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Biochemical Pharmacology (v.74, #8)

Editorial Advisory Board (pp. iii).

Nicotinic acetylcholine receptors as therapeutic targets: emerging frontiers in basic research and clinical science – Editorial comments by Murali Gopalakrishnan; Daniel Bertrand; Michael Williams (pp. 1091-1091).

Neuronal nicotinic receptors: A perspective on two decades of drug discovery research by Stephen P. Arneric; Mark Holladay; Michael Williams (pp. 1092-1101).

Neuronal nicotinic acetylcholine receptors (nAChRs) have been a target for drug discovery efforts, primarily for CNS indications, for the past two decades. While nicotine and related natural products have been used for smoking cessation in various formulations (e.g., gum, spray, patches), it was only in 2006 with the launch of varenicline (Chantix™) by Pfizer for smoking cessation that a new chemical entity (NCE) originating from a rational medicinal chemistry effort targeting neuronal AChRs was approved. The current overview outlines the chronology of drug discovery efforts in nAChRs from the cloning of the receptor family in the 1980s, to initial research efforts at SIBIA, R.J. Reynolds and Abbott, to the current industry-wide interest in nAChR agonists as novel therapeutics for pain, schizophrenia and Alzheimer's Disease. Key events in the evolution of the nAChR field were the development of high throughput electrophysiological screening tools that provided the means to enable lead optimization efforts in medicinal chemistry and the discovery by John Daly at the NIH of the frog alkaloid, epibatidine, that provided the framework for the discovery of ABT-594, an α4β2 agonist that is 200 times more potent than morphine as an analgesic. Over the next decade, it is anticipated that additional NCEs including antagonists and allosteric modulators (both positive and negative), interacting with various nAChR subtypes, will be advanced to the clinic in areas of high unmet medical need, e.g., pain, neurodegeneration, to provide novel medications with improved efficacy.

Keywords: nAChR; Drug discovery; Nicotine

Heterogeneity and complexity of native brain nicotinic receptors by Cecilia Gotti; Milena Moretti; Annalisa Gaimarri; Alessio Zanardi; Franceso Clementi; Michele Zoli (pp. 1102-1111).

Neuronal cholinergic nicotinic receptors (nAChRs) are a heterogeneous class of cationic channels that are widely distributed in the nervous system that have specific functional and pharmacological properties. They consist of homologous subunits encoded by a large multigene family, and their opening is physiologically controlled by the acetylcholine neurotransmitter or exogenous ligands such as nicotine. Their biophysical and pharmacological properties depend on their subunit composition, which is therefore central to understanding receptor function in the nervous system and discovering new subtype-selective drugs.We will review rodent brain subtypes by discussing their subunit composition, pharmacology and localisation and, when possible, comparing them with the same subtypes present in the brain of other mammalian species or chick.In particular, we will focus on the nAChRs present in the visual pathway (retina, superior colliculus and nucleus geniculatus lateralis), in which neurons express most, if not all, nAChR subunits. In addition to the major α4β2 and α7 nAChR subtypes, the visual pathway selectively expresses subtypes with a complex subunit composition. By means of ligand binding and immunoprecipitation and immunopurification experiments on tissues obtained from control and lesioned rats, and wild-type and nAChR subunit knockout mice, we have qualitatively and quantitatively identified, and pharmacologically characterised, the multiple complex native subtypes containing up to four different subunits.

Keywords: Neuronal nicotinic acetylcholine receptors; Subtypes; Pharmacology; Stochiometry

Role of endogenous nicotinic signaling in guiding neuronal development by Zhaoping Liu; Jingming Zhang; Darwin K. Berg (pp. 1112-1119).

Spontaneous nicotinic cholinergic activity is widespread in the developing nervous system. One of the major components mediating this activity is the nicotinic acetylcholine receptor with α7 subunits (α7-nAChR) and high relative calcium permeability. We recently reported that α7-nAChRs co-localize in part with GABA_A receptors during development, and the sites become co-innervated by cholinergic and GABAergic terminals. Patch-clamp recording either from embryonic chick ciliary ganglion neurons or from early postnatal mouse hippocampal interneurons reveals that α7-nAChR activation can impose a rapid and reversible decrease in GABA_A receptor responses. The effect extends to GABAergic synaptic currents, and depends on intracellular calcium, calcium/calmodulin-dependent protein kinase II, and MAP kinase in the postsynaptic cell. Over the longer term, nicotinic activity has a more profound effect: it determines the time during development when GABAergic signaling converts from excitation to inhibition. It does this by changing the pattern of chloride transporters to establish the mature chloride gradient required for inhibitory GABAergic responses. The excitatory phase of GABAergic signaling is critical for proper development and integration of neurons into circuits. By driving the conversion of GABAergic signaling, nicotinic activity not only terminates one set of developmental instructions, but also initiates another by collaborating with GABAergic inhibition to impose new instructions. The results reveal a multi-layered pattern of activity-dependent controls in development and indicate the significance of nicotinic signaling in shaping these events.

Keywords: Abbreviations; αBgt; α-Bungarotoxin; α7-nAChRs; α7-containing nicotinic acetylcholine receptors; α4β2-nAChRs; α4- and β2-containing nicotinic acetylcholine receptors; ACh; acetylcholine; CG; ciliary ganglion; CaMKII; calcium/calmodulin-dependent protein kinase II; DHβE; dihydro-β-erythroidine; GAD; glutamic acid decarboxylase; GFP; green fluorescent protein; IPSCs; inhibitory postsynaptic currents; MLA; methyllycaconitine; SO; stratum oriens; SR; stratum radiatum; VAChT; vesicular ACh transporter; VGCCs; voltage-gated calcium channelsNicotinic; GABAergic; Excitation; Development; Synapse; Activity

Regulation of synaptic transmission and plasticity by neuronal nicotinic acetylcholine receptors by Bruce E. McKay; Andon N. Placzek; John A. Dani (pp. 1120-1133).

Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system and participate in a variety of physiological functions. Recent advances have revealed roles of nAChRs in the regulation of synaptic transmission and synaptic plasticity, particularly in the hippocampus and midbrain dopamine centers. In general, activation of nAChRs causes membrane depolarization and directly and indirectly increases the intracellular calcium concentration. Thus, when nAChRs are expressed on presynaptic membranes their activation generally increases the probability of neurotransmitter release. When expressed on postsynaptic membranes, nAChR-initiated calcium signals and depolarization activate intracellular signaling mechanisms and gene transcription. Together, the presynaptic and postsynaptic effects of nAChRs generate and facilitate the induction of long-term changes in synaptic transmission. The direction of hippocampal nAChR-mediated synaptic plasticity – either potentiation or depression – depends on the timing of nAChR activation relative to coincident presynaptic and postsynaptic electrical activity, and also depends on the location of cholinergic stimulation within the local network. Therapeutic activation of nAChRs may prove efficacious in the treatment of neuropathologies where synaptic transmission is compromised, as in Alzheimer's or Parkinson's disease.

Keywords: Abbreviations; ACh; acetylcholine; AP; action potential; AMPAR; α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor; CaMK; Ca; ²⁺; /calmodulin-dependent protein kinase; CNS; central nervous system; CREB; cAMP response element binding protein; EPSC; excitatory postsynaptic current; EPSP; excitatory postsynaptic potential; GABA; gamma-amino butyric acid; GDP; giant depolarizing potential; HFS; high frequency stimulation; IPSC; inhibitory postsynaptic current; LTD; long-term depression; LTP; long-term potentiation; MAP kinase; mitogen activated protein kinase; mGluR; metabotropic glutamate receptor; nAChR; nicotinic acetylcholine receptor; NMDAR; N; -methyl-; d; -aspartate receptor; PSP; postsynaptic potential; sEPSC; spontaneous EPSC; sIPSC; spontaneous IPSC; STDP; spike timing dependent plasticity; STP; short-term potentiation; VGCC; voltage-gated Ca; ²⁺; channel; VTA; ventral tegmental areaNicotine; Synaptic plasticity; LTP; Development; Hippocampus; Ventral tegmental area

Age-dependent changes in the functional expression of two nicotinic receptor subtypes in CA1 stratum radiatum interneurons in the rat hippocampus by Manickavasagom Alkondon; Edna F.R. Pereira; Edson X. Albuquerque (pp. 1134-1144).

Protein density measurements and mRNA analysis have provided valuable information on age-dependent changes in the distribution of different nicotinic receptor (nAChR) subtypes in various areas of the rat brain, including the hippocampus. However, very little is known regarding the functional expression of nAChRs in individual neuron types at various ages. Likewise, there is paucity of information regarding the functional and pharmacological profile of nAChRs in the mature rat hippocampus. To address these issues, we used the whole-cell patch-clamp technique to record nicotinic responses from CA1 stratum radiatum (SR) interneurons in hippocampal slices from rat pups (5–19 days old) and adult rats (2–5 months old). As previously observed in the hippocampus of rat pups, CA1 SR interneurons in the hippocampus of adult rats responded to choline (10mM, 12s) with whole-cell currents that decayed to the baseline within the agonist pulse, were sensitive to inhibition by methyllycaconitine (10nM) or α-bungarotoxin (50nM), and were, therefore, mediated by α7*¹1According to the nomenclature for nAChRs and their subunits [Lukas RJ, Changeux JP, Le Novere N, Albuquerque, EX, Balfour DJ, Berg, DK et al. International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol Rev 1999; 51: 397–401.], the asterisk next to nAChR subunits throughout text is meant to indicate that the exact subunit composition is not known. nAChRs. Likewise, as previously observed in the hippocampus of young rats, in the adult rat hippocampus excitatory postsynaptic currents (EPSCs) were recorded from SR interneurons in response to a pulse of ACh (0.1mM, 12s) applied in the presence of the GABA_A receptor antagonist bicuculline. ACh-triggered EPSCs were inhibited by mecamylamine (1μM) or choline (1mM) and were, therefore, likely to have resulted from activation of α3β4β2* nAChR. The magnitude of α7* nAChR-mediated responses increased with the age of the animals. In contrast, the magnitude of α3β4β2* nAChR-mediated responses was highest at the second postnatal week. The distinct age dependency of functional expression of α7* and α3β4β2* nAChRs strongly suggests that the excitability of CA1 SR interneurons is differentially regulated by the nicotinic cholinergic system in the hippocampus of rat pups and adult rats.

Keywords: Abbreviations; nAChR; nicotinic acetylcholine receptor; SR; stratum radiatum; SLM; stratum lacunosum moleculare; ACSF; artificial cerebrospinal fluid; QX-314; lidocaine N-ethyl bromide; MLA; methyllycaconitine; α-BGT; α-bungarotoxin; SO; stratum oriens; SP; stratum pyramidaleCholine; Methyllycaconitine; Mecamylamine; Alpha-bungarotoxin; Adult rats; glutamate EPSCs

Cell autonomy, receptor autonomy, and thermodynamics in nicotine receptor up-regulation by Raad Nashmi; Henry Lester (pp. 1145-1154).

