these subtypes. Thus, Oh9-1 interacts with these nAChRs via distinct residues. Unlike α-neurotoxins, the tip of loop-II is not involved. We reveal a novel mode of interaction, where both sides of the β-strand of Oh9-1's loop-II interact with α1β1εδ, but only one side interacts with α3β2. Phylogenetic analysis revealed functional organization of the Ω-neurotoxins independent of α-neurotoxins. Thus, Ω-neurotoxin: Oh9-1 may be a new, structurally distinct class of 3FTxs that, like α-neurotoxins, antagonize nAChRs. However, Oh9-1 binds to the ACh

  18. The α6 nicotinic acetylcholine receptor subunit of Frankliniella occidentalis is not involved in resistance to spinosad.

    PubMed

    Hou, Wenjie; Liu, Qiulei; Tian, Lixia; Wu, Qingjun; Zhang, Youjun; Xie, Wen; Wang, Shaoli; Miguel, Keri San; Funderburk, Joe; Scott, Jeffrey G

    2014-05-01

    Insects evolve resistance which constrains the sustainable use of insecticides. Spinosyns, a class of environmentally-friendly macrolide insecticides, is not an exception. The mode of inheritance and the mechanisms of resistance to spinosad (the most common spinosyn insecticide) in Frankliniella occidentalis (Western flower thrips, WFT) were investigated in this study. Resistance (170,000-fold) was autosomal and completely recessive. Recent studies showed that deletion of the nicotinic acetylcholine receptor α6 subunit gene resulted in strains of Drosophila melanogaster, Plutella xylostella and Bactrocera dorsalis that are resistant to spinosad, indicating that nAChRα6 subunit maybe important for the toxic action of this insecticide. Conversely, a G275E mutation of this subunit in F. occidentalis was recently proposed as the mechanism of resistance to spinosad. We cloned and characterized nAChRα6 from three susceptible and two spinosad resistant strains from China and the USA. The Foα6 cDNA is 1873bp and the open reading frame is 1458bp which encodes 485 amino acid residues with a predicted molecular weight of 53.5-kDa, the 5' and 3' UTRs are 121 and 294bp, respectively. There was no difference in the cDNA sequence between the resistant and susceptible thrips, suggesting the G275E mutation does not confer resistance in these populations. Ten isoforms of Foα6, arising from alternative splicing, were isolated and did not differ between the spinosad-susceptible and resistant strains. Quantitative real time PCR analysis showed Foα6 was highly expressed in the first instar larva, pupa and adult, and the expression levels were 3.67, 2.47, 1.38 times that of the second instar larva. The expression level was not significantly different between the susceptible and resistant strains. These results indicate that Foα6 is not involved in resistance to spinosad in F. occidentalis from China and the USA. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors.

    PubMed

    Marotta, Christopher B; Lester, Henry A; Dougherty, Dennis A

    2015-08-20

    Nicotinic acetylcholine receptors (nAChRs) are vital to neuronal signaling, are implicated in important processes such as learning and memory, and are therapeutic targets for neural diseases. The α7 nAChR has been implicated in Alzheimer's disease and schizophrenia, and allosteric modulators have become one focus of drug development efforts. We investigate the mode of action of the α7-selective positive allosteric modulator, PNU-120596, and show that the higher potency of acetylcholine in the presence of PNU-120596 is not due to an altered agonist binding site. In addition, we propose several residues in the gating interface and transmembrane region that are functionally important to transduction of allosteric properties, and link PNU-120596, the acetylcholine binding region, and the receptor gate. These results suggest global protein stabilization from a communication network through several key residues that alter the gating equilibrium of the receptor while leaving the agonist binding properties unperturbed. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    Mineur, YS; Brunzell, DH; Grady, SR; Lindstrom, JM; McIntosh, JM; Marks, MJ; King, SL; Picciotto, MR

    2009-01-01

    High affinity, β2 subunit-containing (β2*) nAChRs are essential for nicotine reinforcement; however, these nAChRs are found on both GABA and dopaminergic (DA) neurons in the 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 β2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice as compared to tissue from β2 KO mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of β2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low level β2* nAChR expression in the VTA showed no increase in overall levels of 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 was not sufficient to support nicotine place preference in β2tr 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

  1. α6-Containing nicotinic acetylcholine receptors in midbrain dopamine neurons are poised to govern dopamine-mediated behaviors and synaptic plasticity.

    PubMed

    Berry, J N; Engle, S E; McIntosh, J M; Drenan, R M

    2015-09-24

    Acetylcholine (ACh) acts through nicotinic and muscarinic ACh receptors in the ventral midbrain and striatal areas to influence dopamine (DA) transmission. This cholinergic control of DA transmission is important for processes such as attention and motivated behavior, and is manipulated by nicotine in tobacco products. Identifying and characterizing the key ACh receptors involved in cholinergic control of DA transmission could lead to small molecule therapeutics for treating disorders involving attention, addiction, Parkinson's disease, and schizophrenia. α6-Containing nicotinic acetylcholine receptors (nAChRs) are highly and specifically expressed in midbrain DA neurons, making them an attractive drug target. Here, we used genetic, pharmacological, behavioral, and biophysical approaches to study this nAChR subtype. For many experiments, we used mice expressing mutant α6 nAChRs ("α6L9S" mice) that increase the sensitivity of these receptors to agonists such as ACh and nicotine. Taking advantage of a simple behavioral phenotype exhibited by α6L9S mice, we compared the ability of full versus partial α6(∗) nAChR agonists to activate α6(∗) nAChRs in vivo. Using local infusions of both agonists and antagonists into the brain, we demonstrate that neurons and nAChRs in the midbrain are sufficient to account for this behavioral response. To complement these behavioral studies, we studied the ability of in vivo α6(∗) nAChR activation to support plasticity changes in midbrain DA neurons that are relevant to behavioral sensitization and addiction. By coupling local infusion of drugs and brain slice patch-clamp electrophysiology, we show that activating α6(∗) nAChRs in midbrain DA areas is sufficient to enhance glutamatergic transmission in ventral tegmental area (VTA) DA neurons. Together, these results from in vivo studies strongly suggest that α6(∗) nAChRs expressed by VTA DA neurons are positioned to strongly influence both DA-mediated behaviors and the

  2. The association of the alpha-5 subunit of the nicotinic acetylcholine receptor gene and the brain-derived neurotrophic factor gene with different aspects of smoking behavior.

    PubMed

    Breetvelt, Elemi J; Numans, Mattijs E; Aukes, Maartje F; Hoeben, Wopke; Strengman, Eric; Luykx, Jurjen J; Bakker, Steven C; Kahn, René S; Ophoff, Roel A; Boks, Marco P M

    2012-04-01

    Recent studies show that different aspects of smoking behavior are associated with the α-5 subunit of the nicotinic acetylcholine receptor (CHRNA5) gene and the gene coding for brain-derived neurotrophic factor (BDNF). This raises the question whether the amount of cigarettes smoked per day has a different genetic background than smoking initiation and what other smoking phenotypes may be relevant. The aim of this study was to replicate these associations in a large population-based sample. We investigated the association with smoking initiation and the number of cigarettes used per day and additional smoking phenotypes in a population-based sample of 2166 participants of Dutch origin. Rs6265 in BDNF was not associated with smoking initiation. This single nucleotide polymorphism was associated with smoking cessation. Rs16969968 in CHRNA5 was associated with the amount of nicotine used and in particular smoking 25 cigarettes or more per day. Overall, the results confirm the involvement of the CHRNA5 gene in the amount of nicotine use and further suggest involvement of the BDNF gene in smoking behavior.

  3. Primary cultures of rat cortical microglia treated with nicotine increases in the expression of excitatory amino acid transporter 1 (GLAST) via the activation of the α7 nicotinic acetylcholine receptor.

