Understanding the role α7 nicotinic receptors play in dopamine efflux in nucleus accumbens

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.Neuronal nicotinic acetylcholine receptors (NNRs) of the α7 subtype have been shown to contribute to the release of dopamine in the nucleus accumbens. The site of action and the underlying mechanism, however, are unclear. Here we applied a circuit modeling approach, supported by electrochemical in vivo recordings, to clarify this issue. Modeling revealed two potential mechanisms for the drop in accumbal dopamine efflux evoked by the selective α7 partial agonist TC-7020. TC-7020 could desensitize α7 NNRs located predominantly on dopamine neurons or glutamatergic afferents to them or, alternatively, activate α7 NNRs located on the glutamatergic afferents to GABAergic interneurons in the ventral tegmental area. Only the model based on desensitization, however, was able to explain the neutralizing effect of coapplied PNU-120596, a positive allosteric modulator. According to our results, the most likely sites of action are the preterminal α7 NNRs controlling glutamate release from cortical afferents to the nucleus accumbens. These findings offer a rationale for the further investigation of α7 NNR agonists as therapy for diseases associated with enhanced mesolimbic dopaminergic tone, such as schizophrenia and addiction.Peer reviewe

Evaluation of structurally diverse neuronal nicotinic receptor ligands for selectivity at the α6 subtype

Direct comparison of pyridine versus pyrimidine substituents on a small but diverse set of ligands indicates that the pyrimidine substitution has the potential to enhance affinity and/or functional activity at α6 subunit-containing neuronal nicotinic receptors (NNRs) and decrease activation of ganglionic nicotinic receptors, depending on the scaffold. The ramifications of this structure–activity relationship are discussed in the context of the design of small molecules targeting smoking cessation

Structural differences determine the relative selectivity of nicotinic compounds for native α4β2^*-, α6β2^*-, α3β4^*- and α7-nicotine acetylcholine receptors

Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, α4β2^* and α6β2^*-nAChR have the highest affinity for nicotine (where ^* indicates possibility of other subunits). The α4β2^*-nAChRs are widely distributed, while α6β2^*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the mesoaccumbens pathway thought to be essential for reward and addiction. α4β2^*-nAChR also modulate GABA release in these areas. Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics.We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compoundswere screened usingmembrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing hα7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, α4β2^*, α6β2^*, α7 and α3β4^*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at α4b2^* and α6β2^*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for α6β2^*-nAChR

TC299423, a Novel Agonist for Nicotinic Acetylcholine Receptors

(E)-5-(Pyrimidin-5-yl)-1,2,3,4,7,8-hexahydroazocine (TC299423) is a novel agonist for nicotinic acetylcholine receptors (nAChRs). We examined its efficacy, affinity, and potency for α6β2^∗ (α6β2-containing), α4β2^∗, and α3β4^∗ nAChRs, using [^(125)I]-epibatidine binding, whole-cell patch-clamp recordings, synaptosomal ^(86)Rb^+ efflux, [^3H]-dopamine release, and [^3H]-acetylcholine release. TC299423 displayed an EC_(50) of 30–60 nM for α6β2^∗ nAChRs in patch-clamp recordings and [^3H]-dopamine release assays. Its potency for α6β2^∗ in these assays was 2.5-fold greater than that for α4β2^∗, and much greater than that for α3β4^∗-mediated [^3H]-acetylcholine release. We observed no major off-target binding on 70 diverse molecular targets. TC299423 was bioavailable after intraperitoneal or oral administration. Locomotor assays, measured with gain-of-function, mutant α6 (α6L9′S) nAChR mice, show that TC299423 elicits α6β2^∗ nAChR-mediated responses at low doses. Conditioned place preference assays show that low-dose TC299423 also produces significant reward in α6L9′S mice, and modest reward in WT mice, through a mechanism that probably involves α6(non-α4)β2^∗ nAChRs. However, TC299423 did not suppress nicotine self-administration in rats, indicating that it did not block nicotine reinforcement in the dosage range that was tested. In a hot-plate test, TC299423 evoked antinociceptive responses in mice similar to those of nicotine. TC299423 and nicotine similarly inhibited mouse marble burying as a measure of anxiolytic effects. Taken together, our data suggest that TC299423 will be a useful small-molecule agonist for future in vitro and in vivo studies of nAChR function and physiology