Chronic nicotine exposure, in smokers or in experimental rodents administered nicotine, produces elevated levels of nicotinic acetylcholine receptors in several brain regions. However, there are few data on up-regulation of receptors in specific neuronal subtypes. We tested whether functional up-regulation of nicotinic responses occurs in cultured GABAergic neurons of the ventral midbrain. Fura-2 measurements of nicotinic responses were made on ventral midbrain neurons from knock-in mice heterozygous for the α4-M2 domain Leu9′Ala mutation, which confers nicotine hypersensitivity. Chronic nicotine exposure at a concentration (10nM for 3 days) that activates only the hypersensitive α4* (Leu9′Ala) receptors, but not wild-type receptors, resulted in significant potentiation of ACh (100μM)-elicited responses. Experiments were also performed on midbrain neuronal cultures heterozygous for the α4* (Leu9′Ala) mutation as well as for a GFP protein fused to a GABA transporter that reliably reveals GABAergic neurons. In cultures chronically treated with 10nM nicotine, there was significantly increased α4* nicotinic-induced Ca²⁺ influx elicited by low concentration of ACh (3μM). Furthermore, chronic exposure to the competitive antagonist dihydro-β-erythroidine, but not to the noncompetitive antagonist mecamylamine, induced up-regulation of ACh elicited nicotinic responses. These results suggest that occupation of α4* nicotinic receptor binding site(s), at the interface between two subunits, is sufficient to promote assembly and/or up-regulation of functional receptors in GABAergic neurons. Up-regulation in neurons is both “cell-autonomous”, occurring at the cell itself, and “receptor autonomous”, occurring at the receptor itself, and may be a thermodynamic necessity of ligand–protein interactions.

Keywords: Nicotinic receptor; Up-regulation; GABAergic neurons; Ventral midbrain; Fura-2; Neuronal cultures

Allosteric modulation of nicotinic acetylcholine receptors by Daniel Bertrand; Murali Gopalakrishnan (pp. 1155-1163).

Allosteric modulation refers to the concept that proteins could exist in multiple conformational states and that binding of allosteric ligands alters the energy barriers or “isomerization coefficients” between various states. In the context of ligand gated ion channels such as nicotinic acetylcholine receptors (nAChRs), it implies that endogenous ligand acetylcholine binds at the orthosteric site, and that molecules that bind elsewhere on the nAChR subunit(s) acts via allosteric interactions. For example, studies with the homomeric α7 nAChRs indicate that such ligand interactions can be well described by an allosteric model, and that positive allosteric effectors can affect energy transitions by (i) predominantly affecting the peak current response (Type I profile) or, (ii) both peak current responses and time course of agonist-evoked response (Type II profile). The recent discovery of chemically heterogeneous group of molecules capable of differentially modifying nAChR properties without interacting at the ligand binding site illustrates the adequacy of the allosteric model to predict functional consequences. In this review, we outline general principles of the allosteric concept and summarize the profiles of novel compounds that are emerging as allosteric modulators at the α7 and α4β2 nAChR subtypes.

Keywords: Abbreviations; ACh; acetylcholine; nAChRs; neuronal nicotinic acetylcholine receptors; DMPP; dimethylphenylpiperazinium; 5-HI; 5-hydroxyindole; PAM; positive allosteric modulator; NAM; negative allosteric modulator; BSA; bovine serum albumine; LBD; ligand binding domain; CD; channel domain; SLURP-1; secreted mammalian Ly-6/uPAR related protein 1Nicotinic acetylcholine receptor; Allosteric modulation

Structure-guided drug design: Conferring selectivity among neuronal nicotinic receptor and acetylcholine-binding protein subtypes by Palmer Taylor; Todd T. Talley; Zoran Radic’; Scott B. Hansen; Ryan E. Hibbs; Jian Shi (pp. 1164-1171).

Neuronal nicotinic receptors, encoded by nine genes of the α and three of the β type of subunits, and whose gene products assemble in distinct permutations as pentameric molecules, constitute a fertile area for structure-guided drug design. Design strategies are augmented by a wide variety of peptide, alkaloid and terpenoid toxins from various marine and terrestrial species that interact with nicotinic receptors. Also, acetylcholine-binding proteins from mollusks, as structural surrogates of the receptor that mimic its extracellular domain, provide atomic resolution templates for analysis of structure and response. Herein, we describe a structure-guided approach to nicotinic ligand design that employs crystallography of this protein as the basic template, but also takes into consideration the dynamic properties of the receptor molecules in their biological media. We present the crystallographic structures of several complexes of various agonists and antagonists that associate with the agonist site and can competitively block the action of acetylcholine. In so far as the extracellular domain is involved, we identify additional non-competitive sites at those subunit interfaces where agonists do not preferentially bind. Ligand association at these interface sites may modulate receptor function. Ligand binding is also shown by solution-based spectroscopic and spectrometric methods to affect the dynamics of discrete domains of the receptor molecule. The surrogate receptor molecules can then be employed to design ligands selective for receptor subtype through the novel methods of freeze-frame, click chemistry that uses the very structure of the target molecule as a template for synthesis of the inhibitor.

Keywords: Nicotinic receptor structure; Acetylcholine-binding protein; Freeze-frame click chemistry; Crystal structure; Allosteric modulators; Competitive ligand site; Channel vestibule; Fluorescence anisotropy; Isotope exchange

In vitro screening strategies for nicotinic receptor ligands by John Dunlop; Renza Roncarati; Brian Jow; Hendrick Bothmann; Tim Lock; Dianne Kowal; Mark Bowlby; Georg C. Terstappen (pp. 1172-1181).