    PubMed

    Morioka, N; Tokuhara, M; Nakamura, Y; Idenoshita, Y; Harano, S; Zhang, F F; Hisaoka-Nakashima, K; Nakata, Y

    2014-01-31

    Although the clearance of glutamate from the synapse under physiological conditions is performed by astrocytic glutamate transporters, their expression might be diminished under pathological conditions. Microglia glutamate transporters, however, might serve as a back-up system when astrocytic glutamate uptake is impaired, and could have a prominent neuroprotective function under pathological conditions. In the current study, the effect of nicotine, well known as a neuroprotective molecule, on the function of glutamate transporters in cultured rat cortical microglia was examined. Reverse transcription polymerase chain reaction and pharmacological approaches demonstrated that, glutamate/aspartate transporter (GLAST), not glutamate transporter 1 (GLT-1), is the major functional glutamate transporter in cultured cortical microglia. Furthermore, the α7 subunit was demonstrated to be the key subunit comprising nicotinic acetylcholine (nACh) receptors in these cells. Treatment of cortical microglia with nicotine led to a significant increase of GLAST mRNA expression and (14)C-glutamate uptake in a concentration- and time-dependent manner, which were markedly inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The nicotine-induced expression of GLAST mRNA and protein is mediated through an inositol trisphosphate (IP3) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) depend intracellular pathway, since pretreatment with either xestospongin C, an IP3 receptor antagonist, or KN-93, a CaMKII inhibitor, blocked GLAST expression. Together, these findings indicate that activation of nACh receptors, specifically those expressing the α7 subunit, on cortical microglia could be a key mechanism of the neuroprotective effect of nACh receptor ligands such as nicotine. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. Imaging of cerebral α4β2* nicotinic acetylcholine receptors with (−)-[18F]Flubatine PET: Implementation of bolus plus constant infusion and sensitivity to acetylcholine in human brain☆

    PubMed Central

    Hillmer, A.T.; Esterlis, I.; Gallezot, J.D.; Bois, F.; Zheng, M.Q.; Nabulsi, N.; Lin, S.F.; Papke, R.L.; Huang, Y.; Sabri, O.; Carson, R.E.; Cosgrove, K.P.

    2016-01-01

    The positron emission tomography (PET) radioligand (−)-[18F]flubatine is specific to α4β2∗ nicotinic acetylcholine receptors (nAChRs) and has promise for future investigation of the acetylcholine system in neuropathologies such as Alzheimer's disease, schizophrenia, and substance use disorders. The two goals of this work were to develop a simplified method for α4β2∗ nAChR quantification with bolus plus constant infusion (B/I) (−)-[18F]flubatine administration, and to assess the radioligand's sensitivity to acetylcholine fluctuations in humans. Healthy human subjects were imaged following either bolus injection (n = 8) or B/I (n = 4) administration of (−)-[18F]flubatine. The metabolite-corrected input function in arterial blood was measured. Free-fraction corrected distribution volumes (VT/fP) were estimated with modeling and graphical analysis techniques. Next, sensitivity to acetylcholine was assessed in two ways: 1. A bolus injection paradigm with two scans (n = 6), baseline (scan 1) and physostigmine challenge (scan 2; 1.5 mg over 60 min beginning 5 min prior to radiotracer injection); 2. A single scan B/I paradigm (n = 7) lasting up to 240 min with 1.5 mg physostigmine administered over 60 min beginning at 125 min of radiotracer infusion. Changes in VT/fP were measured. Baseline VT/fP values were 33.8 ± 3.3 mL/cm3 in thalamus, 12.9 ± 1.6 mL/cm3 in cerebellum, and ranged from 9.8 to 12.5 mL/cm3 in other gray matter regions. The B/I paradigm with equilibrium analysis at 120 min yielded comparable VT/fP values with compartment modeling analysis of bolus data in extrathalamic gray matter regions (regional means <4% different). Changes in VT/fP following physostigmine administration were small and most pronounced in cortical regions, ranging from 0.8 to 4.6% in the two-scan paradigm and 2.8 to 6.5% with the B/I paradigm. These results demonstrate the use of B/I administration for accurate quantification of (−)-[18F]flubatine VT/fP in 120 min, and

  5. Imaging of cerebral α4β2* nicotinic acetylcholine receptors with (-)-[(18)F]Flubatine PET: Implementation of bolus plus constant infusion and sensitivity to acetylcholine in human brain.

    PubMed

    Hillmer, A T; Esterlis, I; Gallezot, J D; Bois, F; Zheng, M Q; Nabulsi, N; Lin, S F; Papke, R L; Huang, Y; Sabri, O; Carson, R E; Cosgrove, K P

    2016-11-01

    The positron emission tomography (PET) radioligand (-)-[(18)F]flubatine is specific to α4β2(⁎) nicotinic acetylcholine receptors (nAChRs) and has promise for future investigation of the acetylcholine system in neuropathologies such as Alzheimer's disease, schizophrenia, and substance use disorders. The two goals of this work were to develop a simplified method for α4β2(⁎) nAChR quantification with bolus plus constant infusion (B/I) (-)-[(18)F]flubatine administration, and to assess the radioligand's sensitivity to acetylcholine fluctuations in humans. Healthy human subjects were imaged following either bolus injection (n=8) or B/I (n=4) administration of (-)-[(18)F]flubatine. The metabolite-corrected input function in arterial blood was measured. Free-fraction corrected distribution volumes (VT/fP) were estimated with modeling and graphical analysis techniques. Next, sensitivity to acetylcholine was assessed in two ways: 1. A bolus injection paradigm with two scans (n=6), baseline (scan 1) and physostigmine challenge (scan 2; 1.5mg over 60min beginning 5min prior to radiotracer injection); 2. A single scan B/I paradigm (n=7) lasting up to 240min with 1.5mg physostigmine administered over 60min beginning at 125min of radiotracer infusion. Changes in VT/fP were measured. Baseline VT/fP values were 33.8±3.3mL/cm(3) in thalamus, 12.9±1.6mL/cm(3) in cerebellum, and ranged from 9.8 to 12.5mL/cm(3) in other gray matter regions. The B/I paradigm with equilibrium analysis at 120min yielded comparable VT/fP values with compartment modeling analysis of bolus data in extrathalamic gray matter regions (regional means <4% different). Changes in VT/fP following physostigmine administration were small and most pronounced in cortical regions, ranging from 0.8 to 4.6% in the two-scan paradigm and 2.8 to 6.5% with the B/I paradigm. These results demonstrate the use of B/I administration for accurate quantification of (-)-[(18)F]flubatine VT/fP in 120min, and suggest

  6. α7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation

    PubMed Central

    2012-01-01

    Background Although evidence suggests that the prevalence of Parkinson’s disease (PD) is lower in smokers than in non-smokers, the mechanisms of nicotine-induced neuroprotection remain unclear. Stimulation of the α7 nicotinic acetylcholine receptor (α7-nAChR) seems to be a crucial mechanism underlying the anti-inflammatory potential of cholinergic agonists in immune cells, including astrocytes, and inhibition of astrocyte activation has been proposed as a novel strategy for the treatment of neurodegenerative disorders such as PD. The objective of the present study was to determine whether nicotine-induced neuroprotection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model occurs via α7-nAChR-mediated inhibition of astrocytes. Methods Both in vivo (MPTP) and in vitro (1-methyl-4-phenylpyridinium ion (MPP+) and lipopolysaccharide (LPS)) models of PD were used to investigate the role(s) of and possible mechanism(s) by which α7-nAChRs protect against dopaminergic neuron loss. Multiple experimental approaches, including behavioral tests, immunochemistry, and stereology experiments, astrocyte cell cultures, reverse transcriptase PCR, laser scanning confocal microscopy, tumor necrosis factor (TNF)-α assays, and western blotting, were used to elucidate the mechanisms of the α7-nAChR-mediated neuroprotection. Results Systemic administration of nicotine alleviated MPTP-induced behavioral symptoms, improved motor coordination, and protected against dopaminergic neuron loss and the activation of astrocytes and microglia in the substantia nigra. The protective effects of nicotine were abolished by administration of the α7-nAChR-selective antagonist methyllycaconitine (MLA). In primary cultured mouse astrocytes, pretreatment with nicotine suppressed MPP+-induced or LPS-induced astrocyte activation, as evidenced by both decreased production of TNF-α and inhibition of extracellular regulated kinase1/2 (Erk1/2) and p38 activation in astrocytes, and

  7. 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

  8. Administration of the nicotinic acetylcholine receptor agonists ABT-089 and ABT-107 attenuates the reinstatement of nicotine-seeking behavior in rats

    PubMed Central

    Lee, Alycia M.; Arreola, Adrian C.; Kimmey, Blake A.; Schmidt, Heath D.