Differential Sensitivity Of Phosphoinositide Metabolism To Sodium Fluoride And Carbachol Treatments In Pc12 Cells

Exposure to sodium fluoride (NaF) resulted in an increased accumulation (up to 10-fold) of total [3H]inositol phosphates (T-InsP) in rat PC 12 cells. The magnitude of the NaF effect was comparable to that for muscarinic acetylcholine receptor-mediated stimulation of T-InsP accumulation in the presence of saturating concentrations of carbachol, but effects of NaF and muscarinic agonists were additive at subsaturating concentrations. The NaF effect was atropine insensitive; was not mimicked by effects of NaCl (10 mM), aluminum fluoride (1 to 100 μM), forskolin (up to 100 μM), or dibutyryl cyclic AMP (1 mM); and was not altered by treatment with pertussis or cholera toxins (1 μg/ml for 24 h). By contrast, the carbachol response was fully sensitive to atropine and partly sensitive to pertussis toxin. Chelation of extracellular calcium ion following 10 min of pretreatment with EDTA or EGTA (3 mM) inhibited carbachol-stimulated T-InsP accumulation by 50%, but resulted in an enhancement of NaF-stimulated T-InsP accumulation. By contrast, inhibition of the mobilization of intracellular calcium ion with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate inhibited NaF stimulation of T-InsP accumulation by more than 50% but inhibited carbachol-stimulated TInsP accumulation to a much lower extent. Enhanced calcium influx and cell depolarization stimulated by high extracellular concentrations of KCl markedly potentiated carbachol, but not NaF, stimulation of T-InsP accumulation. This differential sensitivity to muscarinic antagonists, cell depolarization, and manipulation of intra- and extracellular calcium ion indicates that different mechanisms underly NaF and carbachol stimulation of T-InsP accumulation. However, stimulation of T-InsP accumulation in the presence of carbachol alone, NaF alone, or carbachol plus NaF was inhibited to a similar extent in the presence of the phorbol ester, phorbol 12-myristate13-acetate. Taken together, these observations suggest that NaF and carbachol effects are mediated through distinct mechanisms but share a common target, perhaps a GTP-binding protein and/or phospholipase C, whose activity is known to be influenced by protein kinase C. © 1991

Heterogeneity And Regulation Of Nicotinic Acetylcholine Receptors

This chapter focuses on nicotinic acetylcholine receptors (nAChRs). This component emphasizes studies done using mammalian tissues or their derivatives, and using vertebrate nonmammalian systems. The chapter foscuses on the bases, manifestations, and functional significance of nAChR diversity. There are at least two populations of putative nAChRs in brain defined based on their ability to interact with 3H-labeled agonists or radiolabeled α-bungarotoxin. These binding sites are differentially regulated during development and exhibit differential anatomical distributions. Studies done on nAChRs in the periphery at the neuromuscular junction or on neural crest-derived cells are largely consistent with this dogma, in that chronic agonist treatment produces a decrease in receptor numbers and usually a comparable or quantitatively larger decrease in functional responsiveness. Chronic administration of two pyrethroid xenobiotics, bioallethrin and deltamethrin, produces an increase in levels of nAChRs. It is also becoming clear that diversity in mechanisms involved in the regulation of nAChR expression and function is built on the diversity of the nAChR family. © 1992, Academic Press Inc

Vanadate Amplifies Receptor-Mediated Accumulation Of Inositol Trisphosphates And Inhibits Inositol Tris- And Tetrakis-Phosphatase Activities

Lithium ion, which inhibits hydrolytic degradation of inositol monophosphates, is the most common therapeutic agent used in the control of bipolar disorder. There exists evidence that elevated elemental vanadium levels may play an etiological role in at least some forms of manic-depression. Here we demonstrate that vanadate treatment of intact cells from several different clonal lines synergistically induces substantial augmentation in neurotransmitter receptor-mediated or growth factor receptor-triggered inositol trisphosphate accumulation in situ. Furthermore, studies done using cellular extracts indicate that effects of vanadate treatment in situ may be due to its ability to inhibit hydrolysis of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate in vitro. These results suggest that vanadate treatment may facilitate characterization of inositol phosphate metabolism and intracellular signaling. © 1992