A common historical strategy to the discovery of nicotinic receptor ligands has involved the use of radioligand-binding assays for ligand identification in combination with two-electrode voltage clamp in Xenopus oocytes for electrophysiological characterization. More recently, higher-throughput methodologies have replaced these approaches to accommodate screening of large compound libraries and to provide increased capacity for electrophysiological profiling in mammalian cell lines. We, and others, have implemented cell-based screening assays using the fluorometric imaging plate reader (FLIPR™) for primary and lead optimization screening of nicotinic receptor agonists and positive allosteric modulators (PAMs). Using GH4C1 cells expressing the rat α7 nicotinic receptor, both acetylcholine and nicotine produced concentration-dependent elevations of intracellular calcium with EC₅₀ values of 5.5 and 1.6μM, respectively. PAM activity was robustly detected using the FLIPR assay; for example, the known α7 receptor PAM 5-hydroxyindole failed to directly activate the receptor but produced a leftward shift of the nicotine concentration–response curve in combination with a potentiation of the maximum evoked response to nicotine. Electrophysiological confirmation of agonist activity was achieved using the Dynaflow rapid perfusion system and patch clamp in the same GH4C1 cell expression system. Estimated EC₅₀ values for acetylcholine-evoked currents in GH4C1/α7 cells were 55 and 576μM for area-under-the-curve (AUC) and maximum peak height calculations, respectively. Similarly, PAM activity was confirmed using electrophysiological recordings while also allowing for the mechanistic discrimination of compounds, not possible using the FLIPR assay. Specifically, PAMs capable of slowing the rapid desensitization of α7 receptors to different extents were discernable in these studies. Further improvements in the capacity to screen compounds using electrophysiology has been achieved by implementation of high-throughput gigaohm quality recording systems such as the QPatch and PatchXpress where agonist EC₅₀ values are highly comparable to those obtained using conventional manual patch clamp.

Keywords: Nicotinic; Electrophysiology; Calcium flux; Membrane potential; Acetylcholine; α7

Nicotinic interactions with antipsychotic drugs, models of schizophrenia and impacts on cognitive function by Edward D. Levin; Amir H. Rezvani (pp. 1182-1191).

People with schizophrenia often have substantial cognitive impairments, which may be related to nicotinic receptor deficits, (α7 and α4β2), documented in the brains of people with schizophrenia. The large majority of people with schizophrenia smoke cigarettes. Thus, nicotinic interactions with antipsychotic drugs are widespread. Complementary co-therapies of novel nicotinic ligands are being developed to add to antipsychotic therapy to treat the cognitive impairment of schizophrenia. Thus, it is critical to understand the interaction between nicotinic treatments and antipsychotic drugs. Nicotinic interactions with antipsychotic drugs, are complex since both nicotine and antipsychotics have complex actions. Nicotine stimulates and desensitizes nicotinic receptors of various subtypes and potentiates the release of different neurotransmitters. Antipsychotics also act on a verity of receptor systems. For example, clozapine acts as an antagonist at a variety of neurotransmitter receptors such as those for dopamine, serotonin, norepinepherine and histamine. In a series of studies, we have found that in normally functioning rats, moderate doses of clozapine impair working memory and that clozapine blocks nicotine-induced memory and attentional improvement. Clozapine and nicotine can attenuate each other's beneficial effects in reversing the memory impairment caused by the psychototmimetic drug dizocilpine. A key to the clozapine-induced attenuation of nicotine-induced cognitive improvement appears to be its 5HT₂ antagonist properties. The selective 5HT₂ antagonist ketanserin has a similar action of blocking nicotine-induced memory and attentional improvements. It is important to consider the interactions between nicotinic and antipsychotic drugs to develop the most efficacious treatment for cognitive improvement in people with schizophrenia.

Keywords: Nicotine; Clozapine; Haloperidol; Risperidone; Memory; Attention; Schizophrenia

Treating schizophrenia symptoms with an α7 nicotinic agonist, from mice to men by Ann Olincy; Karen E. Stevens (pp. 1192-1201).

Current antipsychotic treatments fail to fully address the range of symptoms of schizophrenia, particularly with respect to social and occupational dysfunctions. Recent work has highlighted the role of nicotinie in both cognitive and attentional deficits as well as deficient processing of repetitive sensory information. The predilection for schizophrenia patients to be extremely heavy cigarette smokers may be related to their attempt to compensate for a reduction in hippocampal α7 nicotinic cholinergic receptors by delivering exogenous ligand to the remaining receptors. Studies in rodent models of both learning and memory deficits and deficits in sensory inhibition have confirmed a role for the α7 subtype of the nicotinic cholinergic receptor in these processes. Rodent studies also demonstrated the efficacy of a selective partial α7 nicotinic agonist, DMXBA, to improve these deficits. Subsequent human clinical trials demonstrated improved sensory inhibition in 12 schizophrenia patients and showed improvement in several subtests of the RBANS learning and memory assessment instrument. These data suggest that therapeutic agents selected for α7 nicotinic activity may have utility in treating certain symptoms of schizophrenia.

Keywords: Abbreviations; DMXBA; 3-[2,4-dimethoxybenzylidene]anabaseine; PPI; prepulse startle inhibition; RBANS; repeatable battery for assessment for neuropsychological status; SNP; single nucleotide polymorphismSchizophrenia; Nicotinic receptors; DMXBA; P50 auditory evoked potential; Cognitive deficits; Nicotine

Profile of nicotinic acetylcholine receptor agonists ABT-594 and A-582941, with differential subtype selectivity, on delayed matching accuracy by young monkeys by Jerry J. Buccafusco; Alvin V. Terry Jr.; Michael W. Decker; Murali Gopalakrishnan (pp. 1202-1211).

ABT-594 and A-582941 are high affinity neuronal nicotinic acetylcholine receptor agonists with differential selectivity for the α4β2 and the α7 subtypes, respectively. This study was designed to determine whether either compound, like nicotine also possesses cognitive-enhancing ability. The compounds were administered by intramuscular injection to young adult Rhesus monkeys trained to perform two versions of a computer-assisted delayed matching-to-sample (DMTS) task. ABT-594 (0.115–3.7μg/kg) significantly improved DMTS accuracies, shifting the retention curve (accuracy–delay relationship) to the right in a parallel fashion. DMTS accuracy also was maintained during the sessions initiated 24h after compound administration. Because task accuracy was improved during short delay trials, a separate study was performed in which non-predictable distractors were inserted within the DMTS format to impair accuracy. The 0.115μg/kg dose of ABT-594 almost completely reversed distractor-impaired performance associated with short delay trials. The α7 nAChR agonist, A-582941 (1.14–38μg/kg) also significantly improved DMTS accuracies. The compound produced a significant improvement during long delay trials. The effect was twice as robust for long delay as compared with short delay trials and A-582941 was not as effective as ABT-594 in improving short delay trial accuracy. A-582941 also failed to sustain task improvement during sessions run 24h after dosing. These data are consistent with the ability of subtype-preferring nicotinic receptor agonists to enhance specific components of working memory and cognitive function, and they suggest that differential subtype selectivity could result in varied pharmacological response profiles.