    2014-01-01

    Current smoking cessation pharmacotherapies have modest efficacy, and most smokers relapse within the first few days after a quit attempt. Nicotine withdrawal-induced craving and cognitive impairments predict smoking relapse during abstinence and suggest that cognitive-enhancing drugs may prevent relapse. ABT-089 and ABT-107 are subtype-selective nAChR agonists that improve cognitive performance in laboratory animals. However, there are no studies examining the effects of ABT-089 and ABT-107 on nicotine self-administration and the reinstatement of nicotine-seeking behavior, an animal model of relapse in human smokers. The goal of the present study was to determine the effects of the α4β2*/α6β2* nAChR agonist ABT-089 and the α7 nAChR agonist ABT-107 on nicotine taking and seeking in rats. The effects of acute ABT-089 and ABT-107 pretreatment on nicotine self-administration and reinstatement were tested in male Sprague Dawley rats. Parallel studies of ABT-089 and ABT-107 on sucrose self-administration and reinstatement were tested in separate groups of rats to determine if the effects of these drug treatments generalized to other reinforced behaviors. Nicotine and sucrose self-administration behaviors were not altered following acute administration of ABT-089 (0, 0.12, 1.2 and 12.0 mg/kg) or ABT-107 (0, 0.03 and 0.3 mg/kg). In contrast, both ABT-089 and ABT-107 pretreatment dose-dependently attenuated nicotine reinstatement. These effects were reinforcer-specific as no effects of ABT-089 or ABT-107 pretreatment on sucrose seeking were noted. Taken together, these findings suggest that ABT-089 and ABT-107 do not affect nicotine consumption, but may reduce the likelihood that a smoking lapse will lead to relapse. PMID:25128791

  9. The Effects of Lobeline on α4β2* Nicotinic Acetylcholine Receptor Binding and Uptake of [18F]Nifene in Rats

    PubMed Central

    Hillmer, Ansel T; Wooten, Dustin W; Farhoud, Mohammed; Barnhart, Todd E; Mukherjee, Jogeshwar; Christian, Bradley T

    2013-01-01

    Lobeline is a potential smoking cessation drug with affinity for the α4β2 nicotinic acetylcholine receptor and may inhibit the blood-brain barrier (BBB) amine transporter. The goal of this work was to use PET imaging to evaluate the effects of lobeline on the kinetic properties of [18F]nifene in the rat brain. Methods Direct α4β2* competition of lobeline with [18F]nifene was evaluated using imaging experiments with both displacing and blocking doses of lobeline (1 mg/kg, i.v.) given between two injections of [18F]nifene separated by 50 minutes. Inhibition of the BBB amine transporter was examined using a separate imaging protocol with three injections of [18F]nifene, first at baseline, then following (−)nicotine blocking, and finally following lobeline blocking. Results Rapid displacement of [18F]nifene was observed in the α4β2*-rich thalamus following lobeline administration, suggesting direct competition of the drug at α4β2* sites. Slight decreases in BBB transport of [18F]nifene were observed when the α4β2* system was first saturated with (−)nicotine and then given lobeline. This perturbation may be due to inhibition of the BBB amine transporter by lobeline or reductions in blood flow. Significant cerebellar displacement of [18F]nifene was found following the administration of both lobeline and (−)nicotine, indicating detectable specific binding in the rat cerebellum. Conclusion The competition of lobeline with [18F]nifene is largely dominated at the α4β2* binding site and only small perturbations in BBB transport of [18F]nifene are seen at the 1 mg/kg dose. Similar experiments could be used to study other drugs as therapeutic agents for smoking cessation with PET. PMID:23370310

  10. Natural Variation within the Neuronal Nicotinic Acetylcholine Receptor Cluster on Human Chromosome 15q24: Influence on Heritable Autonomic Traits in Twin Pairs

    PubMed Central

    Rana, Brinda K.; Wessel, Jennifer; Mahboubi, Vafa; Rao, Fangwen; Haeller, Jeannine; Gayen, Jiaur R.; Eskin, Eleazar; Valle, Anne M.; Das, Madhusudan; Mahata, Sushil K.; Taupenot, Laurent; Stridsberg, Mats; Talley, Todd T.; Ziegler, Michael G.; Smith, Douglas W.; Schork, Nicholas J.; O'Connor, Daniel T.

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are combinations of subunits arranged as pentamers encircling a central cation channel. At least nine α and four β subunits are expressed in the central and peripheral nervous systems; their presence in autonomic ganglia, the adrenal medulla, and central nervous system, with accompanying responses elicited by nicotinic agonists, point to their involvement in cardiovascular homeostasis. nAChRs formed by α3, α5, and β4 subunits may regulate blood pressure (BP) by mediating release of catestatin, the endogenous nicotinic antagonist fragment of chromogranin A (CHGA) and potent inhibitor of catecholamine secretion. Genes encoding these subunits (CHRNA3, CHRNA5, and CHRNB4) are clustered on human chromosome 15q24. Because variation in this cluster may alter autonomic regulation of BP, we sequenced ∼15 kilobase pairs in 15q24 containing their coding and 5′- and 3′-untranslated regions in 80 individuals. We identified 63 variants: 25 in coding regions of CHRNA3, CHRNA5, and CHRNB4 and 48 noncoding single-nucleotide polymorphisms (SNPs). Haplotype frequencies varied across ethnic populations. We assessed the contribution of six SNPs in the putative catestatin binding region of CHRNA3 and CHRNB4 to autonomic traits. In twins, catestatin and BP were heritable. CHRNA3 SNPs and haplotypes containing K95K (G285A) associated with circulating plasma catestatin, epinephrine levels, as well as systolic BP, suggesting altered coupling of the nAChRs to BP. Studies of chromaffin cells in vitro reveal that nicotinic agonist stimulation releases catecholamines and CHGA, a process augmented by overexpression of CHRNA3 and blocked by catestatin. These cellular events suggest a homeostatic mechanism underlying the pleiotropic actions of CHRNA3 genetic variation on autonomic function observed in twins. PMID:19671882

  11. Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface.