Differential Regulation Of Nicotinic Acetylcholine Receptor Expression By Human Te671/Rd Cells Following Second Messenger Modulation And Sodium Butyrate Treatments

Effects of second messenger system modulation and sodium butyrate (NaBu) treatment on nicotinic acetylcholine receptor (nAChR) expression by cells of the TE671/RD human clone were established. Treatment with dibutyryl cyclic adenosine 3′:5′-monophosphate (dbcAMP) or other substances that increase cellular cAMP content induces a 70% loss of nAChR per unit of membrane protein as assessed by binding studies using 125I-labeled α-bungarotoxin (I-Bgt). By contrast, phorbol 12-myristate-13-acetate (PMA) treatment induces an initial 50% decrease, and then a later two- to threefold increase, in I-Bgt binding sites. These PMA effects are temporally distinct from a PMA treatment-induced 50% downregulation of membrane-bound phorbol ester binding sites, are blocked by treatment of cells with the putative protein kinase C inhibitors H7 or trifluoroperazine, and are sensitive to the protein synthesis inhibitor cycloheximide and the topoisomerase inhibitor novobiocin. Treatment with both PMA and dbcAMP induces a threefold increase in nAChR expression, whereas treatment with NaBu alone or with PMA induces an 80% decrease in I-Bgt binding site expression. All of these effects are dose and time dependent and reflect changes in the number of binding sites rather than changes in nAChR afnity for I-Bgt. These data indicate involvement of both cAMP and C-kinase pathways in the regulation of nAChR expression in ways that are not simply additive, possibly due to cross-talk between second messenger pathways. In addition, transcriptional and/or translational events are implicated in PMA and NaBu effects. The results indicate a multiplicity in the effects and mechanisms involved in regulation of nAChR expression. © 1991

Cytochalasin Modulation Of Nicotinic Cholinergic Receptor Expression And Muscarinic Receptor Function In Human Te671/Rd Cells: A Possible Functional Role Of The Cytoskeleton

Abstract: Previous studies have shown that cells of the TE671/RD human clonal line express muscleâ€type nicotinic acetylcholine receptors (nAChR) and m3â€type muscarinic acetylcholine receptors (mAChR) whose numbers and function are regulated by agonist treatment and second messenger modulation. Here we show that cytochalasin treatment, which causes disruption of actin networks, induces marked changes in the numbers and distribution of nAChR, but not mAChR. Moreover, whereas cytochalasin treatment fails to alter nAChR function significantly, it acutely potentiates mAChRâ€mediated phosphoinositide hydrolysis. Treatment of TE671/RD cells with different cytochalasin analogues (rank order efficacy at 5 μg/ml is H \u3eJ = B = C = D\u3eA = E) produces a twoâ€to fourfold increase in numbers of membraneâ€bound nAChR (Smax in units of specific 125lâ€labeled αâ€bungarotoxin binding per milligram of membrane protein). nAChR upâ€regulation is evident after 1–2 days of cytochalasin B exposure, is maximal after 3–6 days of drug treatment, and is dominated by an approximately 10â€fold increase (per cell) in an intracellular nAChR pool. Cytochalasinâ€induced nAChR upâ€regulation is similar in magnitude to, but not additive with, upâ€regulation of nAChR following chronic exposure to nicotine or phorbol ester. Northern blot analysis shows a fourâ€to fivefold coordinate increase in levels of mRNA that encode nAChR α, β, γ, or θ subunits in cytochalasinâ€treated cells, suggesting that nAChR upâ€regulation has a possible transcriptional basis. Studies done using a 86Rb+ efflux assay indicate that cytochalasin treatment has no significant effect on nAChR function. By contrast, cytochalasin treatment has no effect on the numbers of mAChR as assessed by binding studies with the radioantagonist 3Hâ€labeled quinuclidinyl benzilate, but it induces marked enhancement of carbacholâ€stimulated, but not basal, phosphoinositide hydrolysis. These studies suggest that presumed modulation by cytochalasin treatment of cytoskeletal microfilament integrity can differentially influence expression and function of mAChR (a prototype of the metabotropic receptor superfamily) and nAChR (a prototype of the ligandâ€gated ionâ€channel superfamily). The results also suggest possible new roles for the cytoskeleton in regulation of membrane receptor expression, function, and cross talk. Copyright © 1993, Wiley Blackwell. All rights reserve