Keywords: Nicotinic acetylcholine receptor agonist; Delayed matching; Operant task; Attention; Memory; Rhesus monkey; ABT-594; A-582491

Neuronal nicotinic receptor agonists for the treatment of attention-deficit/hyperactivity disorder: Focus on cognition by Timothy E. Wilens; Michael W. Decker (pp. 1212-1223).

Attention deficit/hyperactivity disorder (ADHD) is the most commonly diagnosed neurobehavioral disorder in children and adolescents, and in about half of these patients, significant symptomology continues into adulthood. Although impulsivity and hyperactivity are the most salient features of ADHD, cognitive deficits, particularly impairments in attention and executive function, are an important component, particularly in adolescents and adults, with over 90% of adults seeking treatment for ADHD manifesting cognitive dysfunction. Currently available medications treat the core ADHD symptoms but typically do not adequately address cognitive aspects of ADHD, underscoring the need for new therapeutics. Dopamine and norepinephrine are hypothesized to be particularly important in ADHD, but there is emerging evidence that cholinergic neurotransmission, particularly involving neuronal nicotinic acetylcholine receptors (nAChRs), may play a role in the pathophysiology of ADHD. Nicotine has demonstrated procognitive effects in both humans and experimental animals and has produced signals of efficacy in small proof-of-concept adult ADHD trials. Although adverse effects associated with nicotine preclude its development as a therapeutic, a number of novel nAChR agonists with improved safety/tolerability profiles have been discovered. Of these, ABT-418 and ABT-089 have both demonstrated signals of efficacy in adults with ADHD. Notably, tolerability issues that might be expected of a nAChR agonist, such as nausea and emesis, were not observed at efficacious doses of ABT-089. Further understanding of the effects of novel neuronal nAChR agonists on specific aspects of cognitive functioning in ADHD is required to assess the full potential of this approach.

Keywords: ADHD; ABT-418; ABT-089; Children; Adolescents; Cognitive deficits; Treatment

Nicotinic receptors as CNS targets for Parkinson's disease by Maryka Quik; Tanuja Bordia; Kathryn O’Leary (pp. 1224-1234).

Parkinson's disease is a debilitating neurodegenerative movement disorder characterized by damage to the nigrostriatal dopaminergic system. Current therapies are symptomatic only and may be accompanied by serious side effects. There is therefore a continual search for novel compounds for the treatment of Parkinson's disease symptoms, as well as to reduce or halt disease progression. Nicotine administration has been reported to improve motor deficits that arise with nigrostriatal damage in parkinsonian animals and in Parkinson's disease. In addition, nicotine protects against nigrostriatal damage in experimental models, findings that have led to the suggestion that the reduced incidence of Parkinson's disease in smokers may be due to the nicotine in tobacco. Altogether, these observations suggest that nicotine treatment may be beneficial in Parkinson's disease. Nicotine interacts with multiple nicotinic receptor (nAChR) subtypes in the peripheral and central nervous system, as well as in skeletal muscle. Work to identify the subtypes affected in Parkinson's disease is therefore critical for the development of targeted therapies. Results show that striatal α6β2-containing nAChRs are particularly susceptible to nigrostriatal damage, with a decline in receptor levels that closely parallels losses in striatal dopamine. In contrast, α4β2-containing nAChRs are decreased to a much smaller extent under the same conditions. These observations suggest that development of nAChR agonists or antagonists targeted to α6β2-containing nAChRs may represent a particularly relevant target for Parkinson's disease therapeutics.

Keywords: Abbreviations; nAChR; nicotinic acetylcholine receptor; *; indicates the possible presence of other subunits in the receptor complexα-ConotoxinMII; Nicotine; Nicotinic; Parkinson's disease; Nigrostriatal; Striatum

The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum by Sharon R. Grady; Outi Salminen; Duncan C. Laverty; Paul Whiteaker; J. Michael McIntosh; Allan C. Collins; Michael J. Marks (pp. 1235-1246).

This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [¹²⁵I]-α-bungarotoxin and [³H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes. The discovery of epibatidine and α-conotoxin MII (α-CtxMII), and the development of nAChR subunit null mutant mice have been invaluable in determining which nAChR subunits are important for expression and function in mice, as well as allowing validation of the specificity of subunit specific antibodies. These approaches have identified five nAChR subtypes of nAChR that are expressed on dopaminergic nerve terminals. Three of these contain the α6 subunit (α4α6β2β3, α6β2β3, α6β2) and bind α-CtxMII with high affinity. One of these three subtypes (α4α6β2β3) also has the highest sensitivity to nicotine of any native nAChR that has been studied, to date. The two subtypes that do not have high affinity for α-CtxMII (α4β2, α4α5β2) are somewhat more numerous than the α6* subtypes, but do bind nicotine with high affinity. Given that our first studies detected readily measured differences in sensitivity to agonists and antagonists among these five nAChR subtypes, it seems likely that subtype selective compounds could be developed that would allow therapeutic manipulation of diverse nAChRs that have been implicated in a number of human conditions.