    PubMed

    Ahring, Philip K; Olsen, Jeppe A; Nielsen, Elsebet Ø; Peters, Dan; Pedersen, Martin H F; Rohde, Line A; Kastrup, Jette S; Shahsavar, Azadeh; Indurthi, Dinesh C; Chebib, Mary; Gajhede, Michael; Balle, Thomas

    2015-05-01

    The nicotinic acetylcholine receptor α4β2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (α4)2(β2)3 and (α4)3(β2)2. While these are similar in many aspects, the (α4)3(β2)2 stoichiometry differs by harboring a third orthosteric acetylcholine binding site located at the α4-α4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known. The present study was therefore aimed at determining binding affinities of nicotinic ligands to the α4-α4 interface. Given that epibatidine shows large functional potency differences at α4-β2 vs. α4-α4 interfaces, biphasic binding properties would be expected at (α4)3(β2)2 receptors. However, standard saturation binding experiments with [(3)H]epibatidine did not reveal biphasic binding under the conditions utilized. Therefore, an engineered β2 construct (β2(HQT)), which converts the β(-) face to resemble that of an α4(-) face, was utilized to create (α4)3(β2(HQT))2 receptors harboring three α4-α4 interfaces. With this receptor, low affinity binding of epibatidine with a Kd of ∼5 nM was observed in sharp contrast to a Kd value of ∼10 pM observed for wild-type receptors. A strong correlation between binding affinities at the (α4)3(β2(HQT))2 receptor and functional potencies at the wild-type receptor of a range of nicotinic ligands highlighted the validity of using the mutational approach. Finally, large differences in activities at α4-β2 vs. α4-α4 interfaces were observed for structurally related agonists underscoring the need for establishing all binding parameters of compounds at α4β2 receptors. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  12. Design, synthesis, and biological activity of 5'-phenyl-1,2,5,6-tetrahydro-3,3'-bipyridine analogues as potential antagonists of nicotinic acetylcholine receptors.

    PubMed

    Jin, Yafei; Huang, Xiaoqin; Papke, Roger L; Jutkiewicz, Emily M; Showalter, Hollis D; Zhan, Chang-Guo

    2017-09-15

    Starting from a known non-specific agonist (1) of nicotinic acetylcholine receptors (nAChRs), rationally guided structural-based design resulted in the discovery of a small series of 5'-phenyl-1,2,5,6-tetrahydro-3,3'-bipyridines (3a-3e) incorporating a phenyl ring off the pyridine core of 1. The compounds were synthesized via successive Suzuki couplings on a suitably functionalized pyridine starting monomer 4 to append phenyl and pyridyl substituents off the 3- and 5-positions, respectively, and then subsequent modifications were made on the flanking pyridyl ring to provide target compounds. Compound 3a is a novel antagonist, which is highly selective for α3β4 nAChR (K i =123nM) over the α4β2 and α7 receptors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  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.

    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. Evaluation of the sensitivity of the novel α4β2* nicotinic acetylcholine receptor PET radioligand 18F-(−)-NCFHEB to increases in synaptic acetylcholine levels in rhesus monkeys

    PubMed Central

    Gallezot, Jean-Dominique; Esterlis, Irina; Bois, Frederic; Zheng, Ming-Qiang; Lin, Shu-fei; Kloczynski, Tracy; Krystal, John H.; Huang, Yiyun; Sabri, Osama; Carson, Richard E.; Cosgrove, Kelly P.

    2014-01-01

    Objective 18F-(−)-NCFHEB (also known as 18F-(−)-Flubatine) is a new radioligand to image α4β2* nicotinic acetylcholine receptors in vivo with positron emission tomography (PET), with faster kinetics than previous radioligands such as 18F-2-F-A85380. The goal of this study was to assess the sensitivity of 18F-(−)-NCFHEB-PET to increases in synaptic acetylcholine concentration induced by acetylcholinesterase inhibitors. Methods Two rhesus monkeys were scanned four times each on a Focus 220 scanner: first at baseline, then during two bolus plus infusions of physostigmine (0.06–0.28 mg/kg), and finally following a bolus injection of donepezil (0.25 mg/kg). The arterial input function and the plasma free fraction fP were measured. 18F-(−)-NCFHEB volume of distribution VT was estimated using the multilinear analysis MA1 and then normalized by plasma free fraction fP. Results 18F-(−)-NCFHEB fP was 0.89±0.04. At baseline, 18F-(−)-NCFHEB VT/fP ranged from 7.9±1.3 mL plasma/cm3 tissue in the cerebellum to 34.3±8.4 mL plasma/cm3 tissue in the thalamus. Physostigmine induced a dose-dependent reduction of 18F-(−)-NCFHEB VT/fP of 34±9% in the putamen, 32±8% in the thalamus, 25±8% in the cortex, and 23±10% in the hippocampus. With donepezil, 18F-(−)-NCFHEB VT/fP was reduced by 24±2%, 14+3% and 14±5%, 10±6% in the same regions. Conclusion 18F-(−)- NCFHEB can be used to detect changes in synaptic acetylcholine concentration and is a promising tracer to study acetylcholine dynamics with shorter scan durations than previous radioligands. PMID:25043426

  15. Calcium pathways such as cAMP modulate clothianidin action through activation of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors.

    PubMed

    Calas-List, Delphine; List, Olivier; Quinchard, Sophie; Thany, Steeve H

    2013-07-01

    Clothianidin is a neonicotinoid insecticide developed in the early 2000s. We have recently demonstrated that it was a full agonist of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors expressed in the cockroach dorsal unpaired median neurons. Clothianidin was able to act as an agonist of imidacloprid-insensitive nAChR2 receptor and internal regulation of cAMP concentration modulated nAChR2 sensitivity to clothianidin. In the present study, we demonstrated that cAMP modulated the agonist action of clothianidin via α-bungarotoxin-sensitive and insensitive receptors. Clothianidin-induced current-voltage curves were dependent to clothianidin concentrations. At 10 μM clothianidin, increasing cAMP concentration induced a linear current-voltage curve. Clothianidin effects were blocked by 0.5 μM α-bungarotoxin suggesting that cAMP modulation occurred through α-bungarotoxin-sensitive receptors. At 1 mM clothianidin, cAMP effects were associated to α-bungarotoxin-insensitive receptors because clothianidin-induced currents were blocked by 5 μM mecamylamine and 20 μM d-tubocurarine. In addition, we found that application of 1mM clothianidin induced a strong increase of intracellular calcium concentration. These data reinforced the finding that calcium pathways including cAMP modulated clothianidin action on insect nicotinic acetylcholine receptors. We proposed that intracellular calcium pathways such as cAMP could be a target to modulate the mode of action of neonicotinoid insecticides. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. 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

  17. Heme-Oxygenase I and PCG-1α Regulate Mitochondrial Biogenesis via Microglial Activation of Alpha7 Nicotinic Acetylcholine Receptors Using PNU282987.

    PubMed

    Navarro, Elisa; Gonzalez-Lafuente, Laura; Pérez-Liébana, Irene; Buendia, Izaskun; López-Bernardo, Elia; Sánchez-Ramos, Cristina; Prieto, Ignacio; Cuadrado, Antonio; Satrustegui, Jorgina; Cadenas, Susana; Monsalve, Maria; López, Manuela G

    2017-07-10

    A loss in brain acetylcholine and cholinergic markers, subchronic inflammation, and impaired mitochondrial function, which lead to low-energy production and high oxidative stress, are common pathological factors in several neurodegenerative diseases (NDDs). Glial cells are important for brain homeostasis, and microglia controls the central immune response, where α7 acetylcholine nicotinic receptors (nAChR) seem to play a pivotal role; however, little is known about the effects of this receptor in metabolism. Therefore, the aim of this study was to evaluate if glial mitochondrial energetics could be regulated through α7 nAChR. Primary glial cultures treated with the α7 nicotinic agonist PNU282987 increased their mitochondrial mass and their mitochondrial oxygen consumption without increasing oxidative stress; these changes were abolished when nuclear erythroid 2-related factor 2 (Nrf2) was absent, heme oxygenase-1 (HO-1) was inhibited, or peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) was silenced. More specifically, microglia of animals treated intraperitoneally with the α7 nAChR agonist PNU282987 (10 mg/kg) showed a significant increase in mitochondrial mass. Interestingly, LysM cre -Hmox1 Δ/Δ and PGC-1α -/- animals showed lower microglial mitochondrial levels and treatment with PNU282987 did not produce effects on mitochondrial levels. Increases in microglial mitochondrial mass and metabolism can be achieved via α7 nAChR by a mechanism that implicates Nrf2, HO-1, and PGC-1α. This signaling pathway could open a new strategy for the treatment of NDDs, such as Alzheimer's, characterized by a reduction of cholinergic markers. α7 nAChR signaling increases glial mitochondrial mass, both in vitro and in vivo, via HO-1 and PCG-1α. These effects could be of potential benefit in the context of NDDs. Antioxid. Redox Signal. 27, 93-105.