Keywords: Nicotinic acetylcholine receptors; Dopaminergic neurons; Null mutant mice; In situ; hybridization; Agonist and antagonist binding; Neurotransmitter release from synaptosomes

Behavioral modulation of neuronal calcium/calmodulin-dependent protein kinase II activity: Differential effects on nicotine-induced spinal and supraspinal antinociception in mice by M. Imad Damaj (pp. 1247-1252).

Recent studies have implicated the involvement of Ca²⁺-dependent mechanisms, in particular calcium/calmodulin-dependent protein kinase II (CaM kinase II) in nicotine-induced antinociception using the tail-flick test. The spinal cord was suggested as a possible site of this involvement. The present study was undertaken to investigate the hypothesis that similar mechanisms exist for nicotine-induced antinociception in the hot-plate test, a response thought to be centrally mediated. In order to assess these mechanisms, i.c.v. administered CaM kinase II inhibitors were evaluated for their effects on antinociception produced by either i.c.v. or s.c. administration of nicotine in both tests. In addition, nicotine's analgesic effects were tested in mice lacking half of their CaM kinase II (CaM kinase II heterozygous) and compare it to their wild-type counterparts. Our results showed that although structurally unrelated CaM kinase II inhibitors blocked nicotine's effects in the tail-flick test in a dose-related manner, they failed to block the hot-plate responses. In addition, the antinociceptive effects of systemic nicotine in the tail-flick but not the hot-plate test were significantly reduced in CaM kinase II heterozygous mice. These observations indicate that in contrast to the tail-flick response, the mechanism of nicotine-induced antinociception in the hot-plate test is not mediated primarily via CaM kinase II-dependent mechanisms at the supraspinal level.

Keywords: Abbreviations; CaM kinase II; calcium/calmodulin-dependent protein kinase II; nAChR; acetylcholine nicotinic receptor; CNS; central nervous system; %MPE; maximum possible effect; CL; confidence limit for the AD; ₅₀; s.c.; subcutaneous injection; i.c.v.; intracerebroventricular injection; AD; ₅₀; antagonist dose 50%Nicotine; Calmodulin kinase II; Antinociception; Tail-flick; Hot-plate; Mice

A-366833: A novel nicotinonitrile-substituted 3,6-diazabicyclo[3.2.0]-heptane α4β2 nicotinic acetylcholine receptor selective agonist: Synthesis, analgesic efficacy and tolerability profile in animal models by Jianguo Ji; William H. Bunnelle; David J. Anderson; Connie Faltynek; Tino Dyhring; Philip K. Ahring; Lynne E. Rueter; Peter Curzon; Michael J. Buckley; Kennan C. Marsh; Anita Kempf-Grote; Michael D. Meyer (pp. 1253-1262).

5-[(1 R,5 S)-3,6-Diazabicyclo[3.2.0]heptan-6-yl]nicotinonitrile (A-366833) is a novel nicotinic acetylcholine receptor (nAChR) ligand that binds to the agonist-binding site ([³H]-cytisine) with K_i value of 3.1nM and exhibits agonist selectivity at α4β2 nAChR relative to the α3β4 nAChR subtype. The analgesic effects of A-366833 were examined across a variety of animal models including the mouse model of writhing pain (abdominal constriction), the rat models of acute thermal (hot box), persistent chemical (formalin) and neuropathic (spinal nerve ligation, SNL) pain. In the abdominal constriction model, A-366833 was effective at doses ranging from 0.062 to 0.62μmol/kg (i.p.). In addition, A-366833 demonstrated significant effects in acute thermal pain (6.2–19.0μmol/kg, i.p.), formalin (1.9–19μmol/kg i.p.) and SNL (1.9–19μmol/kg i.p.) models. The systemic effects of A-366833 were attenuated by pretreatment with mecamylamine (5μmol/kg i.p.) in both the formalin and SNL models, suggesting that the analgesic effects of A-366833 in models of persistent nociceptive and neuropathic pain are mediated by activation of nAChRs. Pharmacokinetic investigations of A-366833 in rat revealed moderate brain:plasma distribution, half-life of 1.5h and excellent oral bioavailability of 73%. Comparison of peak plasma levels at the minimal effective doses across rat models of acute thermal pain, formalin and SNL with the maximal exposure that does not evoke emesis in ferret revealed therapeutic margins ranging from 6- to 22-fold. These studies indicate that compounds like A-366833 with improved agonist selectivity at α4β2 vs. α3β4 nAChR can elicit a broad spectrum of analgesic efficacy without concurrent adverse effects.

Keywords: nAChR; Nicotine; Epibatidine; A-366833; Pain; Emesis

Multiple brain pathways and receptors underlying tobacco addiction by Jed E. Rose (pp. 1263-1270).

Over the last 20 years much progress has been made in understanding the pharmacologic basis of tobacco addiction. In particular, the role of nicotine in reinforcing smoking behavior has been studied from a variety of perspectives. This article discusses two important aspects of this topic: (1) brain pathways underlying tobacco addiction; and (2) the actions of nicotine at nicotinic cholinergic receptors. Recent evidence will be reviewed indicating that nicotine reinforces smoking behavior by acting on more than one subtype of nicotinic receptor. Similarly, the role of several brain pathways in tobacco addiction will be considered. Tobacco addiction may thus be seen as a complex neuropsychopharmacological disorder; further progress in smoking cessation treatment may require that we address the multiple molecular and brain components of this addiction.

Keywords: Nicotine; Nicotinic; Tobacco; Addiction; Receptor; Brain

Discovery of a novel nicotinic receptor antagonist for the treatment of nicotine addiction: 1-(3-Picolinium)-12-triethylammonium-dodecane dibromide (TMPD) by Linda P. Dwoskin; B. Matthew Joyce; Guangrong Zheng; Nichole M. Neugebauer; Vamshi K. Manda; Paul Lockman; Roger L. Papke; Michael T. Bardo; Peter A. Crooks (pp. 1271-1282).