  18. 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. © 2015 International Society for Neurochemistry.

  19. Desformylflustrabromine Modulates α4β2 Neuronal Nicotinic Acetylcholine Receptor High- and Low-Sensitivity Isoforms at Allosteric Clefts Containing the β2 Subunit

    PubMed Central

    Weltzin, Maegan M.

    2015-01-01

    Alterations in expression patterns of α4β2 nicotinic acetylcholine receptors have been demonstrated to alter cholinergic neurotransmission and are implicated in neurologic disorders, including autism, nicotine addiction, Alzheimer’s disease, and Parkinson’s disease. Positive allosteric modulators (PAMs) represent promising new leads in the development of therapeutic agents for the treatment of these disorders. This study investigates the involvement of the β2-containing subunit interfaces of α4β2 receptors in the modulation of acetylcholine (ACh)-induced responses by the PAM desformylflustrabromine (dFBr). Eight amino acids on the principal face of the β2 subunit were mutated to alanine to explore the involvement of this region in the potentiation of ACh-induced currents by dFBr. ACh-induced responses obtained from wild-type and mutant α4β2 receptors expressed in Xenopus laevis oocytes were recorded in the presence and absence of dFBr using two-electrode voltage clamp electrophysiology. Wild-type and mutant receptors were expressed in both high and low ACh sensitivity isoforms by using biased injection ratios of 1:5 or 5:1 α4 to β2 complementary RNA. Mutations were made in the B, C, and A loops of the principal face of the β2 subunit, which are regions not involved in the binding of ACh. Mutant β2(Y120A) significantly eliminated dFBr potency in both isoform preparations. Several other mutations altered dFBr potentiation levels in both preparations. Our findings support the involvement of the principal face of the β2 subunit in dFBr modulation of ACh-induced responses. Findings from this study will aid in the improved design of dFBr-like PAMs for potential therapeutic use. PMID:26025967

  20. The association between the nicotinic acetylcholine receptor α4 subunit gene (CHRNA4) rs1044396 and Internet gaming disorder in Korean male adults

    PubMed Central

    Rhee, Je-Keun; Kim, Tae-Min; Kwak, Su-Min; Bang, Sol-hee; Cho, Hyun; Cheon, Young-Hoon; Min, Jung Ah; Yoo, Gil Sang; Kim, Kyudong; Choi, Jung-Seok; Choi, Sam-Wook; Kim, Dai-Jin

    2017-01-01

    The primary aim of this study was to investigate the genetic predisposition of Internet gaming disorder (IGD), and the secondary aim was to compare the results to those of alcohol dependence (AD). Two independent case-control studies were conducted. A total of 30 male participants with IGD, diagnosed according to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria, and 30 sex-matched controls participated in study 1. We designed targeted exome sequencing (TES) to test for 72 candidate genes that have been implicated in the pathogenesis of addiction. The genes included seven neurotransmitter (dopamine, serotonin, glutamate, r-aminobutyric acid (GABA), norepinephrine, acetylcholine, and opioid) system genes. A total of 31 male in-patients with AD and 29 normal male controls (NC) were enrolled in study 2. The same 72 genes included in study 1 and ten additional genes related to alcohol-metabolic enzyme were selected as the target genes, and we identified the genetic variants using the same method (TES). The IGD group had a lower frequency of the T allele of rs1044396 in the nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4), and this variant represents a protective allele against IGD. However, we did not find a significant difference in the polymorphisms of the 72 genes that encode neurotransmitter systems between the AD and NC groups. This study demonstrated that rs1044396 of CHRNA4 was significantly associated with IGD. PMID:29240768

  1. The association between the nicotinic acetylcholine receptor α4 subunit gene (CHRNA4) rs1044396 and Internet gaming disorder in Korean male adults.

    PubMed

    Jeong, Jo-Eun; Rhee, Je-Keun; Kim, Tae-Min; Kwak, Su-Min; Bang, Sol-Hee; Cho, Hyun; Cheon, Young-Hoon; Min, Jung Ah; Yoo, Gil Sang; Kim, Kyudong; Choi, Jung-Seok; Choi, Sam-Wook; Kim, Dai-Jin

    2017-01-01

    The primary aim of this study was to investigate the genetic predisposition of Internet gaming disorder (IGD), and the secondary aim was to compare the results to those of alcohol dependence (AD). Two independent case-control studies were conducted. A total of 30 male participants with IGD, diagnosed according to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria, and 30 sex-matched controls participated in study 1. We designed targeted exome sequencing (TES) to test for 72 candidate genes that have been implicated in the pathogenesis of addiction. The genes included seven neurotransmitter (dopamine, serotonin, glutamate, r-aminobutyric acid (GABA), norepinephrine, acetylcholine, and opioid) system genes. A total of 31 male in-patients with AD and 29 normal male controls (NC) were enrolled in study 2. The same 72 genes included in study 1 and ten additional genes related to alcohol-metabolic enzyme were selected as the target genes, and we identified the genetic variants using the same method (TES). The IGD group had a lower frequency of the T allele of rs1044396 in the nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4), and this variant represents a protective allele against IGD. However, we did not find a significant difference in the polymorphisms of the 72 genes that encode neurotransmitter systems between the AD and NC groups. This study demonstrated that rs1044396 of CHRNA4 was significantly associated with IGD.

  2. Expression of Nicotinic Acetylcholine Receptor α4 and β2 Subunits on Direction-Selective Retinal Ganglion Cells in the Rabbit

    PubMed Central

    Lee, Jun-Seok; Kim, Hyun-Jin; Ahn, Chang-Hyun; Jeon, Chang-Jin

    2017-01-01

    The direction selectivity of the retina is a distinct mechanism that is critical function of eyes for survival. The direction-selective retinal ganglion cells (DS RGCs) strongly respond to a preferred direction, but rarely respond to opposite direction or null directional visual stimuli. The DS RGCs are sensitive to acetylcholine, which is secreted from starburst amacrine cells (SACs) to the DS RGCs. Here, we investigated the existence and distribution of the nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits on the dendritic arbors of the DS RGCs in adult rabbit retina using immunocytochemistry. The DS RGCs were injected with Lucifer yellow to identify their dendritic morphology. The double-labeled images of dendrites and nAChR subunits were visualized for reconstruction using high-resolution confocal microscopy. Although our results revealed that the distributional pattern of the nAChR subunits on the dendritic arbors of the DS RGCs was not asymmetric in the adult rabbit retina, the distribution of nAChR α4 and β2 subunits and molecular profiles of cholinergic inputs to DS RGCs in adult rabbit retina provide anatomical evidence for direction selectivity. PMID:28386148

  3. Tryptophan 86 of the alpha subunit in the Torpedo nicotinic acetylcholine receptor is important for channel activation by the bisquaternary ligand suberyldicholine.

    PubMed

    Kapur, Ankur; Davies, Martin; Dryden, William F; Dunn, Susan M J

    2006-08-29

    Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.

  4. Structural and functional studies of the modulator NS9283 reveal agonist-like mechanism of action at α4β2 nicotinic acetylcholine receptors.

    PubMed

    Olsen, Jeppe A; Ahring, Philip K; Kastrup, Jette S; Gajhede, Michael; Balle, Thomas

    2014-09-05

    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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Cloning and characterization of alpha9 subunits of the nicotinic acetylcholine receptor expressed by saccular hair cells of the rainbow trout (Oncorhynchus mykiss).

    PubMed

    Drescher, D G; Ramakrishnan, N A; Drescher, M J; Chun, W; Wang, X; Myers, S F; Green, G E; Sadrazodi, K; Karadaghy, A A; Poopat, N; Karpenko, A N; Khan, K M; Hatfield, J S

    2004-01-01

    alpha9/alpha10 Subunits are thought to constitute the nicotinic acetylcholine receptors mediating cholinergic efferent modulation of vertebrate hair cells. The present report describes the cloning and sequence analysis of subunits of the alpha9-containing receptor of a hair-cell layer from the saccule of the rainbow trout (Oncorhynchus mykiss). A major alpha9 subunit, termed alpha9-I, displayed typical features of a nicotinic alpha subunit, with total coding sequence of 572 amino acids including a 16 amino-acid signal peptide. It possessed an extended cytoplasmic loop between membrane-spanning regions M3 and M4, compared with mammalian homologs. Transcript for alpha9-I was robustly expressed in the saccular hair cell layer and less prominently in trout olfactory mucosa, spleen, pituitary gland, and liver, as determined by reverse transcription-polymerase chain reaction. alpha9-I cDNA was not detected in trout brain, skeletal muscle, retina, and kidney. The alpha9-I nicotinic receptor protein was immunolocalized, with an affinity-purified antibody directed against a trout alpha9-I epitope, to hair-cell and neural sites in the saccular hair-cell layer. Foci were found at basal and basolateral membrane sites on hair cells as well as on afferent nerve. Receptor clustering was observed in hair cells bordering non-sensory epithelium. Since in higher vertebrates the alpha9 is reported to associate with another nicotinic subunit, alpha10, we examined the possibility of expression of additional nicotinic subunits in trout saccular hair cells. Message for another nicotinic subunit, termed alpha9-II, was found to be expressed in the hair cells, although more difficult to amplify than alpha9-I. In contrast to alpha9-I, alpha9-II was expressed in brain, as well as in olfactory mucosa, less prominently in pituitary gland and liver, but not in spleen, skeletal muscle, retina, or kidney. The cloned alpha9-II had a total coding sequence of 550 amino acids, which included a 17-amino

  6. Nicotinic potentiation of frog retinotectal transmission in tectum layer F by α3β2, α4β2, α2β4, α6β2, or α7 acetylcholine receptor subtypes.

    PubMed

    Baginskas, Armuntas; Kuraitė, Vilija; Kuras, Antanas

    2015-01-01

    The aim of the study was to explore the effect of semi-specific antagonists and agonists of the nicotinic acetylcholine receptors on the paired-pulse facilitation and nicotinic tonic and phasic potentiation of the frog retinotectal synaptic transmission. The experiments were performed in vivo on adult frogs, Rana temporaria. An individual retina ganglion cell (or its retinotectal fiber) was stimulated by current pulses delivered through multichannel stimulating electrode positioned on the retina. Responses to a discharge of a single retinal ganglion cell were recorded in the tectum by an extracellular carbon-fiber microelectrode positioned in the terminal arborization of the retinotectal fiber in the tectum layer F. The effect of the antagonists and agonists of the nicotinic acetylcholine receptors on the tectal responses has been tested. We found that the antagonists, MLA and DHβE, and agonists, RJR-2403 and choline, of the nicotinic acetylcholine receptors of the α3β2, α4β2, α2β4, α6β2 or α7 subtypes have had no effect on the phasic and tonic potentiation of the retinotectal transmission. The paired-pulse facilitation of the retinotectal transmission was not appreciably affected by the antagonists, but the choline, agonist of the α7 subtype receptor, has significantly decreased the paired-pulse facilitation. The tonic and phasic potentiation of the retinotectal transmission in the tectum layer F were not mediated by the receptors of α3β2, α4β2, α2β4, α6β2 or α7 subtype. The results suggest that presynaptic nicotinic acetylcholine receptors of the frog optic fibers are different from those of the mammalian optic fibers. Copyright © 2015 Lithuanian University of Health Sciences. Production and hosting by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  7. Synthesis and biological evaluation of novel hybrids of highly potent and selective α4β2-Nicotinic acetylcholine receptor (nAChR) partial agonists.

    PubMed

    Zhang, Han-Kun; Eaton, J Brek; Fedolak, Allison; Gunosewoyo, Hendra; Onajole, Oluseye K; Brunner, Dani; Lukas, Ronald J; Yu, Li-Fang; Kozikowski, Alan P

    2016-11-29

    We previously reported the cyclopropylpyridine and isoxazolylpyridine ether scaffolds to be versatile building blocks for creating potent α4β2 nicotinic acetylcholine receptor (nAChR) partial agonists with excellent selectivity over the α3β4 subtype. In our continued efforts to develop therapeutic nicotinic ligands, seven novel hybrid compounds were rationally designed, synthesized, and evaluated in [ 3 H]epibatidine binding competition studies. Incorporation of a cyclopropane- or isoxazole-containing side chain onto the 5-position of 1-(pyridin-3-yl)-1,4-diazepane or 2-(pyridin-3-yl)-2,5-diazabicyclo[2.2.1]heptane led to highly potent and selective α4β2* nAChR partial agonists with K i values of 0.5-51.4 nM for α4β2 and negligible affinities for α3β4 and α7. Moreover, compounds 21, 25, and 30 maintained the functional profiles (EC 50 and IC 50 values of 15-50 nM) of the parent azetidine-containing compounds 3 and 4 in the 86 Rb + ion flux assays. In vivo efficacy of the most promising compound 21 was confirmed in the mouse SmartCube ® platform and classical forced swim tests, supporting the potential use of α4β2 partial agonists for treatment of depression. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  8. Characterization of a novel α-conotoxin from conus textile that selectively targets α6/α3β2β3 nicotinic acetylcholine receptors.

    PubMed

    Luo, Sulan; Zhangsun, Dongting; Wu, Yong; Zhu, Xiaopeng; Hu, Yuanyan; McIntyre, Melissa; Christensen, Sean; Akcan, Muharrem; Craik, David J; McIntosh, J Michael

    2013-01-11

    α6β2 Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons in the CNS are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine addiction and Parkinson disease. However, recent studies indicate that the α6 subunit can also associate with the β4 subunit to form α6β4 nAChRs that are difficult to pharmacologically distinguish from α6β2, α3β4, and α3β2 subtypes. The current study characterized a novel 16-amino acid α-conotoxin (α-CTx) TxIB from Conus textile whose sequence is GCCSDPPCRNKHPDLC-amide as deduced from gene cloning. The peptide and an analog with an additional C-terminal glycine were chemically synthesized and tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes. α-CTx TxIB blocked α6/α3β2β3 nAChR with an IC(50) of 28 nm. In contrast, the peptide showed little or no block of other tested subtypes at concentrations up to 10 μm. The three-dimensional solution structure of α-CTx TxIB was determined using NMR spectroscopy. α-CTx TxIB represents a uniquely selective ligand for probing the structure and function of α6β2 nAChRs.