Limitations in efficacy and high relapse rates of currently available smoking cessation agents reveal the need for more efficacious pharmacotherapies. One strategy is to develop subtype-selective nicotinic receptor (nAChR) antagonists that inhibit nicotine-evoked dopamine (DA) release, the primary neurotransmitter involved in nicotine reward. Simple alkylation of the pyridino N-atom converts nicotine from a potent agonist into a potent antagonist. The classical antagonists, hexamethonium and decamethonium, differentiate between peripheral nAChR subtypes. Using a similar approach, we interconnected varying quaternary ammonium moieties with a lipophilic linker to provide N, N′-bis-nicotinium analogs, affording a lead compound, N, N′-dodecyl-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), which inhibited nicotine-evoked DA release and decreased nicotine self-administration. The current work describes a novel compound, 1-(3-picolinium)-12-triethylammonium-dodecane dibromide (TMPD), a hybrid of bPiDDB and decamethonium. TMPD completely inhibited (IC₅₀=500nM) nicotine-evoked DA release from superfused rat striatal slices, suggesting that TMPD acts as a nAChR antagonist at more than one subtype. TMPD (1μM) inhibited the response to acetylcholine at α3β4, α4β4, α4β2, and α1β1ɛδ receptors expressed in Xenopus oocytes. TMPD had a 2-fold higher affinity than choline for the blood–brain barrier choline transporter, suggesting brain bioavailability. TMPD did not inhibit hyperactivity in nicotine sensitized rats, but significantly and specifically decreased nicotine self-administration. Together, the results suggest that TMPD may have the ability to reduce the rewarding effect of nicotine with minimal side effects, a pharmacological profile indicative of potential clinical utility for the treatment of tobacco dependence.

Keywords: Dopamine; Nicotine; Nicotinic receptors; Blood–brain barrier choline transporter; Smoking cessation; TMPD

Bupropion inhibits the cellular effects of nicotine in the ventral tegmental area by Huibert D. Mansvelder; Zara M. Fagen; Ben Chang; Robert Mitchum; Daniel S. McGehee (pp. 1283-1291).

Each year, tobacco use causes over 4 million deaths worldwide and billions of dollars are spent on treatment for tobacco-related illness. Bupropion, an atypical antidepressant, improves the rates of successful smoking cessation, however, the mechanisms by which bupropion reduces cigarette smoking and depression are unknown. Here we show that clinical concentrations of bupropion inhibit nicotine's stimulatory effects on brain reward areas. Many drugs of abuse, including nicotine, stimulate dopamine (DA) release in the mesoaccumbens reward system. Nicotinic acetylcholine receptors in the ventral tegmental area (VTA) mediate nicotine's stimulation of DA release, as well as its rewarding effects. Nicotinic receptors are expressed by excitatory and inhibitory neurons that control DA neuron excitability, and by the DA neurons themselves. Bupropion is a broad-spectrum non-competitive nicotinic receptor antagonist. Here we report that pre-treatment of brain slices with a clinically relevant concentration of bupropion dramatically reduces the effects of nicotine on DA neuron excitability. Nicotinic receptors on VTA DA neurons and their synaptic inputs are inhibited by 75 – 95% after bupropion treatment. We also find that bupropion alone reduces GABAergic transmission to DA neurons, thereby diminishing tonic inhibition of these neurons. This increases DA neuron excitability during bupropion treatment in the absence of nicotine, and may contribute to bupropion's antidepressant actions.

Keywords: Acetylcholine; Dopamine; Glutamate; GABA; Synaptic Transmission; Nicotinic

Comparison of the effects of bupropion and nicotine on locomotor activation and dopamine release in vivo by Nimish Sidhpura; Peter Redfern; Helen Rowley; David Heal; Susan Wonnacott (pp. 1292-1298).

Bupropion is an atypical anti-depressant that is approved for smoking cessation. In addition to inhibiting dopamine reuptake, bupropion has been reported to block nicotinic acetylcholine receptors in vitro, and this action might contribute to its efficacy for smoking cessation. In this study we investigated if nicotinic receptor-mediated responses in vivo are decreased in the presence of a behaviorally effective dose of bupropion. In separate experiments we measured locomotor activation and dopamine overflow in the nucleus accumbens core, using in vivo microdialysis in freely moving rats. Bupropion (30mg/kg i.p.) increased locomotor activity, which remained elevated for up to 2h. Nicotine (0.4mg/kg s.c.) also increased locomotor activity but for a shorter duration. When given 20min after bupropion, hyperlocomotion was significantly enhanced, compared to the response to either nicotine or bupropion alone, consistent with the effects of the two drugs being additive. Systemic administration of bupropion (30mg/kg i.p.) also elicited a significant increase in dopamine overflow (113±16% above basal levels). Nicotine (3mM; delivered into the nucleus accumbens core via the microdialysis probe) increased dopamine overflow by 126±35%. Nicotine delivered during the response to bupropion resulted in enhanced dopamine overflow of 294±50%, also consistent with the actions of the two drugs being additive. This study suggests that behaviorally effective concentrations of bupropion in the rat do not diminish the effects of nicotine by blocking nicotinic receptors.

Keywords: Abbreviations; DAT; dopamine transporter; NAc; nucleus accumbens; NAc; _core; nucleus accumbens core; nAChR; nicotinic acetylcholine receptor; VTA; ventral tegmental areaBupropion; Dopamine overflow; Locomotor activation; Microdialysis; Nicotine; Nucleus accumbens

Metabotropic glutamate 2/3 receptor activation induced reward deficits but did not aggravate brain reward deficits associated with spontaneous nicotine withdrawal in rats by Matthias E. Liechti; Athina Markou (pp. 1299-1307).