  9. Characterization of a Novel α-Conotoxin from Conus textile That Selectively Targets α6/α3β2β3 Nicotinic Acetylcholine Receptors*

    PubMed Central

    Luo, Sulan; Zhangsun, Dongting; Wu, Yong; Zhu, Xiaopeng; Hu, Yuanyan; McIntyre, Melissa; Christensen, Sean; Akcan, Muharrem; Craik, David J.; McIntosh, J. Michael

    2013-01-01

    α6β2 Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons in the CNS are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine addiction and Parkinson disease. However, recent studies indicate that the α6 subunit can also associate with the β4 subunit to form α6β4 nAChRs that are difficult to pharmacologically distinguish from α6β2, α3β4, and α3β2 subtypes. The current study characterized a novel 16-amino acid α-conotoxin (α-CTx) TxIB from Conus textile whose sequence is GCCSDPPCRNKHPDLC-amide as deduced from gene cloning. The peptide and an analog with an additional C-terminal glycine were chemically synthesized and tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes. α-CTx TxIB blocked α6/α3β2β3 nAChR with an IC50 of 28 nm. In contrast, the peptide showed little or no block of other tested subtypes at concentrations up to 10 μm. The three-dimensional solution structure of α-CTx TxIB was determined using NMR spectroscopy. α-CTx TxIB represents a uniquely selective ligand for probing the structure and function of α6β2 nAChRs. PMID:23184959

  10. Affinities of bispyridinium non-oxime compounds to [(3)H]epibatidine binding sites of Torpedo californica nicotinic acetylcholine receptors depend on linker length.

    PubMed

    Niessen, K V; Seeger, T; Tattersall, J E H; Timperley, C M; Bird, M; Green, C; Thiermann, H; Worek, F

    2013-12-05

    The toxicity of organophosphorus nerve agents or pesticides arises from accumulation of acetylcholine and overstimulation of both muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) due to inhibition of acetylcholinesterase (AChE). Standard treatment by administration of atropine and oximes, e.g., obidoxime or pralidoxime, focuses on antagonism of mAChRs and reactivation of AChE, whereas nicotinic malfunction is not directly treated. An alternative approach would be to use nAChR active substances to counteract the effects of accumulated acetylcholine. Promising in vitro and in vivo results were obtained with the bispyridinium compounds SAD-128 (1,1'-oxydimethylene bis(4-tert-butylpyridinium) dichloride) and MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)), which were partly attributed to their interaction with nAChRs. In this study, a homologous series of unsubstituted and 4-tert-butyl-substituted bispyridinium compounds with different alkane linker lengths was investigated in competition binding experiments using [(3)H]epibatidine as a reporter ligand. Additionally, the effect of the well-characterised MB327 on the [(3)H]epibatidine equilibrium dissociation (KD) constant in different buffers was determined. This study demonstrated that divalent cations increased the affinity of [(3)H]epibatidine. Since quaternary ammonium molecules are known to inhibit AChE, the obtained affinity constants of the tested bispyridinium compounds were compared with the inhibition of human AChE. In competition experiments, bispyridinium derivatives of longer linker length displaced [(3)H]epibatidine and inhibited AChE strongly. Bispyridinium compounds with short linkers, at most, have an allosteric interaction with the [(3)H]epibatidine binding sites and barely inhibited AChE. In dependence on alkane linker length, the bispyridinium compounds seemed to interact at different binding sites. However, the exact binding sites of the bispyridinium

  11. Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor.

    PubMed

    Halai, Reena; Clark, Richard J; Nevin, Simon T; Jensen, Jonas E; Adams, David J; Craik, David J

    2009-07-24

    Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the alpha9alpha10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp(5)-Arg(7) and Asp(11)-Ile(15), because changes at these positions resulted in the loss of activity at the alpha9alpha10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat alpha9alpha10 and the human alpha9/rat alpha10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the alpha3beta2, alpha3beta4, and alpha7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the alpha9alpha10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the alpha9alpha10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.

  12. Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms

    PubMed Central

    Abongwa, Melanie; Buxton, Samuel K.; Robertson, Alan P.; Martin, Richard J.

    2016-01-01

    Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 μM abamectin in a non-competitive manner (reduced Rmax, no change in EC50). This antagonism increased when abamectin was increased to 0.1 μM. However, when we increased the concentration of abamectin further to 0.3 μM, 1 μM or 10 μM, we found that the antagonism decreased and was less than with 0.1 μM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 μM derquantel alone and in combination with 0.3 μM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex

  13. Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms.

    PubMed

    Abongwa, Melanie; Buxton, Samuel K; Robertson, Alan P; Martin, Richard J

    2016-01-01

    Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 μM abamectin in a non-competitive manner (reduced Rmax, no change in EC50). This antagonism increased when abamectin was increased to 0.1 μM. However, when we increased the concentration of abamectin further to 0.3 μM, 1 μM or 10 μM, we found that the antagonism decreased and was less than with 0.1 μM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 μM derquantel alone and in combination with 0.3 μM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex

  14. Nicotinic acetylcholine receptors containing the α7-like subunit mediate contractions of muscles responsible for space positioning of the snail, Helix pomatia L. tentacle.

    PubMed

    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.

  15. 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

  16. Cross-reactivity of acid-sensing ion channel and Na⁺-H⁺ exchanger antagonists with nicotinic acetylcholine receptors.

    PubMed

    Santos-Torres, Julio; Ślimak, Marta A; Auer, Sebastian; Ibañez-Tallon, Inés

    2011-11-01

    Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the mammalian central and peripheral nervous systems, where they contribute to neuronal excitability and synaptic communication. It has been reported that nAChRs are modulated by BK channels and that BK channels, in turn, are inhibited by acid-sensing ion channels (ASICs). Here we investigate the possible functional interaction between these channels in medial habenula (MHb) neurones. We report that selective antagonists of large-conductance calcium-activated potassium channels and ASIC1a channels, paxilline and psalmotoxin 1, respectively, did not induce detectable changes in nicotine-evoked currents. In contrast, the non-selective ASIC and Na(+)-H(+) exchanger (NHE1) antagonists, amiloride and its analogues, suppressed nicotine-evoked responses in MHb neurones of wild-type and ASIC2 null mice, excluding a possible involvement of ASIC2 in the nAChR inhibition by amiloride. Zoniporide, a more selective inhibitor of NHE1, reversibly inhibited α3β4-, α7- and α4-containing (*) nAChRs in Xenopus oocytes and in brain slices, as well as in PS120 cells deficient in NHE1 and virally transduced with nAChRs, suggesting a generalized effect of zoniporide in most neuronal nAChR subtypes. Independently from nAChR antagonism, zoniporide profoundly blocked synaptic transmission onto MHb neurones without affecting glutamatergic and GABA receptors. Taken together, these results indicate that amiloride and zoniporide, which are clinically used to treat hypertension and cardiovascular disease, have an inhibitory effect on neuronal nAChRs when used experimentally at high doses. The possible cross-reactivity of these compounds with nAChRs in vivo will require further investigation.

  17. Use of Monoclonal Antibodies to Study the Structure and Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons

    DTIC Science & Technology

    1988-03-16

    receptors in muscle is responsible for the muscular weakness characteristic of myasthenia gravis . Some insecticides can act like chemical warfare...expresses muscle-like acetyi-.holine receptor by observing that autoantibodies from myasthenia gravis patients reacted as well with these receptors as...Antibodies in sera from patients with myasthenia gravis do not bind to acetylcholine receptors from human brain. J Neuroimmunol 16:205-213. 21. Whiting

  18. Detection of human neuronal α7 nicotinic acetylcholine receptors by conjugates of snake α-neurotoxin with quantum dots.

    PubMed

    Makarova, Ya V; Shelukhina, I V; Mukherjee, A K; Kuznetsov, D V; Tsetlin, V I; Utkin, Yu N

    2017-07-01

    Fluorescent derivatives are widely used to study the structure and functions of proteins. Quantum dots (QDs), fluorescent semiconductor nanocrystals, have a high quantum yield and are much more resistant to bleaching compared to organic dyes. Conjugates of α-neurotoxins with QDs were used for visualization of human α7 acetylcholine receptors heterologously expressed in GH4C1 pituitary adenoma cells. Specific staining of cells by the conjugated toxins was observed.

  19. Synthesis, in vitro and in vivo studies, and molecular modeling of N-alkylated dextromethorphan derivatives as non-competitive inhibitors of α3β4 nicotinic acetylcholine receptor.

    PubMed

    Jozwiak, Krzysztof; Targowska-Duda, Katarzyna M; Kaczor, Agnieszka A; Kozak, Joanna; Ligeza, Agnieszka; Szacon, Elzbieta; Wrobel, Tomasz M; Budzynska, Barbara; Biala, Grazyna; Fornal, Emilia; Poso, Antti; Wainer, Irving W; Matosiuk, Dariusz

    2014-12-15

    9 N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive inhibitors of α3β4 nicotinic acetylcholine receptors (nAChRs). In vitro activity towards α3β4 nicotinic acetylcholine receptor is determined using a patch-clamp technique and is in the micromolar range. Homology modeling, molecular docking and molecular dynamics of ligand-receptor complexes in POPC membrane are used to find the mode of interactions of N-alkylated dextromethorphan derivatives with α3β4 nAChR. The compounds, similarly as dextromethorphan, interact with the middle portion of α3β4 nAChR ion channel. Finally, behavioral tests confirmed potential application of the studied compounds for the treatment of addiction. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Use of Monoclonal Antibodies to Study the Structural Basis of the Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons

    DTIC Science & Technology

    1989-09-30

    using a modification of the dideoxy - nucleotide chain termination method of Sanger et al. 1 0 9 Cloning and Secuencina of TE671 Receptor A Subunit cDNA. A...Neurosci. 5, 2545-2552. 109. Sanger F., S. Nicklen, and A.R. Coulson (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA...Q rinic acetylcholine receptors of neurons; protein sequencing ; protein __I_ C4 _+Ipurification; RAil; RA.,V,. monoclonal antibodies- 19A&SrRACT

  1. Concentration-response relationship of the α7 nicotinic acetylcholine receptor agonist FRM-17874 across multiple in vitro and in vivo assays.

    PubMed

    Stoiljkovic, Milan; Leventhal, Liza; Chen, Angela; Chen, Ting; Driscoll, Rachelle; Flood, Dorothy; Hodgdon, Hilliary; Hurst, Raymond; Nagy, David; Piser, Timothy; Tang, Cuyue; Townsend, Matthew; Tu, Zhiming; Bertrand, Daniel; Koenig, Gerhard; Hajós, Mihaly

    2015-10-15

    Pharmacological activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) may improve cognition in schizophrenia and Alzheimer's disease. The present studies describe an integrated pharmacological analysis of the effects of FRM-17874, an analogue of encenicline, on α7 nAChRs in vitro and in behavioral and neurophysiological assays relevant to cognitive function. FRM-17874 demonstrated high affinity binding to human α7 nAChRs, displacing [(3)H]-methyllacaconitine (Ki=4.3nM). In Xenopus laevis oocytes expressing human α7 nAChRs, FRM-17874 acted as an agonist, evoking inward currents with an EC50 of 0.42μM. Lower concentrations of FRM-17874 (0.01-3nM) elicited no detectable current, but primed receptors to respond to sub-maximal concentrations of acetylcholine. FRM-17874 improved novel object recognition in rats, and enhanced memory acquisition and reversal learning in the mouse water T-maze. Neurophysiological correlates of cognitive effects of drug treatment, such as synaptic transmission, long-term potentiation, and hippocampal theta oscillation were also evaluated. Modulation of synaptic transmission and plasticity was observed in rat hippocampal slices at concentrations of 3.2 and 5nM. FRM-17874 showed a dose-dependent facilitation of stimulation-induced hippocampal theta oscillation in mice and rats. The FRM-17874 unbound brain concentration-response relationship for increased theta oscillation power was similar in both species, exhibited a biphasic pattern peaking around 3nM, and overlapped with active doses and exposures observed in cognition assays. In summary, behavioral and neurophysiological assays indicate a bell-shaped effective concentration range and this report represents the first attempt to explain the concentration-response function of α7 nAChR-mediated pro-cognitive effects in terms of receptor pharmacology. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. The chaperone protein 14-3-3eta interacts with the nicotinic acetylcholine receptor alpha 4 subunit. Evidence for a dynamic role in subunit stabilization.

    PubMed

    Jeanclos, E M; Lin, L; Treuil, M W; Rao, J; DeCoster, M A; Anand, R

    2001-07-27

    By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) alpha4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3eta, known to interact directly with neuronal AChRs. 14-3-3eta is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3eta interacted with the recombinant alpha4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3eta with recombinant alpha4 subunits was abolished when serine 441 of the alpha4 subunit was mutated to alanine (alpha4(S441A)). The surface levels of recombinant wild-type alpha4beta2 AChRs were approximately 2-fold higher than those of mutant alpha4(S441A)beta2 AChRs. The interaction significantly increased the steady state levels of the alpha4 subunit and alpha4beta2 AChRs but not that of the mutant alpha4(S441A) subunit or mutant alpha4(S441A)beta2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for alpha4beta2 AChRs and alpha4(S441A)beta2 AChRs coexpressed with 14-3-3eta in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native alpha4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of alpha4beta2 AChRs through its interaction with the alpha4 subunit.

  3. [(3)H]Epibatidine photolabels non-equivalent amino acids in the agonist binding site of Torpedo and alpha4beta2 nicotinic acetylcholine receptors.

    PubMed

    Srivastava, Shouryadeep; Hamouda, Ayman K; Pandhare, Akash; Duddempudi, Phaneendra K; Sanghvi, Mitesh; Cohen, Jonathan B; Blanton, Michael P

    2009-09-11

    Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.

  4. [3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors*

    PubMed Central

    Srivastava, Shouryadeep; Hamouda, Ayman K.; Pandhare, Akash; Duddempudi, Phaneendra K.; Sanghvi, Mitesh; Cohen, Jonathan B.; Blanton, Michael P.

    2009-01-01

    Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with [3H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu109 and γTyr117 in Segment E of the complementary (−) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [3H]epibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR. PMID:19620239

  5. Acetylcholine α7 nicotinic and dopamine D2 receptors are targeted to many of the same postsynaptic dendrites and astrocytes in the rodent prefrontal cortex.

    PubMed

    Duffy, Aine M; Fitzgerald, Megan L; Chan, June; Robinson, Danielle C; Milner, Teresa A; Mackie, Kenneth; Pickel, Virginia M

    2011-12-01

    The alpha-7 nicotinic acetylcholine receptor (α7nAChR) and the dopamine D(2) receptor (D(2) R) are both implicated in attentional processes and cognition, mediated in part through the prefrontal cortex (PFC). We examined the dual electron microscopic immunolabeling of α7nAChR and either D(2) R or the vesicular acetylcholine transporter (VAChT) in rodent PFC to assess convergent functional activation sites. Immunoreactivity (ir) for α7nAChR and/or D(2) R was seen in the same as well as separate neuronal and glial profiles. At least half of the dually labeled profiles were somata and dendrites, while most labeled axon terminals expressed only D(2) R-ir. The D(2) R-labeled terminals were without synaptic specializations or formed inhibitory or excitatory-type synapses with somatodendritic profiles, some of which expressed the α7nAChR and/or D(2) R. Astrocytic glial processes comprised the majority of nonsomatodendritic α7nAChR or α7nAChR and D(2) R-labeled profiles. Glial processes containing α7nAChR-ir were frequently located near VAChT-labeled terminals and also showed perisynaptic and perivascular associations. We conclude that in rodent PFC α7nACh and D(2) R activation can dually modulate (1) postsynaptic dendritic responses within the same or separate but synaptically linked neurons in which the D(2) R has the predominately presynaptic distribution, and (2) astrocytic signaling that may be crucial for synaptic transmission and functional hyperemia. Copyright © 2011 Wiley-Liss, Inc.