Glutamate neurotransmission, and particularly metabotropic glutamate (mGlu) 2/3 receptors are implicated in behaviors of relevance to the addictive properties of nicotine. In laboratory animals, the mGlu2/3 receptor agonist LY379268 has been previously shown to decrease intravenous nicotine self-administration and cue-induced reinstatement of nicotine-seeking behavior. Such mGlu2/3 receptor agonists may therefore be useful medications to assist people in smoking cessation. Because of the demonstrated preclinical efficacy of mGlu2/3 receptor agonists in decreasing the primary rewarding and conditioned effects of nicotine in rats, we wished to examine whether such compounds could potentially influence additional aspects of nicotine dependence, such as nicotine withdrawal. We hypothesized that an mGlu2/3 receptor agonist would have negative effects on nicotine withdrawal because mGlu2/3 receptor antagonists have previously been shown to attenuate nicotine withdrawal-induced reward deficits, while an mGlu2/3 receptor agonist precipitated withdrawal-like reward deficits in rats dependent on nicotine. To test this hypothesis, we assessed the effects of the mGlu2/3 receptor agonist LY379268 on brain reward deficits associated with spontaneous nicotine withdrawal in rats. Brain reward function, as assessed by intracranial self-stimulation reward thresholds, was examined after removal of nicotine- or saline-delivering subcutaneous osmotic minipumps. LY379268 administration produced reward deficits in animals “withdrawing” from chronic saline administration and only tended to aggravate nicotine withdrawal-induced reward deficits in rats previously treated with nicotine. Thus, this mGlu2/3 agonist does not appear to significantly influence the affective depression-like aspects of nicotine withdrawal.

Keywords: LY379268; Metabotropic 2/3 glutamate receptor agonist; Nicotine; Withdrawal; Intracranial self-stimulation; Reward; Dependence; Rat

The role of the nicotinic acetylcholine receptors in sleep-related epilepsy by Carla Marini; Renzo Guerrini (pp. 1308-1314).

The role of neuronal acetylcholine receptors (nAChRs) in epilepsy has been clearly established by the finding of mutations in a subset of genes coding for subunits of the nAChRs in a form of sleep-related epilepsy with familial occurrence in about 30% of probands and dominant inheritance, named autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sporadic and familial forms have similar clinical and EEG features. Seizures begin in middle childhood as clusters of sleep-related attacks with prominent motor activity, and sustained dystonic posturing. In addition to nocturnal seizures, psychosis or schizophrenia, behavioral disorders, memory deficits and mental retardation were described in some individuals. Although over hundred families are on record, only a minority of them have been linked to mutations in the genes coding for the α4, α2 and β2 ( CHRNA4, CHRNA2, and CHRNB2) subunits of the nAChRs, indicating that ADNFLE is genetically heterogeneous despite a relatively homogeneous clinical picture. Functional characterization of some mutations suggests that gain of the receptor function might be the basis for epileptogenesis. In vitro and in vivo studies have shown high density of nAChRs in the thalamus, over activated brainstem ascending cholinergic pathway and enhanced GABAergic function, reinforcing the hypothesis that cortico-subcortical networks, regulating arousal from sleep, play a central role in seizure precipitation in ADNFLE.

Keywords: Abbreviations; ACh; acetylcholine; ADNFLE; autosomal dominant nocturnal frontal lobe epilepsy; AEDs; antiepileptic drugs; CBZ; carbamazepine; CHRNA4; α4 subunit of the neuronal acetylcholine receptor gene; CHRNA2; α2 subunit of the neuronal acetylcholine receptor gene; CHRNB2; β2 subunit of the neuronal acetylcholine receptor gene; GABA; gamma-aminobutyric acid; EEG; electroencephalogram; nAChRs; neuronal nicotinic acetylcholine receptors; NFLE; nocturnal frontal lobe epilepsy; NPD; nocturnal paroxysmal dystonia; NREM; non-rapid eye movement; NW; nocturnal wanderings; PA; paroxysmal arousals; PET; positron emission tomography; SPECT; single-photon emission computed tomography; TM2; transmembrane domain 2; TM3; transmembrane domain 3Idiopathic focal epilepsy; ADNFLE; Acetylcholine receptors; CHRNA4; CHRNB2; CHRNA2

Overexpression of SLURP-1 and -2 alleviates the tumorigenic action of tobacco-derived nitrosamine on immortalized oral epithelial cells by Juan Arredondo; Alex I. Chernyavsky; Sergei A. Grando (pp. 1315-1319).

Recent research has demonstrated that mucocutaneous epithelial cells express functional nicotinic acetylcholine receptors (nAChRs) and that tobacco-derived carcinogenic nitrosamines, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein)-1 and -2 can act as non-canonical ligands of these receptors. It was found that recombinant SLURP-1 and -2 can lessen tumorigenic activity of nitrosamines. The immortalized esophageal keratinocytes (Het-1A cells) exhibit low SLURP-1 and -2 mRNA levels that decrease further after treatment with NNK. Based on these observations, we hypothesized that overexpression of full length SLURP proteins may protect Het-1A cells from malignant transformation by NNK. The Het-1A cells transfected with either SLURP-1 or -2 vector produced the highest amounts of respective proteins between 24 and 48h, at which point they were exposed to 1μM NNK for 24h and their tumorigenic activities were subsequently evaluated by plating in soft agar and injecting subcutaneously to Nu/Nu mice. Transfection with either SLURP-1 or -2 cDNA in both cases significantly ( p<0.05) diminished the number of colonies produced by NNK exposed cells. SLURP-1 was more efficient than SLURP-2 in abolishing the tumorigenic effect in nude mice. Thus, the anti-tumorigenic activities of SLURP-1 and -2 were demonstrated both in vitro and in vivo. The obtained results suggest that SLURP-like proteins may become useful for developing novel anti-cancer therapies.

Keywords: Nicotinic acetylcholine receptor; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; Het-1A cells; SLURP-1; SLURP-2

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Biochemical Pharmacology (v.74, #8)